Showing papers in "Biochemistry in 1982"

Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma.

Abstract: We have studied the extractability of type IV collagen, laminin, and heparan sulfate proteoglycan from EHS tumor tissue growth in normal and lathyritic animals. Laminin and heparan sulfate proteoglycan were readily extracted with chaotropic solvents from both normal and lathyritic tissue. The collagenous component was only solubilized from lathyritic tissue in the presence of a reducing agent. These results indicate that lysine-derived cross-links and disulfide bonds stabilize the collagenous component in the matrix but not the laminin or the heparan sulfate proteoglycan. The majority of the collagen present in the extracts had a native triple helix based upon the pattern of peptides resistant to pepsin digestion and visualization in the electron microscope by the rotary shadow technique. This protein was composed of chains (Mr 185000 and 170000) identical in migration to the chains of newly synthesized type IV procollagen. This finding confirms earlier work that indicates that the biosynthetic form, type IV procollagen, is incorporated as such in the basement membrane matrix. Material with smaller chains (Mr 160000 and 140000) appeared on storage in acetic acid solutions. These results indicate that the lower molecular weight collagen in acid extracts of basement membrane arises artifactually due to an endogenous acid-active protease.

Stabilization of protein structure by sugars.

Abstract: The preferential interaction of proteins with solvent components was measured in aqueous lactose and glucose systems by using a high precision densimeter. In all cases, the protein was preferentially hydrated; i.e., addition of these sugars to an aqueous solution of the protein resulted in an unfavorable free-energy change. This effect was shown to increase with an increase in protein surface area, explaining the protein stabilizing action of these sugars and their enhancing effect of protein associations. Correlation of the preferential interaction parameter with the effect of the sugars on the surface tension of water, i.e., their positive surface tension increment, has led to the conclusion that the surface free energy perturbation by sugars plays a predominant role in their preferential interaction with proteins. Other contributing factors are the exclusion volume of the sugars and the chemical nature of the protein surface.

Purification and characterization of liver microsomal cytochromes p-450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or beta-naphthoflavone.

Abstract: Eight different forms of cytochrome P-450 (P-450) were purified to electrophoretic homogeneity by a common procedure from liver microsomes of rats treated with phenobarbital or beta-naphthoflavone. Antibodies were prepared to seven of these forms in rabbits. The eight P-450s were distinguished by spectral properties of the ferric, ferrous, and ferrous carbonyl forms, apparent monomeric molecular weights, peptide mapping, immunological reactivity as discerned by double-diffusion immunoprecipitin analysis and crossed immunoelectrophoresis, and catalytic activities toward the substrates acetanilide, aminopyrine, aniline, benzo[a]-pyrene, d-benzphetamine, N,N-dimethylnitrosamine, 7-ethoxycoumarin, 7-ethoxyresorufin, ethylmorphine, p-nitroanisole, testosterone, and (R)- and (S)-warfarin. Crossed sodium dodecyl sulfate-polyacrylamide gel immunoelectrophoresis was used to estimate the levels of each of the eight forms of P-450 present in the liver microsomes of untreated rats and rats treated with phenobarbital, 5,6-benzoflavone, pregnenolone-16 alpha-carbonitrile, isosafrole, or the polychlorinated biphenyl mixture Aroclor 1254. In each situation, the sum of the levels of these eight P-450s was at least as high as the spectrally determined P-450 content. The results clearly demonstrate that individual forms of P-450 can be induced by different compounds and that a single compound can lower the level of one form of P-450 while inducing one or more other forms of P-450. Catalytic activities toward each of the substrates observed with microsomal preparations are compared to rates predicted on the basis of the content of each of the eight P-450s. These studies provide a basis for further studies on the regulation of individual P-450s, the physical properties of the different P-450s, and the metabolic consequences of changes in the forms of P-450 in rat liver models.

Studies on interaction of anthracycline antibiotics and deoxyribonucleic acid: equilibrium binding studies on interaction of daunomycin with deoxyribonucleic acid.

Abstract: We have used equilibrium dialysis and fluorescence and absorbance titration to study the interaction of daunomycin with DNA. Our data at 200 mM Na+ are best fit by the neighbor exclusion model, with K = 7.0 x 10(5) M-1 and an exclusion parameter of three to four base pairs. The binding is dependent on ionic strength, with d log K/d log [Na+] = -0.84, from which we may estimate quantitatively ion release and the binding free energy corrected for the free energy of counterion release. From the temperature dependence of the binding constant, we find the binding to be exothermic, with a van't Hoff enthalpy of -12.8 kcal/mol. Competition dialysis experiments show that G+C base pairs are slightly preferred as binding sites for the drug and suggest that daunomycin binds preferentially to G+C pairs at low r. Cesium chloride density gradient sedimentation experiments provide an experimental demonstration of this preference. Daunomycin increases the Tm for DNA melting by some 30 degrees C as binding approaches saturation, with biphasic melting at low drug/base pair ratios. The data from these equilibrium studies are consistent with intercalative binding of daunomycin and provide a solid foundation for further structural and kinetic studies.

Preferential interactions of proteins with salts in concentrated solutions

Abstract: The preferential interactions of proteins with solvent components were studied in concentrated salt by densimetric measurements. Proteins were found to be preferentially hydrated in NaCl, NaCH3COO, and Na2SO4. The resulting unfavorable free-energy change was related to the effects of these salts on solubility and stability of the proteins. This unfavorable free-energy change was correlated with the large, positive surface tension increment of these salts, i.e., their perturbation of surface free energy. On the other hand, KSCN, CaCl2, and MgCl2 showed considerable binding to bovine serum albumin, which could be related to their destabilizing and salting-in effects on macromolecules. Since the last two salts have high surface tension increments, it was concluded that this does not necessarily lead to protein preferential hydration and stabilization.

Rat liver thioredoxin and thioredoxin reductase: purification and characterization

Abstract: A reproducible scheme has been developed for the preparation of rat liver thioredoxin and thioredoxin reductase (EC 1.6.4.5) by using assays based on reduction of insulin and 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Both proteins were purified to homogeneity, as judged from polyacrylamide gel electrophoresis. Thioredoxin had a molecular weight of 12 000 and contained about 110 amino acids including 4 half-cystines and an NH2-terminal valine. Peptide maps of reduced and carboxymethylated thioredoxin showed that the protein had the active center sequence -Cys-Gly-Pro-Cys-Lys-Met- characteristic of thioredoxins also from procaryotes. Prolonged air oxidation of fully reduced thioredoxin created inactive, aggregated disulfide-containing molecules. Thioredoxin reductase showed a subunit molecular weight of 58 000 and a native molecular weight of 116 000. The enzyme was highly specific for NADPH with a Km of 6 microM. It contained FAD as prosthetic group and was sensitive to inhibition by arsenite. Thioredoxin reductase had a Km of 2.5 microM for rat and calf liver thioredoxin and a Kcat of 3000 min-1.

Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model.

Abstract: The subunit structure of islet-activating protein (IAP), pertussis toxin, has been analyzed to study a possibility that this protein is one of the A-B toxins [Gill, D. M. (1978) in Bacterial Toxins and Cell Membranes (Jeljaszewicz, J., & Wadstrom, T., Eds.) pp 291-332, Academic Press, New York]. Heating IAP with 1% sodium dodecyl sulfate caused its dissociation into five dissimilar subunits named S-1 (with a molecular weight of 28 000), S-2 (23 000), S-3 (22 000), S-4 (11 700), and S-5 (9300), as revealed by polyacrylamide gel electrophoresis; their molar ratio in the native IAP was 1:1:1:2:1. The molecular weight of IAP estimated by equilibrium ultracentrifugation was 117 000 which was not at variance with the value obtained by summing up molecular weights of the constituent subunits. The preparative separation of these IAP subunits was next undertaken; exposure of IAP to 5 M ice-cold urea for 4 days followed by column chromatography with carboxymethyl-Sepharose caused sharp separation of S-1 and S-5, leaving the other subunits as two dimers. These dimers were then dissociated into their constituent subunits, i.e., S-2 and S-4 for one dimer and S-3 and S-4 for the other, after 16-h exposure to 8 M urea; these subunits were obtained individually upon further chromatography on a diethylaminoethyl-Sepharose column. Subunits other than S-1 were adsorbed as a pentamer by a column using haptoglobin as an affinity adsorbent. The same pentamer was obtained by adding S-5 to the mixture of two dimers. Neither this pentamer nor other oligomers (or protomers) exhibited biological activity in vivo. Recombination of S-1 with the pentamer at the 1:1 molar ratio yielded a hexamer which was identical with the native IAP in electrophoretic mobility and biological activity to enhance glucose-induced insulin secretion when injected into rats. In the broken-cell preparation, S-1 was biologically as effective as the native IAP; both catalyzed ADP-ribosylation of a protein in membrane preparations from rat C6 glioma cells. In conclusion, IAP is an oligomeric protein consisting of an A (active) protomer (the biggest subunit) and a B (binding) oligomer which is produced by connecting two dimers by the smallest subunit in a noncovalent manner. Rationale for this terminology is discussed based on the A-B model.

N-hydroxysulfosuccinimide active esters: bis(N-hydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane-impermeant, protein cross-linkers.

Abstract: We have synthesized and characterized N-hydroxysulfosuccinimide, a new hydrophilic ligand for the preparation of active esters. We have incorporated this ligand into two new protein cross-linking reagents, 3,3'-dithiobis(sulfosuccinimidyl propionate) and bis(sulfosuccinimidyl) suberate. In experiments with rabbit muscle aldolase, it is demonstrated that both of these reagents are highly efficient protein cross-linkers at physiological pH and that 3,3'-dithiobis(sulfosuccinimidyl propionate) is quantitatively cleavable by reduction under mild conditions. In experiments with intact human erythrocytes and erythrocyte membranes, it is shown that both reagents are membrane impermeant and that when erythrocytes are treated with either reagent, both cross-link subunits of the anion channel (band 3) to covalent dimers at the extracytoplasmic membrane face.

Adenosine cyclic 3',5'-monophosphate dependent protein kinase: kinetic mechanism for the bovine skeletal muscle catalytic subunit.

Abstract: The kinetic mechanism for adenosine cyclic 3',5'-monophosphate dependent protein kinase was determined from initial velocity studies in the absence and presence of the product MgADP and dead-end inhibitors. Data are consistent with random addition of MgATP and Ser-peptide and ordered release of phospho-Ser-peptide and MgADP with a dead-end E-MgADP-Ser-peptide complex. In addition to the metal required for the nucleotide, we also characterized the binding of Mg2+ to a second site. Increasing the Mg2+ results in a 5-6-fold decrease in V,,, in the presence or absence of 0.1 M KCl. There is a 5-fold increase in V/KMgATP in the absence of KCl and a 13-fold increase in V/KMdTP at 0.1 M KC1. The effect of increasing free Mg2+ on V,,, and V/K was also obtained with MgITP (20% the V,,, with MgATP) and MgGTP (10% the V,,, with MgATP) as substrates. The dissociation constant for Mg2+ from E-Ser-peptide-Mg2+ and central complexes is 2-3 mM. At low concentrations of free Adenosine cyclic 3',5'-monophosphate dependent protein kinase catalyzes the phorphorylation of a variety of proteins according to

Reversible Denaturation of Aequorea Green-Fluorescent Protein: Physical Separation and Characterization of the Renatured Protein

Abstract: The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75 The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 70 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 122 (excitation lambda max = 476 nm; emission lambda max = 505 nm) With its peak position red-shifted 78 nm at pH 122, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 70 are also identical, suggesting that the chromophore binding site has returned to its native state Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure

Synthesis of the antibacterial peptide cecropin A (1-33).

Abstract: Cecropin A(1-33) was synthesized by an improved stepwise solid-phase method. The synthesis was designed to give high coupling yields and minimal amounts of byproducts. All coupling steps were monitored for completion by a new ninhydrin procedure, and the fully protected peptide-resin was analyzed for deletion peptides by the solid-phase Edman preview technique. Both methods indicated that the average coupling yield was greater than 99.8%. The unpurified peptide mixture resulting from HF cleavage and extraction into 10% acetic acid was analyzed by reverse-phase high-pressure liquid chromatography, and 93% of the total product was shown to be the desired [Trp(For)2]cecropin A(1-33), indicating an average yield per synthetic cycle of 99.8%. Removal of the formyl group at pH 9, followed by ion-exchange chromatography, gave the purified product. Cecropin A(1-33) showed antibacterial activity against both Gram-positive and Gram-negative bacteria. Against Escherichia coli, the activity was only slightly lower than that of the natural 37-residue cecropin A when tested over a 100-fold concentration range; the minimum inhibitory concentration was approximately 1 microM. The formyl derivative was somewhat less effective in killing E. coli than the free 1-33 peptide. The antibacterial activity was discussed in terms of an amphipathic alpha-helix structure and the binding of the peptide to bacterial membranes.

Determination of the rate constant of enzyme modification by measuring the substrate reaction in the presence of the modifier

Abstract: On the basis of the equations derived previously [Tsou, C. L. (1965) Sheng Wu Hua Hsueh Yu Sheng Wu Wu Li Hsueh Pao 5, 398-408, 409-417] for the substrate reaction during the course of enzyme modification, the kinetic behavior of the system chymotrypsin-substrate-modifier has been studied. The kinetics of benzoyltyrosine ester hydrolysis during the course of irreversible inhibition of the enzyme has been found to be in satisfactory agreement with equations obtained previously. The apparent rate constant between the enzyme and an irreversible inhibitor can be easily obtained in one single experiment by following the course of substrate hydrolysis in the presence of the inhibitor. The results are also in accord with the assumption that diisopropyl fluorophosphate can be classified as an irreversible competitive inhibitor. For both phenylmethanesulfonyl fluoride and L-1-[(p-toluene-sulfonyl)amino]-2-phenylethyl chloromethyl ketone, the inhibition has been found to be in agreement with the kinetics of the complexing type; i.e., a noncovalent enzyme-inhibitor complex is formed before irreversible enzyme modification. Both the equilibrium constants for the complex formation and the first-order rate constants for the irreversible modification step have been determined also by following the course of substrate hydrolysis in the presence of the irreversible inhibitor.

Calcium-dependent .alpha.-helical structure in osteocalcin

Abstract: Osteocalcin is an abundant Ca2+-binding protein of bone containing three residues of vitamin K dependent gamma-carboxyglutamic acid (Gla) among its 49 (human, monkey, cow) or 50 (chicken) amino acids. Gla side chains participate directly in the binding of Ca2+ ions and the adsorption of osteocalcin to hydroxylapatite (HA) surfaces in vivo and in vitro. Osteocalcin exhibits a major conformational change when Ca2+ is bound. Metal-free chicken osteocalcin is a random coil with only 8% of its residues in the alpha helix as revealed by circular dichroism. In the presence of physiological levels of Ca2+, 38% of the protein adopts the alpha-helical conformation with a transition midpoint at 0.75 mM Ca2+ in a rapid, reversible fashion which (1) requires an intact disulfide bridge, (2) is proportionally diminished when Gla residues are decarboxylated to Glu, (3) is insensitive to 1.5 m NaCl, and (4) can be mimicked by other cations. Tyr fluorescence, UV difference spectra, and Tyr reactivity to tetranitromethane corroborate the conformational change. Homologous monkey osteocalcin also exhibits Ca2+-dependent structure. Integration of predictive calculations from osteocalcin sequence has yielded a structural model for the protein, the dominant features of which include two opposing alpha-helical domains of 9-12 residues each, connected by a bea turn and stabilized by the Cys23-Cys29 disulfide bond. Cation binding permits realization of the full alph a-helical potential by partial neutralization of high anionic charge in the helical domains. Periodic Gla occurrence at positions 17, 21, and 24 has been strongly conserved throughout evolution and places all Gla side chains on the same face of one alpha helix spaced at intervals of approximately 5.4 A, closely paralleling the interatomic separation of Ca2+ in the HA lattice. Helical osteocalcin has greatly increased affinity for HA; thus, the Ca2+-induced structural transition may perform an informational role related to bone metabolism.

Identification of myosin-binding sites on the actin sequence

Abstract: The rigor complex of actin and trypsin-treated myosin subfragment 1 (S1) whose heavy chain was cleaved into three fragments (20K, 25K, and 50K) was cross-linked with a zero-length cross-linker, 1-ethyl-3-[3-(dimethyl-amino) propyl]carbodiimide. The cross-linking reaction generated three types of cross-linked products with apparent molecular weights of 65K, 68K, and 95K. The 65K, 68K, and 95K products were covalently linked complexes of actin-20K fragment of the S1 heavy chain, actin-alkaline light chain 1, and actin-50K fragment of the S1 heavy chain, respectively. Cross-linking sites of S1 heavy and light chains on the actin sequence have been determined by digesting the cross-linked products with cyanogen bromide or with hydroxylamine and then mapping resulting peptides on sodium dodecyl sulfate gels. The result indicates that some of the N-terminal acidic residues of actin at positions 1, 2, 3, 4, and 11 are cross-linking sites of the 20K and 50K fragments of the S1 heavy chain while some of its C-terminal acidic residues at positions 360, 362, and 363 are cross-linking sites of the alkaline light chain 1.

Purification of zymogen to plasminogen activator from human glioblastoma cells by affinity chromatography with monoclonal antibody.

Abstract: Incorporation of the serine protease active site reagent diisopropyl fluorophosphate (DFP) into a plasminogen activator with an Mr of approximately 52000 released from cultured human glioblastoma cells was strongly enhanced by incubation with plasmin. This observation led to the isolation of an inactive form of the enzyme from serum-free conditioned culture fluid by affinity chromatography on a column of a Sepharose-bound monoclonal antibody raised against urokinase. An 831-fold purification was obtained with a yield of 41%. The purified molecule was homogeneous as evaluated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (NaDodSO4), having one stainable band under nonreducing as well as reducing conditions with an Mr of approximately 52000. It was unable to activate plasminogen, but catalytic amounts of plasmin converted it into active enzyme. After NaDodSO4-polyacrylamide gel electrophoresis, the active enzyme showed one band under nonreducing conditions, but after reduction, two bands with Mr values of approximately 20000 and 32000 were observed. The active enzyme incorporated [3H]DFP into the approximately Mr 32000 band, while no incorporation was observed into the inactive form. These findings show that the Mr 52000 human plasminogen activator exists in a proenzyme form consisting of a single polypeptide chain that by proteolysis between half-cystine residues is converted into the active enzyme consisting of two chains with molecular weights of approximately 20000 and 32000, the active site being on the latter chain. The results are consistent with the active form of the enzyme being identical with the higher molecular weight form of urokinase, and together with recent observations that a murine plasminogen activator is released from sarcoma virus transformed cells as an inactive proenzyme, they suggest that zymogens to plasminogen activators are of more general occurrence.

Selfassociation of daunomycin

Abstract: Daunomycin, a potent anthracycline antibiotic, self-associates in aqueous solution at concentrations greater than 10 microM. We report here visible absorbance, sedimentation equilibrium, and proton nuclear magnetic resonance (NMR) experiments that characterize this self-association. In contrast to earlier reports that the process is a simple dimerization, we find that an indefinite association model best fits our data, with an intrinsic association constant, Ki, equal to 1500 M-1. From the temperature dependence of the observed NMR spectra, an enthalpy of approximately -8.0 kcal/mol is calculated. The NMR data show that the aromatic protons of the anthracycline portion of the drug are most affected by aggregation, probably due to stacking of the anthracycline rings. Knowledge of the applicable model for the self-association process, and the equilibrium constant that describes the process, enables us to assess quantitatively the possible effects of drug aggregation on the interpretation of drug-DNA binding data. For the ionic conditions most commonly used in such studies, the amount of aggregated daunomycin will be slight and may safely be ignored.

Lipid selectivity of the calcium and magnesium ion dependent adenosine triphosphatase, studied with fluorescence quenching by a brominated phospholipid

Abstract: 1,2-Bis(9,10-dibromooleoyl)phosphatidylcholine (BRPC) has been prepared from dioleoylphosphatidylcholine (DOPC). It is shown that the gel to liquid-crystalline phase transition for BRPC occurs below ca. 5 degrees C and that the motional properties of bilayers of BRPC and DOPC as detected by spin-labeled fatty acids are similar. The ATPase activities of the (Ca2+-Mg2+)-ATPase from rabbit muscle sarcoplasmic reticulum reconstituted with BRPC and DOPC are similar. The brominated lipid quenches the fluorescence of the ATPase and can be used to determine selectivity of lipid binding to the ATPase. We show that there is little selectivity on the basis of fatty acyl chain length. Binding constants for phosphatidylcholines and phosphatidylserines are similar in the absence of calcium, although that for phosphatidylserine decreases in the presence of calcium. Phosphatidylethanolamines binds less strongly than phosphatidylcholines, although the difference is small. The largest difference in binding constants is seen between phosphatidylcholines in the gel and liquid-crystalline phases, with a distribution coefficient of 30 in favor of the liquid-crystalline phase. It is shown that the distribution of the ATPase in mixtures of dipalmitoylphosphatidylcholine and BRPC can be understood in terms of the phase diagram for this mixture of lipids. Activities of the ATPase in the presence of mixtures of lipids can be explained in terms of the relative binding constants obtained from the fluorescence experiments.

Characteristics of the isolated purine nucleotide binding protein from brown fat mitochondria.

Abstract: The isolation of the purine nucleotide binding protein (NbP), the putative uncoupling protein, from hamster brown adipose tissue mitochondria and some of its functional characteristics are described. (1) Among various detergents tested, Triton is the most suitable; the total GDP binding capacity can be recovered after solubilization by Triton and is rather stable in this extract. (2) For separation of NbP from the ADP/ATP carrier, differences in the solubilizing conditions and the stability at room temperature between both proteins are exploited. The preparation is substantially free of ADP/ATP carrier. (3) The purified NbP has a binding capacity for 16 mumol of GDP/g of protein, corresponding to a 16-fold purification from mitochondria. (4) In sodium dodecyl sulfate-polyacrylamide gel electrophoresis in single band of Mr 32 000 is found. A dimer structure is suggested from chemical cross-linking, from the binding capacity for GDP, and from the previously reported centrifugation equilibrium. (5) The isolated NbP preparation consists of Triton-protein-phospholipid mixed micelles with a Stokes radius of 60.5 A as determined by gel filtration. The Triton binding is 1.9 g/g of protein, and the phospholipid binding is 0.2 g/g of protein. (6). The amino acid composition has a polarity index of 43.5%. The N-terminal peptide has the sequence Val-Asp-Pro-Thr-Thr-Ser-Glu-Val. (7) The affinity of NbP for different purine nucleotides decreases in the order GTP greater than GDP greater than ATP greater than ITP greater than ADP greater than IDP. The affinity for the monophosphates is 100 time lower. (8) Photooxidation and the lysine reagent 2,4,6-trinitrobenzenesulfonic acid decrease the binding capacity without influencing the affinity of the unaffected sites. GDP protects against photooxidation.

Oxidation of trichloroethylene by liver microsomal cytochrome P-450: evidence for chlorine migration in a transition state not involving trichloroethylene oxide.

Abstract: Trichloroethylene (TCE) was metabolized by cytochrome P-450 containing mixed-function oxidase systems to chloral (2,2,2,-trichloroacetaldehyde), glyoxylic acid, formic acid, CO, and TCE oxide. TCE oxide was synthesized, and its breakdown products were analyzed. Under acidic aqueous conditions the primary products were glyoxylic acid and dichloracetic acid. The primary compounds formed under neutral or basic aqueous conditions were formic acid and CO. TCE oxide did not form chloral in any of these or other aqueous systems, even when iron salts, ferriprotoporphyrin IX, or purified cytochrome P-450 was present. Ferric iron salts catalyzed the rearrangement of TCE oxide to chloral only in CH2Cl2 or CH3CN. A 500-fold excess of iron was required for complete conversion. A kinetic model involving the zero-order oxidation of TCE to TCE oxide by cytochrome P-450 and the first-order degradation of the epoxide was used to test the hypothesis that TCE oxide is an obligate intermediate in the conversion of TCE to other metabolites. Kinetic constants fo the breakdown of TCE oxide and for the oxidative metabolism of TCE to stable metabolites were used to predict epoxide concentrations required to support the obligate intermediacy of TCE oxide. The maximum levels of TCE oxide detected in systems using microsomal fractions and purified cytochrome P-450 were 5-28-fold lower than those predicted from the model. The kinetic data and the discrepancies between the observed metabolites and TCE oxide breakdown products support the view that the epoxide is not an obligate intermediate in the formation of chloral, and an alternative model is presented in which chlorine migration occurs in an oxygenated TCE-cytochrome P-450 transition state.

Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase

Abstract: A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.

Role of lipid phase separations and membrane hydration in phospholipid vesicle fusion

Abstract: The relationship between lipid phase separation and fusion of small unilamellar phosphatidylserine-containing vesicles was investigated. The kinetics of phase separation were monitored by following the increase of self-quenching of the fluorescent phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, which occurs when the local concentration of the probe increases upon Ca2+-induced phase separation in phosphatidylserine (PS) bilayers [Hoekstra, D. (1982) Biochemistry 21, 1055-1061]. Fusion was determined by using the resonance energy transfer fusion assay [Struck, D. K., Hoekstra, D., & Pagano, R. E. (1981) Biochemistry 20, 4093-4099], which monitors the mixing of fluorescent lipid donor and acceptor molecules, resulting in an increase in energy transfer efficiency. The results show that in the presence of Ca2+, fusion proceeds much more rapidly (t 1/2 less than 5 s) than the process of phase separation (T 1/2 congruent to 1 min). Mg2+ also induced fusion, albeit at higher concentrations than Ca2+. Mg2+-induced phase separation were not detected, however. Subthreshold concentrations of Ca2+ (0.5 mM) or Mg2+ (2 mM) induced extensive fusion of PS-containing vesicles in poly(ethylene glycol) containing media. This effect did not appear to be a poly(ethylene glycol)-facilitated enhancement of cation binding to the bilayer, and consequently Ca2+-induced phase separation was not observed. The results suggest that macroscopic phase separation may facilitate but does not induced the fusion process and is therefore, not directly involved in the actual fusion mechanism. The fusion experiments performed in the presence of poly(ethylene glycol) suggest that the degree of bilayer dehydration and the creation of "point defects" in the bilayer without rigorous structural rearrangements in the membrane are dominant factors in the initial fusion events.

Purification of individual components of the neurofilament triplet: filament assembly from the 70 000-dalton subunit.

Abstract: Mammalian neurofilaments are composed of three subunit polypeptides with approximate molecular weights of 200 000, 150 000, and 70 000 (P200, P150, and P70). These subunits were separated by ion-exchange chromatography in the presence of 8 M urea. The P200 polypeptide was differentially eluted on a diethylaminoethyl (DEAE) column. The P70 and P150 polypeptides obtained after the DEAE column were separable on a hydroxylapatite column. Under neurofilament assembly conditions, only the P70 polypeptide was able to reassemble into an intermediate filament in the absence of the other two polypeptides. The P150 and P70 polypeptides copolymerized into an intermediate filament, only if P70 was present. These results suggest that the P70 polypeptide forms the core of the filament and the other two polypeptides are tightly associated accessory proteins.

Psoralen-deoxyribonucleic acid photoreaction. Characterization of the monoaddition products from 8-methoxypsoralen and 4,5'8-trimethylpsoralen.

Abstract: The isolation and structural characterization are described of the major monoaddition products formed in the photoreaction of two naturally occurring psoralens, 8-methoxypsoralen and 4,5',8-trimethylpsoralen, with high molecular weight, double-stranded DNA. Hydrolysis of the psoralen-modified DNA and subsequent chromatography resulted in the isolation of four modified nucleosides from each psoralen. Structural characterization was accomplished by mass spectrometry and 1H NMR analysis. The major products, accounting for 44-52% of the covalently bound psoralen, are two diastereomeric thymidine adducts formed by cycloaddition between the 5,6 double bond of the pyrimidine and the 4',5' (furan) double bond of the psoralen. A minor product, less than 2% of the covalently bound psoralen, is a furan-side adduct to deoxyuridine, derived from an initially formed deoxycytidine adduct by hydrolytic deamination. A fourth product is a thymidine adduct where cycloaddition has taken place between the 5,6 double bond of the pyrimidine and the 3,4 (pyrone) double bond of the psoralen. This pyrone-side adduct accounts for 19% of the covalently bound 8-methoxypsoralen but for less than 3% of the covalently bound 4,5'8-trimethylpsoralen. All of the isolated adducts have cis-syn stereochemistry. The stereochemistry and product distribution of the adducts are determined in part by the constraints imposed by the DNA helix on the geometry of the noncovalent intercalation complex formed by psoralen and DNA prior to irradiation.

Protein dynamics in solution and in a crystalline environment: a molecular dynamics study.

Abstract: The effect of a solvent and a crystalline environment on the dynamics of proteins is investigated by the method of computer simulation. Three 25-ps molecular dynamics simulations at 300 K of the bovine pancreatic trypsin inhibitor (BPTI), consisting of 454 heavy atoms, are compared: one of BPTI in vacuo, one of BPTI in a box with 2647 spherical nonpolar solvent atoms, and one of BPTI surrounded by fixed crystal image atoms. Both average and time-dependent molecular properties are examined to determine the effect of the environment on the behavior of the protein. The dynamics of BPTI in solution or in the crystal environment are found to be very similar to that found in the vacuum calculation. The primary difference in the average properties is that the equilibrium structure in the presence of solvent or the crystal field is significantly closer to the X-ray structure than is the vacuum result; concomitantly, the more realistic environment leads to a number density closer to experiment. The presence of solvent has a negligible effect on the overall magnitude of the positional or dihedral angle fluctuations in the interior of the protein; however, there are changes in the decay times of the fluctuations of interior atoms. For surface residues, both the magnitude and the time course of the motions are significantly altered by the solvent. There tends to be an increase in the displacements of long side chains and the flexible parts of the main chain that protrude into the solvent. Further, these motions tend to have a more diffusive character with longer relaxation times than in vacuo. The crystal environment has a specific effect on a number of side chains which are held in relatively fixed positions through hydrogen-bond and electric interactions with the neighboring protein atoms. Most of the effects of the solution environment seem to be sufficiently nonspecific that it may be possible to model them by applying a mean field and stochastic dynamic methods.