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Showing papers in "Biochemistry in 1985"


Journal ArticleDOI
TL;DR: Using lattice statistical mechanics, theory is developed to account for the folding of a heteropolymer molecule such as a protein to the globular and soluble state and the number of accessible conformations is calculated to be an exceedingly small fraction of the number available to the random coil.
Abstract: Using lattice statistical mechanics, we develop theory to account for the folding of a heteropolymer molecule such as a protein to the globular and soluble state. Folding is assumed to be driven by the association of solvophobic monomers to avoid solvent and opposed by the chain configurational entropy. Theory predicts a phase transition as a function of temperature or solvent character. Molecules that are too short or too long or that have too few solvophobic residues are predicted not to fold. Globular molecules should have a largely solvophobic core, but there is an entropic tendency for some residues to be "out of place", particularly in small molecules. For long chains, molecules comprised of globular domains are predicted to be thermodynamically more stable than spherical molecules. The number of accessible conformations in the globular state is calculated to be an exceedingly small fraction of the number available to the random coil. Previous estimates of this number, which have motivated kinetic theories of folding, err by many tens of orders of magnitude.

1,222 citations


Journal ArticleDOI
TL;DR: Deglycosylation studies with PNGase F revealed that many proteins in their native conformation were susceptible to this enzyme but that prior denaturation in sodium dodecyl sulfate greatly decreased the amount of enzyme required for complete carbohydrate removal.
Abstract: Endo-beta-N-acetylglucosaminidase F (Endo F) and peptide:N-glycosidase F (PNGase F) were purified from cultures of Flavobacterium meningosepticum by ammonium sulfate precipitation followed by gel filtration on TSK HW-55(S). This system separated the two enzymes and provided PNGase F in a high state of purity, but the basis for the resolution appeared to be hydrophobic interaction and not molecular size. Studies using purified Endo F and PNGase F with defined glycopeptides demonstrated that Endo F was somewhat similar to Endo H in that it hydrolyzed many, but not all, high-mannose and hybrid oligosaccharides, as well as complex biantennary oligosaccharides. PNGase F, in contrast, hydrolyzed all classes of asparagine-linked glycans examined, provided both the alpha-amino and carboxyl groups of the asparagine residue were in peptide linkage. Deglycosylation studies with PNGase F revealed that many proteins in their native conformation were susceptible to this enzyme but that prior denaturation in sodium dodecyl sulfate greatly decreased the amount of enzyme required for complete carbohydrate removal.

1,091 citations


Journal ArticleDOI
TL;DR: In kinetic studies on the formation of the various adducts, a clear preference of the Pt compound to react with guanines occurring in the base sequence d(pGpG) was established and the method was used to optimize the digestion conditions for cis-DDP-treated DNA.
Abstract: Salmon sperm DNA, treated with the antitumor agent cis-diamminedichloroplatinum(II) (cis-DDP), was enzymatically degraded to (oligo)nucleotides. Four Pt-containing products were identified by 1H NMR after preparative chromatography on a diethylaminoethyl-Sephacel column at pH 8.8. In all identified adducts, comprising approximately 90% of the total Pt in the DNA, Pt was linked to the N7 atoms of the nucleobases guanine and adenine. The two major adducts were cis-Pt(NH3)2d(pGpG) and cis-Pt-(NH3)2d(pApG), both derived from intrastrand cross-links of cis-DDP on neighboring nucleobases. Only the d(pApG) but not the d(pGpA) adduct could be detected. Two minor adducts were Pt(NH3)3dGMP, resulting from monofunctionally bound cis-DDP to guanine, and cis-Pt(NH3)2d(GMP)2, originating from interstrand cross-links on two guanines as well as from intrastrand cross-links on two guanines separated by one or more bases. For analytical purposes we developed an improved method to determine cis-DDP adducts. Routinely, 40-micrograms samples of enzymatically degraded cis-DDP-treated DNA are now analyzed by separation of the mononucleotides and Pt-containing (oligo)nucleotides on the anion-exchange column Mono Q (FPLC) at pH 8.8 (completed within 14 min) and subsequent determination of the Pt content in the collected fractions by atomic absorption spectroscopy. The method was used to optimize the digestion conditions for cis-DDP-treated DNA. In kinetic studies on the formation of the various adducts, a clear preference of the Pt compound to react with guanines occurring in the base sequence d(pGpG) was established.

979 citations


Journal ArticleDOI
TL;DR: The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin.
Abstract: The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.

881 citations


Journal ArticleDOI
TL;DR: Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe and found five overlapping lambda phages were identified that contained the genes for factor IX.
Abstract: Two different human genomic DNA libraries were screened for the gene for blood coagulation factor IX by employing a cDNA for the human protein as a hybridization probe. Five overlapping lambda phages were identified that contained the gene for factor IX. The complete DNA sequence of about 38 kilobases for the gene and the adjacent 5' and 3' flanking regions was established by the dideoxy chain termination and chemical degradation methods. The gene contained about 33.5 kilobases of DNA, including seven introns and eight exons within the coding and 3' noncoding regions of the gene. The eight exons code for a prepro leader sequence and 415 amino acids that make up the mature protein circulating in plasma. The intervening sequences range in size from 188 to 9473 nucleotides and contain four Alu repetitive sequences, including one in intron A and three in intron F. A fifth Alu repetitive sequence was found immediately flanking the 3' end of the gene. A 50 base pair insert in intron A was found in a clone from one of the genomic libraries but was absent in clones from the other library. Intron A as well as the 3' noncoding region of the gene also contained alternating purine-pyrimidine sequences that provide potential left-handed helical DNA or Z-DNA structures for the gene. KpnI repetitive sequences were identified in intron D and the region flanking the 5' end of the gene. The 5' flanking region also contained a 1.9-kb HindIII subfamily repeat. The seven introns in the gene for factor IX were located in essentially the same position as the seven introns in the gene for human protein C, while the first three were found in positions identical with those in the gene for human prothrombin.

640 citations


Journal ArticleDOI
TL;DR: Direct measurements of the full interbilayer force laws between bilayers of various phosphatidylcholines andosphatidylethanolamine in aqueous solutions concluded that bilayer fusion is not simply related to the interbilayers force law.
Abstract: We report direct measurements of the full interbilayer force laws (force vs. distance) between bilayers of various phosphatidylcholines and phosphatidylethanolamine in aqueous solutions. Bilayers were first deposited on molecularly smooth (mica) surfaces and the interbilayer forces then measured at a resolution of 1 A. Three types of forces were identified: attractive van der Waals forces, repulsive electrostatic (double-layer) forces, and (at short range) repulsive steric hydration forces. Double-layer forces, which arise from ion binding, were insignificant in monovalent salt solutions, e.g., NaCl up to 1 M, but were already present in solutions containing millimolar levels of CaCl2 and MgCl2, giving rise to forces in excellent agreement with theory. Ca2+ binds more strongly than Mg2+, and both bind less to lecithin bilayers in the fluid state (T greater than Tc). The plane of charge coincides with the location of the negative phosphate groups, while the effective plane of origin of the van der Waals force is 4-5 A farther out. In water, the adhesion energies are in the range 0.10-0.15 erg/cm2 for lecithins and approximately 0.8 erg/cm2 for phosphatidylethanolamine. The adhesion energies vary on addition of salt due to changes in the repulsive double-layer and hydration forces rather than to a change in the attractive van der Waals force. The short-range repulsive forces which balance the van der Waals force at separations of 10-30 A are due to a combination of hydration and steric repulsions, the latter arising from thermal motions of head groups and thickness fluctuations of fluid bilayers (above Tc). It is also concluded that bilayer fusion is not simply related to the interbilayer force law.

584 citations


Journal ArticleDOI
TL;DR: Investigation of the influence of age, sex, and hormonal status on the expression of eight rat hepatic cytochrome P-450 (P-450) isoenzymes established that the response of these latter five isoenZymes to the P- 450 inducers phenobarbital, beta-naphthoflavone, pregnenolone-16 alpha-carbonitrile, and isosafrole is qualitatively and quantitatively equivalent in females as in males.
Abstract: The influence of age, sex, and hormonal status on the expression of eight rat hepatic cytochrome P-450 (P-450) isoenzymes was evaluated by both catalytic and immunochemical methods. The male specificity of P-450 2c(male)/UT-A, the major microsomal steroid 16 alpha-hydroxylase of uninduced rat liver [Waxman, D.J. (1984) J. Biol. Chem. 259, 15481-15490], was shown to reflect its greater than or equal to 30-fold induction at puberty in male but not in female rats. The female specificity of P-450 2d(female)/UT-I was shown to reflect its developmental induction in females. P-450 PB-2a/PCN-E was shown to mediate greater than or equal to 85% of microsomal steroid 6 beta-hydroxylase activity; the male specificity of this P-450 largely reflects its developmental suppression in female rats. Neonatal gonadectomy and hormonal replacement experiments established that neonatal androgen "imprints" or programs the male rat for developmental induction of P-450 2c(male)/UT-A, for maintenance of P-450 PB-2a/PCN-E, and for suppression of P-450 2d(female)/UT-I, all of which occur in male rats at puberty. By contrast, the expressed levels of P-450 isoenzymes PB-1/PB-C, 3/UT-F, PB-4/PB-B, ISF-G, and beta NF-B were mostly unaffected by the rats' age, sex, and hormonal status. Studies on the sex specificity of P-450 induction established that the response of these latter five isoenzymes to the P-450 inducers phenobarbital, beta-naphthoflavone, pregnenolone-16 alpha-carbonitrile, and isosafrole is qualitatively and quantitatively equivalent in females as in males.

580 citations



Journal ArticleDOI
TL;DR: It is shown that H+ causes lipid mixing in this pH range but not mixing of aqueous contents, which affirms the necessity of using both aqueously space and lipid bilayer assays to comprehend the fusion event between two liposomes.
Abstract: A new liposome fusion assay has been developed that monitors the mixing of aqueous contents at neutral and low pH. With this assay we have investigated the ability of H+ to induce membrane destabilization and fusion. The assay involves the fluorophore 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and its quencher N,N'-p-xylylenebis(pyridinium bromide) (DPX). ANTS is encapsulated in one population of liposomes and DPX in another, and fusion results in the quenching of ANTS fluorescence. The results obtained with the ANTS/DPX assay at neutral pH give kinetics for the Ca2+-induced fusion of phosphatidylserine large unilamellar vesicles (PS LUV) that are very similar to those obtained with the Tb3+/dipicolinic acid (DPA) assay [Wilschut, J., & Papahadjopoulos, D. (1979) Nature (London) 281, 690-692]. ANTS fluorescence is relatively insensitive to pH between 7.5 and 4.0. Below pH 4.0 the assay can be used semiquantitatively by correcting for quenching of ANTS due to protonation. For PS LUV it was found that, at pH 2.0, H+ by itself causes mixing of aqueous contents, which makes H+ unique among the monovalent cations. We have shown previously that H+ causes a contact-induced leakage from liposomes composed of phosphatidylethanolamine and the charged cholesteryl ester cholesteryl hemisuccinate (CHEMS) at pH 5.0 or below, where CHEMS becomes protonated. Here we show that H+ causes lipid mixing in this pH range but not mixing of aqueous contents. This result affirms the necessity of using both aqueous space and lipid bilayer assays to comprehend the fusion event between two liposomes.

458 citations


Journal ArticleDOI
TL;DR: This labeling method has been used to provide material for chemical analysis of the tritiated moiety that is posttranslationally added to flagellar alpha-tubulin, and analysis of complete proteolytic digests by thin-layer chromatography has revealed that this acetyl group is located on the epsilon-amino group of a flagella-induced lysine residue.
Abstract: Previous work has shown that the principal alpha-tubulin within Chlamydomonas reinhardtii flagellar axonemes differs from the major alpha-tubulin in the cell body. These two variants of alpha-tubulin are related to one another since posttranslational modification of the cell body form converts it to the axonemal form. When flagella are induced to assemble in the absence of de novo protein synthesis, tritiated acetate can be used to posttranslationally label alpha-tubulin in vivo, and under these conditions, no other flagellar polypeptides exhibit detectable labeling [L'Hernault, S. W., & Rosenbaum, J. L. (1983) J. Cell Biol. 97, 258-263]. In the present report, this labeling method has been used to provide material for chemical analysis of the tritiated moiety that is posttranslationally added to flagellar alpha-tubulin. This radioactivity was volatile after acid hydrolysis, suggesting that the posttranslational modification is the addition of neither an amino acid nor carbohydrate. Treatment of posttranslationally 3H-labeled alpha-tubulin with hydrazine yields radioactive acetylhydrazine, indicating that the moiety involved in posttranslational modification is an acetyl group. Analysis of complete proteolytic digests by thin-layer chromatography has revealed that this acetyl group is located on the epsilon-amino group of a flagellar alpha-tubulin lysine residue.

444 citations


Journal ArticleDOI
TL;DR: All the poly(ethylene glycols) studied, when used at levels of 10-30%, decreased the thermal stability of beta-lactoglobulin, suggesting that caution must be exercised in the use of this additive at extreme conditions such as high temperature.
Abstract: Poly(ethylene glycol) (PEG) is one of the most useful protein salting-out agents. In this study, it has been shown that the salting-out effectiveness of PEG can be explained by the large unfavorable free energy of its interaction with proteins. Preferential interaction measurements of beta-lactoglobulin with poly(ethylene glycols) with molecular weights between 200 and 1000 showed preferential hydration of the protein for those with Mr greater than or equal to 400, the degree of hydration increasing with the increase in poly(ethylene glycol) molecular weight. The preferential interaction parameter had a strong cosolvent concentration dependence, with poly(ethylene glycol) 1000 having the sharpest decrease with an increase in concentration. The preferential hydration extrapolated to zero cosolvent concentration increased almost linearly with increasing size of the additive, suggesting steric exclusion as the major factor responsible for the preferential hydration. The poly(ethylene glycol) concentration dependence of the preferential interactions could be explained in terms of the nonideality of poly(ethylene glycol) solutions. All the poly(ethylene glycols) studied, when used at levels of 10-30%, decreased the thermal stability of beta-lactoglobulin, suggesting that caution must be exercised in the use of this additive at extreme conditions such as high temperature.

Journal ArticleDOI
TL;DR: The spectral properties of a series of new potentiometric membrane probes have been explored and the most potential sensitive dye has been designated di-4-ANEPPS and has a 6-amino-2-naphthyl group in place of the p-anilino on the parent chromophore.
Abstract: The properties of a series of new potentiometric membrane probes have been explored. The probes all contain an (aminostyryl)pyridinium chromophore or a more highly conjugated analogue. The spectral properties of the dyes are discussed in terms of the excitation-induced charge shift from the pyridine to the aniline; this charge shift also provides the basis for the voltage dependence of the spectra according to an electrochromic mechanism. The spectral responses to a membrane potential on a hemispherical bilayer have been obtained and, grossly, are quite similar for all probes tested. The more subtle variations from dye to dye can be partially rationalized by consideration of binding parameters, the depth within the membrane, and structural factors. The most potential sensitive dye in this collection has been designated di-4-ANEPPS and has a 6-amino-2-naphthyl group in place of the p-anilino on the parent chromophore. Both the relative fluorescence emission and excitation responses have maxima of 8% per 100 mV, and these two spectra display a striking symmetry.

Journal ArticleDOI
TL;DR: The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material.
Abstract: The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Journal ArticleDOI
TL;DR: The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.
Abstract: The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively. These fluoro ketones are 10(4)-10(5) times better as inhibitors than the corresponding methyl ketone. Since nucleophiles readily add to fluoro ketones, it is likely that these compounds inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogues are also inhibitors of zinc metallo- and aspartylproteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is an inhibitor of carboxypeptidase A (Ki = 2 X 10(-7) M). Trifluoromethyl ketone dipeptide analogues are good inhibitors of angiotensin converting enzyme. An analogue of pepstatin that contains a difluorostatone residue in place of statine has been prepared and found to be an extremely potent inhibitor of pepsin (Ki = 6 X 10(-11) M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.

Journal ArticleDOI
TL;DR: Results add to the growing evidence that specific Ca2+-protein-lipid interactions are important in determining both the structure and function of extracellular lung surfactant fractions.
Abstract: Previous studies have demonstrated that lung-specific proteins are associated with surfactant lipids, particularly the highly surface active subfraction known as tubular myelin We have isolated a surfactant-associated protein complex with molecular weight components of 36 000, 32 000, and 28 000 and reassembled it with protein-free lung surfactant lipids prepared as small unilamellar liposomes The effects of divalent cations on the structure and surface activity of this protein-lipid mixture were investigated by following (1) the state of lipid dispersion by changes in turbidity and by electron microscopy and (2) the ability of the surfactant lipids to form a surface film from an aqueous subphase at 37 degrees C The protein complex markedly increased the rate of Ca2+-induced surfactant-lipid aggregation Electron microscopy demonstrated transformation of the small unilamellar liposomes (median diameter 440 A) into large aggregates The threshold Ca2+ concentration required for rapid lipid aggregation was reduced from 13 to 05 mM by the protein complex This protein-facilitated lipid aggregation did not occur if Mg2+ was the only divalent cation present Similarly, 5 mM Ca2+ but not 5 mM Mg2+ improved the ability of the protein-lipid mixture to form a surface film at 37 degrees C Extensive aggregation of the surfactant lipids without protein by 20 mM Ca2+ or 20 mM Mg2+ did not promote rapid surface film formation These results add to the growing evidence that specific Ca2+-protein-lipid interactions are important in determining both the structure and function of extracellular lung surfactant fractions

Journal ArticleDOI
TL;DR: It is concluded that, in addition to glucocorticoids, macrolide antibiotics are specific inducers of P-450p.
Abstract: We administered triacetyloleandomycin (TAO) to rats and found that this macrolide antibiotic is the most efficacious inducer of liver microsomal cytochrome P-450 (P-450) examined to date. Liver microsomes prepared from TAO-treated rats contained greater than 5.0 nmol of P-450/mg of protein and a single induced protein as judged by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein comigrated with P-450p, the major form of P-450 induced in liver microsomes of rats treated with pregnenolone-16 alpha-carbonitrile (PCN) or dexamethasone (DEX). On immunoblots of such gels developed with antibodies to P-450p, the TAO-induced protein reacted strongly as a single band. There was strict parallelism between the amount of immunoreactive P-450p in liver microsomes prepared from untreated rats or from rats treated with phenobarbital, TAO, DEX, or PCN, the ability of these microsomes to catalyze conversion of TAO to a metabolite which forms a spectral complex, and the ethylmorphine and erythromycin demethylase activities. Antibodies to P-450p specifically blocked microsomal TAO metabolite complex formation and ethylmorphine and erythromycin demethylase activities. Moreover, anti-P-450p antibodies completely immunoprecipitated solubilized TAO metabolite complexes prepared by detergent treatment of liver microsomes obtained from TAO-treated rats. Finally, we found that the major form of P-450 isolated from liver microsomes of TAO-treated rats and purified to homogeneity was indistinguishable from purified P-450p as judged by molecular weights, spectral characteristics, enzymatic activities, ability to bind TAO, peptide maps, and amino-terminal amino acid sequences. We concluded that, in addition to glucocorticoids, macrolide antibiotics are specific inducers of P-450p.

Journal ArticleDOI
TL;DR: The absorption changes that occur in reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides during the initial photochemical electron-transfer reaction have been examined and no evidence was found for the formation of a bacteriochlorophyll anion, Bchl-, prior to the formationOf Bph-.
Abstract: The absorption changes that occur in reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides during the initial photochemical electron-transfer reaction have been examined. Measurements were made between 740 and 1300 nm at 295 and 80 K by using a pulse-probe technique with 610-nm, 0.8-ps flashes. An excited singlet state of the bacteriochlorophyll dimer P* was found to give rise to stimulated emission with a spectrum similar to that determined previously for fluorescence from reaction centers. The stimulated emission was used to follow the decay of P*; its lifetime was 4.1 +/- 0.2 ps at 295 K and 2.2 +/- 0.1 ps at 80 K. Within the experimental uncertainty, the absorption changes associated with the formation of a bacteriopheophytin anion, Bph-, develop in concert with the decay of P* at both temperatures, as does the absorption increase near 1250 nm due to the formation of the cation of P, P+. No evidence was found for the formation of a bacteriochlorophyll anion, Bchl-, prior to the formation of Bph-. This is surprising, because in the crystal structure of the Rhodopseudomonas viridis reaction center [Deisenhofer, J., Epp, O., Miki, K., Huber, R., & Michel, H. (1984) J. Mol. Biol. 180, 385-398] a Bchl is located approximately in between P and the Bph. It is possible that Bchl- (or Bchl+) is formed but, due to kinetic or thermodynamic constraints, is never present at a sufficient concentration for us to observe. Alternatively, a virtual charge-transfer state, such as P+Bchl-Bph or PBchl+Bph-, could serve to lower the energy barrier for direct electron transfer between P* and the Bph.

Journal ArticleDOI
TL;DR: A systematic study of the translational diffusion of the phospholipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) in liquid crystalline phase phosphatidylcholine bilayers suggests that a "free volume" model may be appropriate for the description of lipid diffusion in lipid bilayers.
Abstract: A systematic study of the translational diffusion of the phospholipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE) has been undertaken in liquid crystalline phase phosphatidylcholine bilayers by using the fluorescence recovery after photobleaching technique. This work was done with the intention of comparing the experimental results with the predictions of theoretical models for diffusion in membranes. The following is shown. For NBD-PE, the dependence of the translational diffusion coefficient (Dt) upon the acyl chain length of the diffusant is not that predicted by continuum fluid hydrodynamic models for diffusion in membranes [Saffman, P.G., & Delbrueck, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113; Hughes, B. D., Pailthorpe, B. A., & White, L. R. (1981) J. Fluid Mech. 110, 349-372]. Plots of Dt vs. 1/T (Arrhenius plots) are nonlinear in dilauroyl-phosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers where the acyl chain composition of the NBD-PE is matched with that of the host bilayer lipid. This suggests that a "free volume" model may be appropriate for the description of lipid diffusion in lipid bilayers. In bilayers of phosphatidylcholines with saturated acyl chains at the same "reduced temperature", the magnitude of Dt follows the order distearoylphosphatidylcholine greater than DPPC greater than DMPC greater than DLPC. This is the inverse of what may be expected from the hydrodynamic model but is in agreement with the free volume in these bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)

Journal ArticleDOI
TL;DR: The determination of the complete primary structure for phenylalanine hydroxylase represents the first among mixed-function oxidases.
Abstract: A full-length human phenylalanine hydroxylase complementary DNA (cDNA) clone was isolated from a human liver cDNA library, and the nucleotide sequence encoding the entire enzyme was determined. The cDNA clone contains an inserted DNA fragment of 2448 base pairs, including 19 base pairs of poly(A) at the 3' end. The first methionine codon occurs at nucleotide position 223, followed by an open reading frame of 1353 base pairs, encoding 451 amino acids. Translation of the nucleotide sequence in the open reading frame predicts the amino acid sequence of human phenylalanine hydroxylase. The human protein shows a 96% amino acid sequence homology with the corresponding rat enzyme. The determination of the complete primary structure for phenylalanine hydroxylase represents the first among mixed-function oxidases.

Journal ArticleDOI
TL;DR: High-resolution 31P NMR studies on the detergent-solubilized ADP/ATP carrier from beef heart mitochondria revealed narrow signals from phosphatidylcholine andosphatidylethanolamine and a broadened signal of 30-40-Hz line width, suggestive of cardiolipin.
Abstract: An unusual binding of cardiolipin to the ADP/ATP carrier has been found, which is distinguished by the relatively large amount and by the tightness of binding. High-resolution 31P NMR studies on the detergent-solubilized ADP/ATP carrier from beef heart mitochondria revealed narrow signals from phosphatidylcholine and phosphatidylethanolamine and a broadened signal of 30-40-Hz line width, suggestive of cardiolipin. Line broadening of this magnitude is to be expected when tumbling of the whole protein-detergent micelle is the only source of phosphorus spin-spin relaxation. Thus a strong immobilization of the protein-bound cardiolipin is inferred. By sucrose density gradient centrifugation phosphatidylcholine and phosphatidylethanolamine were removed, while approximately six +/- one molecules of cardiolipin remained tightly bound in the dimeric protein molecule. The cardiolipin binding was stable against treatment with sodium dodecyl sulfate although release of the inhibitor carboxyatractyloside revealed at least partial protein denaturation. Ca2+ ions did not readily interact either with the bound cardiolipin. Complete detachment of the bound phospholipid was achieved by a short heat pulse in the presence of sodium dodecyl sulfate. Denaturation of the carrier protein by guanidinium chloride or NaClO4 also led to release of the bound phospholipid. Thus different stages of protein denaturation must be envisaged.

Journal ArticleDOI
TL;DR: Curve fitting of the peptide spectra to estimate the secondary structure fractions has shown that the two proteins assume a similar structure at an early stage of folding and that the intermediate has a structure similar to that of partially unfolded species produced by heat and a moderate concentration of guanidine hydrochloride.
Abstract: Refolding kinetics of two homologous proteins, lysozyme and alpha-lactalbumin, were studied by following the time-dependent changes in the circular dichroism spectra in the aromatic and the peptide regions The refolding was initiated by 20-fold dilution of the protein solutions originally unfolded at 6 M guanidine hydrochloride, at pH 15 for lysozyme and pH 70 for alpha-lactalbumin at 45 degrees C In the aromatic region, almost full changes in ellipticity that were expected from the equilibrium differences in the spectra between the native and unfolded proteins were observed kinetically The major fast phase of lysozyme folding has a decay time of 15 s The decay time of alpha-lactalbumin depends on the presence or absence of bound Ca2+: 10 s for the holoprotein and 100 s for the apoprotein In the peptide region, however, most of the ellipticity changes of the two proteins occur within the dead time (less than 3 s) of the present measurements This demonstrates existence of an early folding intermediate which is still unfolded when measured by the aromatic bands but has folded secondary structure as measured by the peptide bands Extrapolation of the ellipticity changes to zero time at various wavelengths gives a spectrum of the folding intermediate Curve fitting of the peptide spectra to estimate the secondary structure fractions has shown that the two proteins assume a similar structure at an early stage of folding and that the intermediate has a structure similar to that of partially unfolded species produced by heat and, for alpha-lactalbumin, also by acid and a moderate concentration of guanidine hydrochloride(ABSTRACT TRUNCATED AT 250 WORDS)

Journal ArticleDOI
TL;DR: The amino acid sequence of human angiogenin is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonUClease are also conserved in angiogenesis factor.
Abstract: Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin This provocative finding is thought to have important physiological implications

Journal ArticleDOI
TL;DR: The present results are consistent with the existence of a Mg2+- and ATP-dependent protein in erythrocytes that selectively translocates aminophospholipids to the membrane inner monolayer engendering amin phospholipid asymmetry.
Abstract: Cell morphology changes are used to examine the interaction of exogenous phosphatidylserine and phosphatidylethanolamine with human erythrocytes. Short-chain saturated lipids transfer from liposomes to cells, inducing shape changes that are indicative of their incorporation into, and in some cases translocation across, the cell membrane bilayer. Dioleoylphosphatidylserine and low concentrations of dilauroyl- and dimyristoylphosphatidylserine induce stomatocytosis. At higher concentrations, dilauroylphosphatidylserine and dimyristoylphosphatidylserine induce a biphasic shape change: the cells crenate initially but rapidly revert to a discocytic and eventually stomatocytic shape. The extent of these shape changes is dose dependent and increases with increasing hydrophilicity of the phospholipid. Cells treated with dilauroylphosphatidylethanolamine and bovine brain lysophosphatidylserine exhibit a similar biphasic shape change but revert to discocytes rather than stomatocytes. These shape changes are not a result of vesicle--cell fusion nor can they be accounted for by cholesterol depletion. The reversion from crenated to stomatocytic forms is dependent on intracellular ATP and Mg2+ concentrations and the state of protein sulfhydryl groups. The present results are consistent with the existence of a Mg2+- and ATP-dependent protein in erythrocytes that selectively translocates aminophospholipids to the membrane inner monolayer engendering aminophospholipid asymmetry.

Journal ArticleDOI
TL;DR: The results suggest that at least some of the mitogenic, angiogenic, and neovascularizing activities described as being present in the retina are due to the existence of FGF in this tissue.
Abstract: Two retina-derived growth factors have been isolated on the basis of their ability to stimulate the proliferation of capillary endothelial cells in vitro. Gas-phase sequence analysis identified the amino-terminal sequence of the major form of the mitogen as being identical with residues 1-35 of bovine basic fibroblast growth factor (FGF). Amino-terminal sequence analysis of the second form identified 28 residues that are indistinguishable from those of brain acidic FGF (residues 1-28). The possibility that these retina-derived endothelial cell growth factors are related to, if not identical with, basic and acidic FGF is supported by observations that they have similar molecular weights (15000-16000), similar retention behavior on all steps of chromatography (ion-exchange, heparin-Sepharose), and similar amino acid compositions and that they cross-react with antibodies to basic and acidic FGF. The eye-derived growth factors, like FGF, are potent stimulators of capillary endothelial cell growth in vitro. The results identify the major retina-derived endothelial cell growth factor as indistinguishable from basic FGF and demonstrate the presence of an acidic FGF in the eye. They suggest that at least some of the mitogenic, angiogenic, and neovascularizing activities described as being present in the retina are due to the existence of FGF in this tissue. The implications of this finding on the etiology and pathophysiology of vasoproliferative diseases of the eye are discussed.

Journal ArticleDOI
TL;DR: Comparing the difference spectra in the region of the CO2- stretching vibrations of labeled and unlabeled BR indicates that ionized aspartic acids are influenced during the photocycle, the earliest effect being observed already at the K610 intermediate.
Abstract: The molecular events during the photocycle of bacteriorhodopsin have been studied by the method of time-resolved and static infrared difference spectroscopy Characteristic spectral changes involving the C=O stretching vibration of protonated carboxylic groups were detected To identify the corresponding groups with either glutamic or aspartic acid, BR was selectively labeled with [4-13C]aspartic acid An incorporation of ca 70% was obtained The comparison of the difference spectra in the region of the CO2- stretching vibrations of labeled and unlabeled BR indicates that ionized aspartic acids are influenced during the photocycle, the earliest effect being observed already at the K610 intermediate Taken together, the results provide evidence that four internal aspartic acids undergo protonation changes and that one glutamic acid, remaining protonated, is disturbed The results are discussed in relation to the various aspects of the proton pumping mechanism, such as retinal isomerization, charge separation, pK changes, and proton pathway

Journal ArticleDOI
TL;DR: Evidence for incorporating a new binary complex, RPi, in the pathway was provided by experiments that distinguished between stably bound species and active promoter after temperature-jump perturbations, and measurement of the rate of reequilibration between the stably Bound species and determination of the corresponding equilibrium constant.
Abstract: The forward and reverse kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter have been studied in the temperature range of 15-42 degrees C. The standard two-step model, involving the formation of a closed intermediate, RPc, followed by an isomerization that leads to the active complex RPo, could not account for the present data. The promoter-enzyme lifetime measurements showed an inverse temperature dependence (apparent activation energy, -35 kcal/mol). A third step, which is very temperature dependent and which is very rapid at 37 degrees C, was postulated to involve the unstacking of DNA base pairs that immediately precedes open complex formation. Evidence for incorporating a new binary complex, RPi, in the pathway was provided by experiments that distinguished between stably bound species and active promoter after temperature-jump perturbations. These experiments allowed measurement of the rate of reequilibration between the stably bound species and determination of the corresponding equilibrium constant. They indicated that the third step became rate limiting below 20 degrees C; this prediction was checked by an analysis of the forward kinetics. A quantitative evaluation of the parameters involved in this three-step model is provided. Similar experiments were performed on a negatively supercoiled template: in this case the third equilibrium was driven toward formation of the open complex even at low temperature, and the corresponding step was not rate limiting.

Journal ArticleDOI
TL;DR: These two residues must be the major contributors to the binding and must be linked to the biologic activity of the octasaccharide.
Abstract: The importance of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide iduronic acid(1)----N-acetylglucosamine 6-O-sulfate(2)----glucuronic acid(3)----N-sulfated glucosamine 3,6-di-O-sulfate(4)----iduronic acid 2-O-sulfate(5)----N-sulfated glucosamine 6-O-sulfate(6)----iduronic acid 2-O-sulfate(7)----anhydromannitol 6-O-sulfate(8) was determined by comparing with synthetic tetra- and penta-saccharides its ability to bind human antithrombin. The octasaccharide had an affinity for antithrombin of 1 X 10(-8) M (10.2 kcal/mol) measured by intrinsic fluorescence enhancement at 6 degrees C. The synthetic pentasaccharide, consisting of residues 2-6, had an affinity of 3 X 10(-8) M (9.6 kcal/mol). The same pentasaccharide, except lacking the 3-O-sulfate on residue 4, had an affinity of 5 X 10(-4) M (4.5 kcal/mol) measured by equilibrium dialysis. The tetrasaccharide, consisting of residues 2-5, bound antithrombin with an affinity of 5 X 10(-6) M (6.8 kcal/mol). The tetrasaccharide, consisting of residues 3-6, had an affinity of 5 X 10(-5) M (5.5 kcal/mol). Since the loss of either the 6-O-sulfated residue 2 or the 3-O-sulfate of residue 4 results in a 4-5 kcal/mol or a 40-50% loss in binding energy of the pentasaccharide, these two residues must be the major contributors to the binding and must be linked to the biologic activity of the octasaccharide.

Journal ArticleDOI
TL;DR: 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase.
Abstract: The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.

Journal ArticleDOI
TL;DR: Consideration of several possible mechanisms for the reaction, taking into account the biochemical and crystallographic facts presented above, suggests that the cleavage involves removal of the proton from the 2'-OH of ribose-17 by a Pb(II)-bound hydroxyl group.
Abstract: X-ray diffraction data from monoclinic crystals of yeast tRNAPhe soaked in dilute lead(II) acetate solutions at pH 5.0 and at pH 7.4 have been collected to a resolution of 3 A, and the Pb(II) binding sites have been obtained by difference Fourier analyses. The same three Pb(II) binding sites are observed at both of these pH values. At pH 7.4 an extra peak of negative electron density appears on the difference map close to one of the Pb(II) binding sites and at the position of phosphate-18, indicating cleavage of the sugar-phosphate-chain between residues D-17 and G-18 of the tRNAPhe molecule in this derivative. Chain scission does not occur to any observable extent in the structure at pH 5.0, and we have, therefore, a picture of the reactants (at pH 5.0) and products (at pH 7.4) of this cleavage reaction. Polyacrylamide gel electrophoresis as well as sequencing experiments confirms the cleavage of the tRNAPhe molecule into one-fourth and three-fourth fragments, with the shorter fragment consisting essentially of residues G-1 through D-17 while the larger fragment contains residues G-18 through A-76. End-group analyses suggest a ribose cyclic 2',3'-phosphate at D-17 of the one-fourth fragment with a 5'-OH at G-18 of the three-fourth fragment. Cleavage of the tRNAPhe molecule does not occur in the absence of Pb(II), and the proximity of one of these metal ions to the cleavage site strongly implicates this metal ion in the cleavage reaction. Consideration of several possible mechanisms for the reaction, taking into account the biochemical and crystallographic facts presented above, suggests that the cleavage involves removal of the proton from the 2'-OH of ribose-17 by a Pb(II)-bound hydroxyl group. Subsequent nucleophilic attack of the resulting 2'-O- on the phosphorus atom of phosphate-18, presumably through a pentacoordinate phosphorus cyclic intermediate (as in the action of pancreatic ribonuclease A), results in chain scission. It cannot be decided whether the displacement, within the pentacoordinate intermediate, proceeds via an in-line or adjacent pathway, but an exploration of the likelihood of either pathway is presented. Strand cleavage at the particular site occurs fortuitously because the aquo Pb(II) ion binds at the correct distance and presumably in such a manner as to present a hydroxyl group in the correct orientation to effect the proton abstraction.(ABSTRACT TRUNCATED AT 400 WORDS)

Journal ArticleDOI
TL;DR: It is demonstrated that phasing is, in fact, essential for the observed abnormal electrophoretic behavior of DNA molecules, thus ruling out alternative models which invoke any form of isotropic or centrosymmetric flexibility as the source of the phenomenon.
Abstract: Certain DNA molecules derived from both prokaryotic and eukaryotic organisms display markedly abnormal electrophoretic behavior on polyacrylamide gels. These molecules share a common element of sequence which involves collections of A/T residues that are approximately in phase with the helix repeat. This sequence periodicity has led to the suggestion that such phasing is important in generating the abnormal behavior. We have demonstrated that such phasing is, in fact, essential, thus ruling out alternative models which invoke any form of isotropic or centrosymmetric flexibility as the source of the phenomenon. We have also shown that the abnormal behavior is not a simple consequence of marginal thermodynamic stability. The most plausible explanation for the observed behavior is that stable, local distortions of the helix axis result in macroscopic curvature when such distortions are propagated in phase with the helix repeat.