Showing papers in "Biochemistry in 1977"

RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination.

Abstract: RNA molecular weight measurements were carried out by gel electrophoresis under four different denaturing conditions including 99% formamide, 10 mM methyl mercury, 2.2 M formaldehyde, and 6 M urea at pH 3.8. Electrophoresis a t a series of gel concentrations and at least two different voltage gradients resulted in some RNA species exhibiting apparent molecular weights that vary with both gel concentration and voltage gradient. Three different deviations from the requirement for hydrodynamically equivalent conformations were observed: (1) deformation of the random coil structure of very large RNAs at moderately high gel concentrations and voltage gradients resulting, in extreme cases, in a molecular weight independent migration of RNA molecules; (2) incomplete denaturation of RNA molecules with very GC rich helical regions; and (3) varying charge/mass ratio due to G e l electrophoresis has become the major analytical procedure for characterizing charged macromolecules both because of the high resolution it provides and the relative simplicity of the technology it requires. The applicability of this procedure to molecular weight determination of nucleic acids and NaDodS04-protein complexes is based both on the fact that these macromolecules are polymers with a constant charge-mass ratio and hydrodynamically equivalent conformations, and on the linearity of the empirical log molecular weight-mobility relation used to determine molecular weights from mobilities. When these assumptions are fulfilled, molecular weights can be measured in a single experiment using standards of known molecular weight. Moreover, since RNA molecules do not have hydrodynamically equivalent conformations in aqueous solutions (Boedtker, 1968; Groot et al., 1970; MacLeod, 1975), several methods were developed in which R N A molecular weights could be determined by gel electrophoresis under denaturing conditions. Denaturing conditions first used included reaction with formaldehyde (Boedtker, 1971), 8 M urea at 60 'C (Rejinders et al., 1973) and 99% formamide at room temperature (Pinder et al., 1974), the latter being by far the most widely used. In addition to the denaturing gel systems first developed, three others have been reported more recently: 6 M urea at pH 3.5 run at 2 "C (Rosen et ai., 1975), 5 mM methyl mercury run at room temperature (Bailey and Davidson, 1976), and 99% formamide run at 45-55 'C (Spohr et al., 1976). This proliferation of denaturing gel systems arose because it was recognized that some of the denaturants first used were not able to denature very GC rich RNA and could not be used in the dilute agarose gels required t From the Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02 138. Receiued March 30, 1977. H. Lehrach was the recipient of a postdoctoral fellowship from the Jane Coffin Childs Memorial Fund for Medical Research. John M. Wozney was awarded a Camille and Henry Dreyfus Foundation Summer Research Fellowship. This investigation was supported by National Institutes of Health Grant HD-01229. differential protonation at pH 3.8. Reliable molecular weight measurements of RNA molecules as large as 4.0 X lo6 containing G C rich helical regions could only be made on dilute (0.5-1.0%) agarose gels after reaction with either 2.2 M formaldehyde or 10 mM methyl mercury hydroxide. A theoretical justification for the use of the empirical log molecular weight-mobility relation is presented. It is also demonstrated that the gel electrophoretic behavior of a homologous series of random coils can be approximated by that of a series of spheres with radii proportional to the square root of radius of gyration of a random coil. Consequently, molecular weight determinations of denatured RNAs, especially those obtained by extrapolation, are more reliable if the square root of the molecular weight is plotted vs. log mobility. for molecular weight determinations of very large RNA molecules. We report here a comparison of the behavior of high molecular weight R N A molecules on four different denaturing gels: polyacrylamide in 99% formamide, agarose in 10 mM methyl mercury hydroxide, 2.2 M formaldehyde, and 6 M urea, pH 3.8. In addition, we offer a rationale for the use of the empirical log molecular weight-mobility relation and suggest an alternate method of obtaining RNA molecular weights from mobilities based on the finding that the migration of denatured RNA molecules depends on the square root of the radius of gyration, or the fourth root of the molecular weight. Materials and Methods RNA Samples. TMV and chicken ribosomal RNA were prepared as described previously (Boedtker, 1960; Boedtker et al., 1973). E. coli 23s and 16s rRNA was prepared by phenol-CHC13 extraction (Perry et al., 1972). followed by fractionation on linear 5-20% sucrose gradients. Mouse 28s rRNA was donated by Jesse F. Scott of the Harvard Medical School. Sindbis virus, a gift from Michelene McCarthy of the Department of Biochemistry and Molecular Biology, was extracted with phenol-CHC13 (Perry et al., 1972). Polyacrylamide Gel Electrophoresis in 99% Formamide. To achieve reproducible mobilities, pure reasonably dry deionized formamide is essential. 99% formamide (Eastman Chemical Co.) was deionized following the procedure of Pinder et al. (1 974) by stirring with 40 g /L mixed bed resin (Bio-Rad AG501-X8, 20-50 mesh). After about 5 h, the conductivity should decrease to about 70 pmho. We have found, however, that some batches of formamide are not deionized under these conditions even when more resin is used. Such formamide is unsuitable for gel electrophoresis because irreproducible polymerization of gels occurs. The deionized formamide was filtered through a sintered-glass filter and then distilled under B I O C H E M I S T R Y , V O L . 1 6 , N O . 2 1 , 1 9 7 7 4743 L E H R A C H E T A L electrophoresis buffer (E buffer): 0.05 M boric acid, 0.005 M NazB407.10H~0, 0.01 M sodium sulfate, and 0.001 M Na3EDTA, pH 8.2, without CH3HgOH. The suspension was heated for 5 min in an autoclave and diluted to the appropriate concentration with hot E buffer. CH3HgOH was added to the hot buffered agarose by syringe pipet and stirred rapidly at 60 “C. The gels were poured immediately using 2.5 mL per tube. Before samples were applied, the top of the gel was sliced off with a razor blade to provide a flat surface. RNA samples were prepared by dissolving the RNA in E buffer containing 5 or 10 mM methyl mercury hydroxide as indicated. One-half volume of a 1:l mixture of glycerol-HzO solution containing 0.004% bromophenol blue was added and the sample applied to the gel. Electrophoresis was carried out for various times as indicated at 2.5 or 3 mA per tube, at room temperature inside the closed hood. The latter was clearly designated as being hazardous both because of high voltage and CH3HgOH. All operations involving methyl mercury hydroxide were carried out in the hood, with the operator wearing gloves. The tops of the gels, leftover agarose, used gels, and any material contaminated with CH3HgOH (gloves, paper, disposable items) were placed in a disposable plastic bag and stored in the hood. CH3HgOH waste and contaminated items were disposed of every 3 months by the Department of Chemistry’s hazardous chemical disposal service, the Radiac Corp. The operator was monitored for CH3HgOH accumulation every 6 months by the Harvard Health Services. Agarose Gel Electrophoresis in 2.2 M Formaldehyde. RNA samples were heated in 2.2 M formaldehyde (prepared from 37% Mallinckrodt formaldehyde) in 50% formamide, 0.01 8 M NazHP04-0.002 M NaHzPO4, for 5 min at 60 “C. Agarose gels were prepared by heating a “3%” agarose-water suspension in the autoclave for 5 min and diluting wi th either 1 volume of 4.4 M formaldehyde in 0.036 M NazHP04-0.004 M NaH2P04 to make 1.5% gels or with 2 volumes of the latter plus 1 volume of water to make 0.75% gels. The gels were poured immediately using 2.5 mL per tube. Electrophoresis was carried out at 2 mA per tube at room temperature for the times indicated. The electrophoresis buffer was 2.2 M formaldehyde-0.018 M Na2HP04-0.002 M NaH2P04. Agarose Gel Electrophoresis in 6 M Urea at pH 3.8. Electrophoresis was performed following the procedure described by Rosen et al. ( I 975) with the following modifications. Three times agarose (usually 3 g/lOO mL of HzO) was heated in the autoclave and then diluted with 2 volumes of warm I .5X buffer to give the final concentration of agarose and 6.0 M urea, 0.025 M citric acid (pH 3.8), heated at 65 “ C for 20 s, and fast cooled. One-half volume of the glycerol dye was added as described above. Electrophoresis was carried out for 12 h at 0.38 mA per gel at 4 “C. Staining Gels with Ethidium Bromide. Agarose and polyacrylamide gels were stained overnight in 1 yg/mL ethidium bromide in 0.1 M ammonium acetate (Bailey and Davidson, 1976). After staining, the gels were photographed under short-wave U V light with a Polaroid M P 3 Land camera and high-speed type 57 film (Polaroid Corp.) using a yellow f i l ter.

A simple method for the preparation of homogeneous phospholipid vesicles.

Abstract: A new method is described for the preparation of homogeneously sized, single-lamellar phospholipid vesicles. This method, wich is based on differential high-speed ultracentrifugation, has the advantages of a higher vesicle yield without dilution and rapidity of preparation when compared to the molecular-sieve technique. The homogeneity of vesicle dispersions, prepared by this new method, is examined by several physical techniques and found to be comparable to the best samples prepared by molecular-sieve chromatography.

A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S.

Abstract: Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.

Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids

Abstract: The mechanism of the enhancement of the fluorescence of ethidium bromide on binding to double helical RNA and DNA has been investigated. From an examination of the effect of different solvents on the fluorescence lifetime, quenching of fluorescence by proton acceptors, and the substantial lengthening of lifetime observed upon deuteration of the amino protons, regardless of the medium, we conclude that proton transfer from the excited singlet state is the process primarily responsible for the approximately equal to 3.5-fold increase in the lifetime of free ethidium bromide in going from H2O to D2O; the fact that addition of small amounts of water to nonaqueous solvents decreases the fluorescence whereas addition of small amounts of D2O enhances the fluorescence; and the enhancement of the ethidium bromide triplet state yield on binding to DNA. Other proposed mechanisms are shown to be inconsistent with our findings.

Conformational transition accompanying the binding of Ca2+ to the protein activator of 3',5'-cyclic adenosine monophosphate phosphodiesterase.

Abstract: The Ca2+-dependent protein activator of 3':5'-cyclic adenosine monophosphate phosphodiesterase is shown to undergo a conformational transition upon binding of 2 mol of Ca2+/mol of activator. Circular dichroic studies indicate that Ca2+ induces an increase of 5-8% in alpha-helix content with a concomitant decrease in the amount of random coil. In the absence of Ca2+ and in the presence of [ethylenebis(oxoethylenenitrilo)]tetraacetic acid (EGTA), the protein contains 30-35% alpha helix, 50% random coil, and 15-20% beta-pleated sheat. Spectrophotometric titration indicates that the two tyrosyl residues have pK's of 10.4 and 11.9 and are therefore in different environments. The Ca2+-induced conformational change is accompanied by an increased exposure to protons of the partially exposed tyrosine, as shown by a shift in its pK from 10.4 to 10.). Increased solvation is also consistent with a negative difference spectrum at 287 and 279 nm as seen upon Ca2+ binding. Modification in the environment of all or some of the phenylalanine residues also is part of the conformational change accompanying Ca2+ binding. A new and rapid purification procedure which yields large amounts (25-30% yields) of homogenous protein activator and a direct and sensitive assay procedure for cAMP phosphodiesterase and its activator are also described.

Dynamic Structure of Lipid Bilayers Studied by Nanosecond Fluorescence Techniques

Abstract: Molecular motions in liposomes of dipalmitoyl- phosphatidylcholine (DPPC) were studied by nanosecond fluorescence techniques. As a fluorescent probe for the hy- drocarbon region, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used. Time courses of fluorescence intensity ZT(t) and emission anisotropy r(t) of DPH embedded in DPPC liposomes were measured at various temperatures. The value of the fluores- cence lifetime 7 obtained from a single exponential decay of Ir(t) was somewhat higher than that in liquid paraffin below the transition temperature TI and decreased above TI. Higher values of 7 below TI, indicate the almost complete hydrophobic environment. The decay curves of r(t) were separated into two phases: an initial fast decreasing phase of the order of one nanosecond and a second almost constant phase. This indicates Structure and dynamics of lipids in biological membranes have been recognized as the essential factors in their functions and organization (Inesi et al., 1973; Racker and Hinkle, 1974). Phospholipids in model membranes such as aqueous disper- sions or liposomes are known to be in a bilayer structure and the cooperative melting of their hydrocarbon chains, the crystalline-liquid-crystalline phase transition, takes place at a certain temperature (Trauble and Eibl, 1974; Sackmann et al., 1973; Chapman, 1975; Jacobson and Papahadjopoulos, 1975). Studies of dynamic properties or molecular motion of lipids in model bilayers, however, have not resolved several important questions. Although the use of spin-label techniques for the dynamic studies of lipid bilayers is widespread, the information re- garding molecular motion is restricted in most investigations. In describing the characteristics of molecular motion of that the orientational motion of DPH in the hydrocarbon re- gion is described by a wobbling diffusion restricted by a certain anisotropic potential. The results were analyzed on the model that the wobbling diffusion is confined in a cone with a uniform diffusion constant. Though temperature dependence of the cone angie was sigmoidal, that of the wobbling diffusion con- stant was like the exponential function. The change in the cone angle at TI was sharper than that in the wobbling diffusion constant at TI. Estimated values of the viscosity in the cone were an order of magnitude smaller than the values of "mi- croviscosity" which were estimated from the steady-state emission anisotropy without considering the restrictions on the rotational motion.

Human skin collagenase: isolation of precursor and active forms from both fibroblast and organ cultures.

Abstract: Human skin procollagenase has been isolated, in pure form, from the medium of fibroblasts cultured in the presence or absence of added serum. Purification was achieved using a combination of cation-exchange (phosphocellulose or carboxymethylcellulose) and gel-filtration chromatography. Two forms (60 000 and 55 000 daltons) of the procollagenase were detected by electrophoresis in sodium dodecyl sulfatepolyacrylamide gels and could be separated by chromatography on Ultrogel AcA-44. Each form was converted to active enzyme by trypsin, producing species of 50 000 and 45 000 daltons, respectively. An autoactivation process also occurred, which yielded active enzyme without a detectable change in molecular weight. Procollagenase also was found in organ cultures of human skin but only when serum was added to the medium. This suggests that a serum-inhibitable proteolytic system is present in these cultures which, like trypsin, converts procollagenase to the active enzyme forms that can be isolated from serum-free organ culture medium. The collagenase species obtained from either fibroblast or organ culture medium were chromatographically and electrophoretically identical.

In vitro reconstitution of calf brain microtubules: effects of solution variables.

Abstract: The effects of solution variables on the in vitro reconstitution of calf brain tubulin, purified by the method of Weisenberg et al. (Weisenberg, R. C., Borisy, G. G., and Taylor, E. W. (1968), Biochemistry 7, 4466-4479; Weisenberg, R. C., and Timasheff, S. N. (1970), Biochemistry 9, 4110-4116), as modified by Lee et al. (Lee, J. C., Frigon, R. P., and Timasheff, S. N. (1973), J. Biol. Chem. 248, 7253-7262), were investigated at pH 7.0. Reconstitution of microtubules was successful in a variety of buffer systems, the free energy of the propagation step of microtubule formation being little dependent on the buffer. Microtubule formation is promoted by magnesium ions and guanosine triphosphate, but inhibited by calcium ions. The dependence of the apparent association constant for microtubule formation on ligand concentration was analyzed by the linked function theory of Wyman (Wyman, J. (1964), Adv. Protein Chem. 19, 224-286), leading to the conclusion that the formation of a tubulin-tubulin contact involves the binding of one additional magnesium ion per tubulin dimer. Microtubule formation is also accompanied by the apparent binding of one additional proton and the release of water molecules, as suggested by the thermodynamic parameters determined. The reaction is entropy driven with an apparent heat capacity change, deltaCp, of -1500 +/- 500 cal/deg-mol. The enhancement of tubulin reassembly by glycerol is most likely due to nonspecific protein-solvent general thermodynamic interactions.

Binding of chloromethyl ketone substrate analogs to crystalline papain

Abstract: Papain (EC 3.4.22.2) is a proteolytic enzyme, the three-dimensional structure of which has been determined by x-ray diffraction at 2.8 A resolution (Drenth, J., Jansonius, J.N., Koekoek, R., Swen, H. M., and Wothers, B.G. (1968), Nature (London) 218, 929-932). The active site is a groove on the molecular surface in which the essential sulfhydryl group of cysteine-25 is situated next to the imidazole ring of histidine-159. The main object of this study was to determine by the difference-Fourier technique the binding mode for the substrate in the groove in order to explain the substrate specificity of the enzyme (P2 should have a hydrophobic side chain (Berger and Schechter, 1970) and to contribute to an elucidation of the catalytic mechanism. To this end, three chloromethyl ketone substrate analogues were reacted with the enzyme by covalent attachment to the sulfur atom of cysteine-25. The products crystallized isomorphously with the parent structure that is not the native, active enzyme but a mixture of oxidized papain (probably papain-SO2-) and papain with an extra cysteine attached to cysteine-25. Although this made the interpretation of the difference electron density maps less easy, it provided us with a clear picture of the way in which the acyl part of the substrate binds in the active site groove. The carbonyl oxygen of the P1 residue is near two potential hydrogen-bond donating groups, the backbone NH of cysteine-25 and the NH2 of glutamine-19. Valine residues 133 and 157 are responsible for the preference of papain in its substrate splitting. By removing the methylene group that covalently attaches the inhibitor molecules to the sulfur atom of cysteine-25 we obtained acceptable models for the acyl-enzyme structure and for the tetrahedral intermediate. The carbonyl oxygen of the P1 residue, carrying a formal negative charge in the tetrahedral intermediate, is stabilized by formation of two hydrogen bonds with the backbone NH of cysteine-25 and the NH2 group of glutamine-19. This situation resembles that suggested for the proteolytic serine enzymes (Henderson, R., Wright, C. S., Hess, G. P., and Blow, D. M. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 63-70; Robertus, J. D., Kraut, J., Alden, R. A., and Birktoft, J. J. (1972b), Biochemistry 11, 4293-4303). The nitrogen atom of the scissile peptide bond was found close to the imidazole ring of histidine-159, suggesting a role for this ring in protonating the N atom of the leaving group (Lowe, 1970). This proton transfer would be facilitated by a 30 degrees rotation of the ring around the C beta-Cgamma bond from an in-plane position with the sulfur atom to an in-plane position with the N atom. The possibility of this rotation is derived from a difference electron-density map for fully oxidizied papain vs. the parent protein.

Synthesis and characterization of new psoralen derivatives with superior photoreactivity with DNA and RNA

Abstract: The synthesis of five new psoralen derivatives is described. Three of these, 4'-hydroxymethyl-4,5',8-trimethylpsoralen, 4'-methoxymethyl-4,5',8-trimethylpsoralen, and 4'-aminomethyl-4,5',8-trimethylpsoralen hydrochloride, and characterized with respect to their photoreactivity with DNA and RNA. They are found to be greatly superior to 4,5',8-trimethylpsoralen and 8-methoxypsoralen, the two commonly used psoralens, in their abilities to saturate the photoreactive sites on DNA and RNA without repeated addition of reagent. A simplified mechanism for the photoreaction of psoralens with nucleic acids is presented and provides a basis for understanding the superior properties of these compounds. The compounds have superior reactivity not only with isolated DNA and RNA but also in viruses and in cells. Psoralens are shown for the first time to cross-link RNA double helices.

Conjugated Polyene Fatty Acids as Fluorescent Probes: Synthetic Phospholipid Membrane Studies

Abstract: The preparation of polyene fatty acid membrane probes cis- and trans-parinaric acid and parinaroylphosphatidylcholines and their use in studies of several one- and two- component lipid systems are described. The fluorescence quantum yield of trans-parinaric acid in dipalmitoylphosphatidylcholine at 20 degrees C is approximately 0.3; the quantum yield in aqueous solution is negligibly small. Thermal-phase transitions in single-component phospholipid dispersions are monitored with absorption and fluorescence excitation peak position, fluorescence intensity, lifetime, and polarization. The transition temperatures observed are consistent with previous determinations. Shifts in the absorption peak position are related to the bilayer expansion as it undergoes the gel to liquid-crystalline transition, while fluorescence depolarization provides semiquantitative information concerning molecular motion of the probe in the bilayer. A long fluorescence lifetime component is observed for parinaric acid in the solid phase (up to 50 ns), and a short lifetime component is observed (ca. 5 ns) in the fluid phase of dipalmitoylphosphatidylcholine; both lifetime components are observed in the transition region. In most phospholipids, cis-parinaric acid detects the melting transition at about 1 degree C lower than trans-parinaric acid. Partitioning experiments involving mixed populations of phospholipid vesicles show that trans-parinaric acid preferentially associates with solid-phase lipids, while cis-parinaric acid shows a more equal distribution between solid and fluid lipids. The binding of cis-parinaric acid to dipalmitoylphosphatidylcholine at 25 degrees C is described as partitioning of parinaric acid between lipid vesicles and the aqueous phase with a partition coefficient of 5 X 10(5). Several rates are observed in the binding process which are interpreted as rapid outer monolayer uptake and a much slower process of interlamellar exchange. The phase diagram of the binary lipid mixture dipalmitoylphosphatidylcholine-dipalmitoylphosphatidylethanolamine has also been examined and found to be essentially identical to the one constructed using a nitroxide probe.

Oxidation of spermidine and spermine in rat liver: purification and properties of polyamine oxidase.

Abstract: A novel enzyme responsible for the oxidation of spermidine and spermine has been found in rat liver. Spermidine is shown to be degraded to putrescine and 3-aminopropionaldehyde, and spermine to be cleaved to spermidine and 3-aminopropionaldehyde. A single enzyme catalyzing both reactions and designated as polyamine oxidase has been purified 4000-fold to electrophoretic homogeneity. Polyamine oxidase appears to be a flavoprotein, containing flavin adenine dinucleotide (FAD) as a prosthetic group. Hydrogen peroxide is evolved in the reaction and no other electron acceptors except molecular oxygen have been found. The molecular weight of the enzyme was approximately 60 000 and the sedimentation coefficient 4.5 S. The enzyme appears to be a single polypeptide chain since no evidence for structural subunits was obtained. Polyamine oxidase was sensitive to sulfhydryl and carbonyl group reagents. The optimum pH value for the oxidation of polyamines was close to 10. The reaction velocities were enhanced by various aldehydes, especially certain aromatic aldehydes. Polyamine oxidase appears to be localized in peroxisomes of liver cells, although the existence of an isoenzyme in the cytosolic fraction was not definitively ruled out. No marked changes were observed in the activity of polyamine oxidase in rat liver after partial hepatectomy, carbon tetrachloride poisoning, and after treatment with growth hormone or thioacetamide, conditions which are known to alter profoundly the metabolism and accumulation of polyamines.

The progeny of rabbit articular chondrocytes synthesize collagen types I and III and type I trimer, but not type II. Verifications by cyanogen bromide peptide analysis.

Abstract: The radioactive collagens synthesized by the fourth subculture progeny of rabbit articular chondrocytes were extracted and purified after limited pepsin digestion by neutral and acid salt precipitation. In order to identify the different types of collagen present, denatured collagen chains were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 5% gels, electrophoretically eluted, and cleaved with cyanogen bromide, and the resultant peptides were fractionated by a new sodium dodecyl sulfate electrophoresis system (tris(hydroxymethyl)aminomethane-borate buffer, 15% gels). Comparison of these separate peptide profiles with those from alpha1(I) and alpha1(III) collagen chains permitted the unambiguous identification of these chains in the radioactive collagen synthesized by chondrocytes. Although cartilage slices predominantly synthesized alpha1(II) chains, only alpha1(I) chains were made by cells in fourth subculture. A large fraction of these alpha1(I) chains could not be accounted for by the presence of type I collagen. While in a native, triple-helical conformation, some of these extra alpha1(I) chains were completely separated from type I collagen by their solubility at pH 8.0 in 2.6 M NaCl and therefore identified as [alpha1(I)]3, type I trimer. In addition to type I collagen and type I trimer, these chondrocyte progeny also synthesized type III collagen and two new collagen chains, X and Y. Each collagen type was further characterized by carboxymethylcellulose chromatography and its distribution between the medium and the cell layer. These findings support the idea that cultured chondrocytes assume a collagen phenotype similar to that of their undifferentiated mesenchymal cell precursors.

Exposure of tryptophanyl residues and protein dynamics

Abstract: The acrylamide quenching reaction is shown to be very discriminating in sensing the exposure of fluorescing tryptophanyl residues in globular proteins. The quenching rate constants for some proteins, such as aldolase and human serum albumin, are reported to be independent of the solvent viscosity, indicating that the reaction is limited by penetration of the quencher through the protein matrix. Temperature-dependent studies are performed to determine the activation energy and entropy for the penetration of acrylamide into these proteins. The tryptophanyl residues in aldolase are shown to be shielded by a large activation energy barrier, while the single residue in human serum albumin is shielded by a large activation entropy barrier. These parameters characterize the nature of the protein matrix enveloping the fluorophors.

Carbon-13 nuclear magnetic resonance probe of active-site ionizations in human carbonic anhydrase B.

Abstract: Human carbonic anhydrase B (HCAB), prepared by a new affinity chromatography procedure, was carboxymethylated exclusively at NT of its active-site histidine-200 using 90% [1-13C]bromoacetate. The 13C nuclear magnetic resonance signal of the covalently attached carboxylate was easily detected over the natural abundance background due to the other carbonyl and carboxyl carbons of this 29 000 molecular weight zinc metalloenzyme. Its chemical shift proved very sensitive to the presence of inhibitors in the active site and to variations in pH. Two perturbing groups with pKa values of 6.0 and 9.2 were assigned to the modified histidine-200 itself and the zinc-bound water ligand, respectively, making use of 13C NMR titration data on Nr- and Nr-carboxymethyl-L-histidine model compounds. The results rule out histidine-200 as the critical group whose ionization controls the catalytic activity. They also strongly suggest an interaction of the carboxylate of the carboxymethyl group with either the zinc or its water ligand around pH 8, possibly explaining the basis for the major differences between HCAB and CmHCAB.

Resonance Raman studies of the purple membrane

Abstract: The individual resonance Raman spectra of the PM568 and M412 forms of light-adapted purple membrane from Halobacterium halobium have been measured using the newly developed flow technique. For comparison purposes, the Raman spectra of the model chromophores, all-trans- and 13-cis retinal n-butylamine, both as protonated and unprotonated Schiff bases, have also been obtained. In agreement with previous work, the Raman data indicate that the retinal chromophore is linked to the purple membrane protein via a protonated. Schiff base in the case of the PM568 and an unprotonated Schiff base for the M412 form. The basic mechanism for color regulation in both forms appears to be electron delocalization. The spectral features of the two forms are different from each other and different from the model compound spectra.

Effect of a single cis double bond on the structures of a phospholipid bilayer.

Abstract: The ordering of the hydrocarbon chains and the rates of lipid motion are two independent parameters characterizing the structure and the dynamics, respectively, of a bilayer membrane. In this work, deuterium magnetic resonance has been used to elucidate the influence of a single cis double bond on the hydrocarbon chain ordering of a phospholipid bilayer. 1-Palmitoyl-2-oleoyl-3-sn-phosphatidylcholine was specifically deuterated at various segments of the palmitic acyl chain and at the 9, 10 position of the oleic acyl chain, and the segmental order parameters were deduced from the quadrupole splittings of the unsonicated bilayer phases. The shape of the order profile of the palmitic acyl chain is similar to that observed for the corresponding fully saturated membrane, but the magnitude of the order parameters is distinctly smaller in the unsaturated system. This demonstrates that the presence of a double bond in a membrane causes a more disordered conformation of the hydrocarbon chains. Considering the relative flexibility within the palmitic acyl chain, the deuterium resonance data indicate a local stiffening of those segments which are located in the vicinity of the double bond. The membrane fluidity was investigated using a nitroxide-labeled stearic acid spin probe. The smaller electron paramagnetic resonance line width in bilayers of 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine demonstrates an increased fluidity compared to bilayers of 1,2-dipalmitoyl-3-sn-phosphatidylcholine.

Conjugated polyene fatty acids on fluorescent probes: spectroscopic characterization.

Abstract: This paper is the first in a series which extends introductory studies of parinaric acid and its phospholipid derivatives as membrane probes (Sklar, L.A., Hudson, B., and Simoni, R.D. (1975), Proc. Natl. Acad. Sci. after U.S.A. 72, 1649; (1976), J. Supramol. Struct. 4, 449). Parinaric acid has a conjugated tetraene chromophore and exhibits many spectroscopic properties common to linear polyenes. Its absorption spectrum is characterized by a strong near-ultraviolet transition with vibronic structure, which is strongly affected by solvent polarizability. The fluorescence emission occurs at considerably lower energy than the absorption and the wavelength of the emission is nearly independent of the solvent. The fluorescence quantum yield and lifetime are strongly affected by temperature and solvent. These spectral features are interpreted in terms of an excited electronic-state order such that a weak transition occurs at longer wavelengths than the strongly allowed transition which dominates the absorption. The sensitivity of the fluorescence quantum yield an lifetime to environment is shown to be due primarily to variations in the nonradiative rate, although changes in the radiative rate constant are also observed and interpreted. The absorption spectrum (epsilon max greater than 65 000) is in the 300-320-nm range, a region relatively free of absorption due to intrinsic biological chromophores. Shifts of several nanometers are characteristic of different environments. These shifts are compared to similar effects observed for a series of diphenylpolyenes for which new data are given and are correlated using a simple but adequate theory of solvent shifts. The intrinsic (or radiative) fluorescence lifetime is near 100 ns in a wide variety of environments. This is much longer than the intrinsic lifetime calculated from the absorption spectrum and strongly supports the proposed excited-state order.

Nonspecific interaction of lac repressor with DNA: an association reaction driven by counterion release

Abstract: We have investigated the nonspecific interaction of lac repressor protein with DNA by a quantitative application of DNA-cellulose chromatography (deHaseth, P.L., et al. (1977), Biochemistry 16 (third of five papers in a series in this issue)). The observed association constant for the interaction, KRD obsd, is a sensitive function of ion concentrations and pH. Application of binding theory to interpret these effects gives the results that 11 +/- 2 monovalent ions are released in the interaction and two groups on repressor must be protonated for repressor to bind to DNA. We argue that much of the ion release results from the displacement of cations from the DNA, and estimate on this basis that 12 +/- 2 phosphates are involved in ionic interactions with the protein. Ion release drives the protonation reaction and the overall repressor-DNA interaction. The major role of low molecular weight ions in the repressor-DNA interaction suggests that ion concentration changes must be considered in discussing mechanisms of control of gene expression.

Lateral diffusion in phospholipid multibilayers measured by fluorescence recovery after photobleaching

Abstract: The method of fluorescence recovery after photobleaching has been used to measure the temperature dependence of the lateral diffusion coefficients (D) of two fluorescent lipid analogues in phospholipid multibilayers of various compositions. The probes employed were 3,3-dioctadecyloxocarbocyanine (diO-C18(3) and N-4-nitrobenz-2-oxa-1,3-diazole phosphatidylethanolamine (NBD-PE). In fluid egg phosphatidylcholine multibilayers at 25 degrees C, D was about 4 X 10(-8) cm2/s for NBD-PE and 1.5 X 10(-7) cm2/s for diO-C18(3) and was moderately temperature dependent (2-fold change over 10 degrees C). Equimolar cholesterol reduced D for NBD-PE in these multibilayers by a factor of 2. A greater than 100-fold decrease in D was detected in dimyristoylphosphatidylcholine multibilayers at approximately 23 degree C, which coincides with the gel-to-liquid-crystalline transition temperature, Tm (D 5 X 10(-8) cm2/s at T greater than Tm to D less than 5 X 10(-10) cm2/s at T less than Tm). Equimolar cholesterol abolished this transition behavior, raising D below Tm and decreasing D above Tm. These results confirm and extend previous studies of lateral diffusion employing magnetic resonance and other optical techniques and give additional confidence in the fluorescence methods.

Isolation and properties of the rabbit skeletal muscle protein inhibitor of adenosine 3',5'-monophosphate dependent protein kinases.

Abstract: The heat-stable protein inhibitor (Walsh, D. A., et al. (1971), J. Biol. Chem. 246, 1977--1985) of the cyclic adenosine 3',5'-monophosphate dependent protein kinase has been isolated in pure form from rabbit skeletal muscle after a 430 000-fold purification with a 47% yield. The four-step procedure involves sequentially a heat treatment, batchwise anion and cation exchange, and affinity chromatography on protein kinase catalytic subunit covalently coupled to Sepharose 4B. The inhibitor is an acidic protein (pI = 4.24) of molecular weight 11 300. It contains 98 amino acid residues none of which contains sulfur and only 2 (phenylalanine and tyrosine) are aromatic. The NH2-terminus is blocked. The muscle content is ca. 0.6 mg of inhibitor per L of intracellular water. The inhibitor is tightly bound to the catalytic subunit of protein kinase (Ki congruent to 2 X 10(-9) M) and acts competitively with respect to the protein substrates. Protein kinase recognizes a short stretch of the inhibitor sequence, in which arginyl side chains play a crucial role. A study of various competitive inhibitors of the kinase confirms the importance of guanidino groups and hydrophobic side chains in the specific interaction with the substrate binding site.

3'-Phosphatase activity in T4 polynucleotide kinase.

Abstract: The purification of T4 polynucleotide kinase results in the copurification of an activity which will specifically remove the 3'-terminal phosphate from a variety of deoxyribonucleotides and ribonucleotides in the absence of ATP. This phosphatase activity requires magnesium, has a pH optiumum of 6.0, and is more active with deoxyribonucleotides than ribonucleotides. T4 polynucleotide kinase and the 3'-phosphatase activity copurify by gradient elution column chromatography on DEAE-cellulose, phosphocellulose, and hydroxylapatite. The two activities are included in and comigrate on Sephadex G-200. Polyacrylamide gel electrophoresis at PH 9.2 results in conigration of the two activities together with the major protein band. The two activities respond in parallel to heat inactivation at 35 degrees C and ATP, a substrate for the kinase only, protects both activities from heat inactivation. It is therefore suggested that the two activities are functions of the same protein molecule.

Interpretation of monovalent and divalent cation effects on the lac repressor-operator interaction.

Abstract: We have investigated the effects of mixed Na+: Mg2+ ionic solutions on the stability of the nonspecific lac repressor-DNA complex The effects of Mg2+ are simply interpreted in terms of its role as a competitor (with repressor) for DNA sites From these studies, the binding constant of the Mg-DNA complex can be determined as a function of the concentration of Na+ We have used this information to interpret the data of Riggs and collaborators (Riggs, AD, et al (1970), J Mol Biol 48, 67-83; 53, 401-417) on the ion dependence of the repressor-operator interaction We find that there are approximately 70% as many ionic interactions in the repressor-operator complex as in the nonspecific complex Our best estimate is that 8 +/- 1 ion pairs are formed We calculate that the release of counterions in the formation of the specific complex contributes approximately 40% of the favorable free energy change in the association reaction under in vivo ionic conditions Implications of these findings for the control of the lac operon and for the molecular relationship between the specific and nonspecific complexes are considered