Abstract: A new set of hydrophilicity high-performance liquid chromatography (HPLC) parameters is presented. These parameters were derived from the retention times of 20 model synthetic peptides, Ac-Gly-X-X-(Leu)3-(Lys)2-amide, where X was substituted with the 20 amino acids found in proteins. Since hydrophilicity parameters have been used extensively in algorithms to predict which amino acid residues are antigenic, we have compared the profiles generated by our new set of hydrophilic HPLC parameters on the same scale as nine other sets of parameters. Generally, it was found that the HPLC parameters obtained in this study correlated best with antigenicity. In addition, it was shown that a combination of the three best parameters for predicting antigenicity further improved the predictions. These predicted surface sites or, in other words, the hydrophilic, accessible, or mobile regions were then correlated to the known antigenic sites from immunological studies and accessible sites determined by X-ray crystallographic data for several proteins.

Abstract: Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80 000 to 210 000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210 000 and 80 000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90 000-180 000 polypeptides are derived from the Mr 210 000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80 000-210 000 proteins and the generation of polypeptides of Mr 73 000, 50 000, and 43 000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90 000-210 000 proteins with the generation of an Mr 45 000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80 000 protein was not degraded by APC. Factor Xa cleaved the Mr 80 000-210 000 factor VIII proteins, resulting in the generation of fragments of Mr 73 000, 67 000, 50 000, 45 000, and 43 000. Factor Xa was found to initially activate and subsequently inactivate factor VIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Intrastrand cross-links represent the majority of modifications in DNA resulting from interaction with the cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) (cis-DDP). These adducts were recently characterized although several discrepancies remained to be resolved. In these studies, [3H]-cis-dichloro(ethylenediamine)platinum(II) (cis-DEP) was used because of the convenience of the radiolabel; this analogue produces adducts at identical sites in DNA as cis-DDP. Both drugs platinate the following sequences in DNA: GG, 65%; AG, 25%; GNG, 6%. The adduct at AG sequences invariably has adenine on the 5'-terminus of the dimer. The present enzyme digestion protocol included P1 nuclease, which produced complete digestion rather than as previously reported. The frequency of platination at GG was too high to be explained by an initial monofunctional platination at any guanine. However, direct bifunctional attack preferentially at GG was obviated because monofunctional adducts could be trapped with thiourea at short time periods. After short incubations, with cis-DEP and removal of unreacted drug, the monofunctional adducts slowly rearranged to bifunctional adducts. It is suggested that this evolution of adducts may result from the drug "walking" along the double helix, a phenomenon that does not appear to occur in single-stranded DNA.

Abstract: A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.

Abstract: The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.

Abstract: Protein-lipid interactions were studied by using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into membranes containing various synthetic lipids and native lipids. AChR function was determined by measuring two activities at 4 degrees C: (1) low to high agonist affinity-state transition of AChR in the presence of an agonist (carbamylcholine) in either membrane fragments or sealed vesicles and (2) ion-gating activity of AChR-containing vesicles in response to carbamylcholine. Sixteen samples were examined, each containing different lipid compositions including phosphatidylcholine, cholesterol, phosphatidic acid, phosphatidylethanolamine, asolectin, neutral lipid depleted asolectin, native lipids, and cholesterol-depleted native lipids. Phosphatidylcholines with different configurations of fatty acyl chains were used. The dynamic structures of these membranes were probed by incorporating spin-labeled fatty acid into AChR-containing vesicles and measuring the order parameters. It was found that both aspects of AChR function were highly dependent on the lipid environment even though carbamylcholine binding itself was not affected. An appropriate membrane fluidity was necessarily required to allow the interconversion between the low and high affinity states of AChR. An optimal fluidity hypothesis is proposed to account for the conformational transition properties of membrane proteins. In addition, the conformational change was only a necessary, but not sufficient, condition for the AChR-mediated ion flux activity. Among membranes in which AChR manifested the affinity-state transition, only those containing both cholesterol and negatively charged phospholipids (such as phosphatidic acid) retained the ion-gating activity.

Abstract: The binding of factor VII and tissue factor produces a membrane-associated proteolytic complex which may be the primary biological initiator of coagulation Homogeneous tissue factor, a glycoprotein purified from bovine brain, was reconstituted into phospholipid vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (40% phosphatidylserine) with octyl glucoside The vesicles were characterized with respect to size and tissue factor content and orientation Employing data from protease digestion, we deduced that tissue factor is randomly oriented; thus, its effective concentration in these vesicles was half its total concentration In all binding experiments, 1 mol of enzyme was bound per mole of available activator at saturation This stoichiometry was not affected by the form of the enzyme employed or the phospholipid composition of the vesicles With tissue factor incorporated into phosphatidylcholine vesicles, the Kd was 132 +/- 072 nM for factor VII and 454 +/- 137 nM for factor VIIa Thus, the one-chain zymogen binds to the activator with only slightly less affinity than the more active two-chain enzyme Active-site modification of factor VII and factor VIIa with diisopropyl fluorophosphate resulted in tighter binding of the derivatized molecules Inclusion of phosphatidylserine in the vesicles altered the binding both quantitatively and qualitatively With increasing acidic phospholipid, the concentration of enzyme required to occupy half the activator sites was decreased In addition, positive cooperativity was observed, the degree of which depended on the vesicle charge and the form of the enzyme An explicit two-site cooperative binding model is presented which fits these complex data In this model, tissue factor is at least a dimer with two interacting enzyme binding sites

Abstract: A protein kinase phosphorylation site in chicken gizzard myosin light chain kinase (MLCK) has been identified, and a synthetic peptide analogue of this site has been shown to be a high-affinity calmodulin binding peptide as well as a substrate for cyclic AMP dependent protein kinase. Phosphorylation of the site in MLCK is diminished when reactions are done in the presence of calmodulin. A fragment of MLCK containing the phosphorylation site was shown to have the amino acid sequence Ala-Arg-Arg-Lys-Trp-Gln-Lys-Thr-Gly-His-Ala-Val-Arg-Ala-Ile-Gly-Arg-Leu- Ser-Ser. The interaction of calmodulin with a synthetic peptide based on this sequence was characterized by using circular dichroism and fluorescence spectroscopies and inhibition of calmodulin activation of MLCK. The peptide-calmodulin complex had an estimated dissociation constant in the range of 1 nM, underwent spectroscopic changes in the presence of calmodulin consistent with the induction of an alpha-helical structure, and interacted with calmodulin with an apparent 1:1 stoichiometry. Studies with other synthetic peptide analogues indicated that the phosphorylation of the serine residues diminished the ability of the peptide to interact with calmodulin even though the serines are not required for calmodulin binding. On the basis of the primary and secondary structural characteristics of these peptide analogues, a potential calmodulin binding region in another calmodulin binding protein, the gamma subunit of rabbit skeletal muscle phosphorylase kinase, was identified.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: A new nitrogenase from Azotobacter vinelandii has been isolated and characterized. It consists of two proteins, one of which is almost identical with the Fe protein (component 2) of the conventional enzyme. The second protein (Av1'), however, has now been isolated and shown to differ completely from conventional component 1, i.e., the MoFe protein. This new protein consists of two polypeptides with a total molecular weight of around 200,000. In place of Mo and Fe it contains V and Fe with a V:Fe ratio of 1:13 +/- 3. The ESR spectrum of Av1' also differs from conventional component 1 in that lacks the g = 3.6 resonance that arises from the FeMo cofactor but contains an axial signal with gav less than 2 as well as inflections in the g = 4-6 region possibly arising from an S = 3/2 state. This new enzyme can reduce dinitrogen, protons, and acetylene but is only able to utilize 10-15% of its electrons for the reduction of acetylene.

Abstract: The hydration repulsive force between lipid bilayers and the deformability of both gel and liquid-crystalline bilayers have been quantitated by an X-ray diffraction analysis of osmotically stressed liposomes. Both sampling theorem reconstructions and electron density distributions were calculated from diffraction data obtained from multilayers with applied osmotic pressures of 0-50 atm. The bilayer thickness and area per lipid molecule remain nearly constant (to within about 4%) in this pressure range, as adjacent bilayers move from their equilibrium separation in excess water to within 2-4 A of each other. This analysis indicates that the bilayers are relatively incompressible. This results differs from previously published X-ray diffraction studies of bilayer compressibility but agrees with direct mechanical measurements of the bilayer compressibility modulus. It is also found that the hydration repulsive force decays exponentially with separation between bilayers with a decay constant of 1.4 A for gel-state dipalmitoylphosphatidylcholine and 1.7 A for liquid-crystalline egg phosphatidylcholine bilayers. This implies that the exponential decay constant is not necessarily equal to the diameter of a water molecule, as has been previously suggested on experimental and theoretical grounds.

Abstract: A lambda gtll cDNA library prepared from human liver poly(A) RNA has been screened with affinity-purified antibody to human factor XI, a blood coagulation factor composed of two identical polypeptide chains linked by a disulfide bond(s) A cDNA insert coding for factor XI was isolated and shown to contain 2097 nucleotides, including 54 nucleotides coding for a leader peptide of 18 amino acids and 1821 nucleotides coding for 607 amino acids that are present in each of the 2 chains of the mature protein The cDNA for factor XI also contained a stop codon (TGA), a potential polyadenylation or processing sequence (AACAAA), and a poly(A) tail at the 3' end Five potential N-glycosylation sites were found in each of the two chains of factor XI The cleavage site for the activation of factor XI by factor XIIa was identified as an internal peptide bond between Arg-369 and Ile-370 in each polypeptide chain This was based upon the amino acid sequence predicted by the cDNA and the amino acid sequence previously reported for the amino-terminal portion of the light chain of factor XI Each heavy chain of factor XIa (369 amino acids) was found to contain 4 tandem repeats of 90 (or 91) amino acids plus a short connecting peptide Each repeat probably forms a separate domain containing three internal disulfide bonds The light chains of factor XIa (each 238 amino acids) contain the catalytic portion of the enzyme with sequences that are typical of the trypsin family of serine proteases The amino acid sequence of factor XI shows 58% identity with human plasma prekallikrein

Abstract: Peptidyl fluoromethyl ketones that are specific inhibitors of the serine proteases ..cap alpha..-chymotrypsin and porcine pancreatic elastase were synthesized. By analogy with the corresponding aldehydes it is assumed that the fluoromethyl ketones react with the ..gamma..-OH group of the active site serine to form a stable hemiacetal. /sup 19/F NMR studies of the chymotrypsin-bound trifluoromethyl ketone inhibitors Ac-Leu-ambo-Phe-CF/sub 3//sup 1/ and Ac-ambo-Phe-CF/sub 3/ clearly indicate that the carbonyl carbon is tetrahedral at the active site of the enzyme. The inhibitor is bound as either the stable hydrat or the hemiacetal, involving the active site serine. The effect of varying the number of amino acid residues in the peptidyl portion of the inhibitor and the number of fluorines in the fluoromethyl ketone moiety is examined. In the series of trifluoromethyl ketone elastase inhibitors, the lowering of K/sub i/ concomitant with the change from a dipeptide analogue to a tetrapeptide analogue correlates well with the variation in V/K for hydrolysis of the corresponding amide substrates. This trend is indicative of the inhibitors acting as transition-state analogues. In addition to chain length, the number of fluorine substituents also affects the K/sub i/. In the case of chymotrypsin, the K/sub i/ for Ac-Leu-ambo-Phe-CF/sub 3/more » is 30-fold lower than that for Ac-Leu-ambo-Phe-CF/sub 2/H. With elastase this trend is not as profound. In all cases, however, the difluoro- and trifluoromethyl ketones are better inhibitors than the monofluoromethyl and nonfluorinated analogues. This improvement must be associated with both the degree of hydration of the fluoromethyl ketones and the significant effect that fluorine substitution has on lowering the first pK/sub a/ of the hemiacetal hydroxyl. The monofluoromethyl ketone inhibitor of chymotrypsin, Ac-Leu-ambo-Phe-CFH/sub 2/, is a weak competitive inhibitor.« less

Abstract: A fast algorithm that detects internal cavities in proteins and predicts the positions of buried water molecules is described. The cavities are characterized in terms of volume, surface area, polarity, and the presence of bound waters. The algorithm is applied to 12 proteins whose structures are known to high resolution and successfully predicts the locations of over 80% of internal water molecules. Most proteins are found to have a number of internal cavities ranging in volume from 10 to 180 A3. Some of these cavities contain water and some do not, with the probability of containing a buried water increasing with cavity size. However, many large cavities are found to be empty (i.e., they do not contain a crystallographically determined water). For multidomain proteins over half of the total cavity volume is at the interdomain interface. Possible implications for the energetics of cavity formation and for the functional role of internal cavities are discussed.

Abstract: Preferred conformations, orientations, and accumulations of 26 opioid peptides on lipid membranes were estimated and compared with pharmacologic and selective binding data taken from the literature. Interaction with mu-receptors was governed by the net positive charge effective at the message domain of the agonist peptides z(eff) as the Boltzmann term ez(eff) that determines relative accumulation on anionic biologic membranes. Selection for delta-receptors was reduced by z(eff) and correlated with e-z(eff). Selection for kappa-receptors was governed by the peptide amphiphilic moment A. A pronounced scalar magnitude A and almost perpendicular orientation of the N-terminal message domain as an alpha-helix were favorable for kappa-site selection. Potencies as kappa-agonists and binding affinities correlated with A X ez(eff). The classical site selectivity caused by the receptor requirements for a complementary fit of the agonist to the discriminator site is thus crucially supplemented by a selection mechanism based on peptide membrane interactions (membrane requirements). In the model presented here, the delta-site is exposed to the aqueous compartment surrounding the target cell at a distance comparable to or greater than the Debye-Huckel length and is in a cationic vicinity. The mu-site is exposed to the anionic fixed-charge compartment of the membrane in aqueous surroundings. The kappa-site is buried in a more hydrophobic membrane compartment close to the fixed-charge compartment. The relative accumulation of the opioid message domains in these compartments is determined by the address domains and constitutes a major part of the site selection mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The area per lipid molecule for fully hydrated dilauroylphosphatidylethanolamine (DLPE) has been obtained in both the gel and liquid-crystalline states by combining wide-angle X-ray diffraction, electron density profiles, and previously published dilatometry results [Wilkinson, D. A., & Nagle, J. F. (1981) Biochemistry 20, 187-192]. The molecular area increases from 41.0 +/- 0.2 to 49.1 +/- 1.2 A2 upon melting from the gel to liquid-crystalline phase. The thickness of the bilayer, as measured from the electron density profiles, decreases about 4 A upon melting, from 45.2 +/- 0.3 to 41.0 +/- 0.6 A. A somewhat unexpected result is that the fluid layer between fully hydrated bilayers is the same in both gel and liquid-crystalline phases and is only about 5 A thick. From these data, plus the volume of the anhydrous DLPE molecule, it is possible to determine the number of water molecules per lipid and their approximate distribution relative to the lipid molecule. Our analysis shows that there are about 7 and 9 waters per DLPE molecule in the gel and liquid-crystalline phases, respectively. About half of the water is located in the fluid space between adjacent bilayers, and the remaining waters are intercalated into the bilayer, presumably in the head group region. There are significantly fewer water molecules in the fluid spaces between DLPE bilayers than in the fluid spaces in gel- or liquid-crystalline-phase phosphatidylcholine bilayers. This small fluid space in PE bilayers could arise from interbilayer hydrogen bond formation through the water molecules or electrostatic interactions between the amine and phosphate groups on apposing bilayers.

Abstract: We report a rapid procedure for the large-scale purification of the Escherichia coli encoded single-strand binding (SSB) protein, helix-destabilizing protein which is essential for replication, recombination, and repair processes in E. coli. To facilitate the isolation of large quantities of the ssb gene product, we have subcloned the ssb gene into a temperature-inducible expression vector, pPLc28 [Remaut, E., Stanssens, P., & Fiers, W. (1981) Gene 15, 81-93], carrying the bacteriophage lambda PL promoter. A large overproduction of the ssb gene product results upon shifting the temperature of E. coli strains which carry the plasmid and also produce the thermolabile lambda cI857 repressor. After 5 h of induction, the ssb gene product represents approximately 10% of the total cell protein. The overexpression of the ssb gene and the purification protocol reported here enable one to isolate SSB protein (greater than 99% pure) with final yields of approximately 3 mg of SSB protein/g of cell paste. In fact, very pure (greater than 99%) SSB protein can be obtained after approximately 8 h, starting from frozen cells in the absence of any columns, although inclusion of a single-stranded DNA-cellulose column is generally recommended to ensure that the purified SSB protein possesses DNA binding activity. The ability to easily purify 1 g of SSB protein from 300-350 g of induced cells will facilitate physical studies requiring large quantities of this important protein.

Abstract: The refolding kinetics of alpha-lactalbumin at different concentrations of guanidine hydrochloride have been investigated by means of kinetic circular dichroism and stopped-flow absorption measurements. The refolding reaction consists of at least two stages, the instantaneous accumulation of the transient intermediate that has peptide secondary structure and the subsequent slow process associated with formation of tertiary structure. The transient intermediate is compared with the well-characterized equilibrium intermediate observed during the denaturant-induced unfolding. Stabilities of the secondary structures against the denaturant, affinities for Ca2+, and tryptophan absorption properties of the transient and equilibrium intermediates were investigated. In all of these respects, the transient intermediate is identical with the equilibrium one, demonstrating the validity of the use of the equilibrium intermediate as a model of the folding intermediate. Essentially the same transient intermediate was also detected in the folding of lysozyme, the protein known to be homologous to alpha-lactalbumin but whose equilibrium unfolding is represented as a two-state reaction. The stability and cooperativity of the secondary structure of the intermediate of lysozyme are compared with those of alpha-lactalbumin. The results show that the protein folding occurring via the intermediate is not limited to the proteins that show equilibrium intermediates. Although the unfolding equilibria of most proteins are well approximated as a two-state reaction, the two-state hypothesis may not be applicable to the folding reaction under the native condition. Two models of protein folding, intermediate-controlled folding model and multiple-pathway folding model, which are different in view of the role of the intermediate in determining the pathway of folding, are also discussed.

Abstract: Factor X is one of six vitamin K dependent proteins known to be involved in blood coagulation, the others being factor VII, factor IX, prothrombin, protein S, and protein C. In the present studies, recombinant bacteriophage containing overlapping DNA inserts coding for the gene for human factor X have been isolated and characterized. These DNA inserts code for almost the entire gene for factor X, extending from the prepro leader peptide through the 3' noncoding region of the transcription product. The organization of the gene for factor X was established by DNA sequencing to identify the location of the introns and exons in the gene. Seven introns and eight exons were identified and their intron/exon boundaries established. The seven introns interrupt the coding sequence at essentially identical locations in the amino acid sequence as the introns in the genes for human factor IX and protein C. In addition, the introns in the gene for factor X divide the coding sequence into discrete exons that code for potential structural and functional domains of the protein. This information provides strong evidence to support the suggestion that the vitamin K dependent proteins present in plasma have evolved from a single, common gene and that this ancestral gene arose through a process that involved the assembly of small protein coding units of DNA into a single gene.

Abstract: The effects of incorporating diacylglycerol (DG) derived from egg phosphatidylcholine (PC) into PC, egg phosphatidylethanolamine (PE), and bovine phosphatidylserine (PS) have been measured. In excess solution DG induces a multilamellar-to-hexagonal (L-H) structural transition in PE and PC that is temperature dependent. At 37 degrees C it begins at about 3 and 30 mol%, respectively. In PC at lower DG concentrations a modified lamellar phase is formed; at about 70 mol% DG a single primitive cubic phase forms. An L-H transition induced by 20-30 mol% DG in PS is dependent on ionic strength and degree of lipid hydration, with the appearance of crystalline acyl chains at the higher DG levels. Calcium precipitates of DG/PS (1/1) mixtures have melted chains. Structural parameters were derived for the lamellar phases at subtransition levels of DG in PE and PC. The area per polar group is increased, but by contrast with cholesterol, the polar group spreading is not accompanied by an increase in bilayer thickness. DG does not affect the equilibrium separation of PC or PE bilayers. Measured interbilayer forces as they vary with bilayer separation show that DG at 20 mol% does not effect closer apposition of PC bilayers at any separation. Spreading the polar groups may effect the binding of protein kinase C or the activation of phospholipases; the nonlamellar phases may be linked to the biochemical production of DG in cellular processes involving membrane fusion.

Abstract: X-ray structural information provides the opportunity to explore quantitatively the relation between the microenvironments of heme proteins and their redox potentials. This can be done by considering the protein as a "solvent" for its redox center and calculating the difference between the electrostatic energy of the reduced and oxidized heme. Such calculations are presented here, applying the protein dipoles-Langevin dipoles (PDLD) model to cytochrome c. The calculations focus on an evaluation of the difference between the redox potentials of cytochrome c and the octapeptide-methionine complex formed by hydrolysis of cytochrome c. The corresponding difference (approximately 7 kcal/mol) is accounted for by the PDLD calculations. It is found that the protein provides basically a low dielectric environment for the heme, which destabilizes the oxidized heme (relative to its energy in water). The effect of the charged propionic acids on the heme is examined in a preliminary way. It is found that the negative charges of these groups are in a hydrophilic rather than a hydrophobic environment and that the protein-water system provides an effective high dielectric constant for their interaction with the heme. The dual nature of the dielectric effect of the cytochrome (a low dielectric constant for the self-energy of the heme and a high dielectric constant for charge-charge interactions) is discussed. The findings of this work are consistent with the difference between the folding energies of the reduced and oxidized cytochrome c.

Abstract: Primary cultures of neonatal human foreskin keratinocytes converted 25-hydroxyvitamin D in high yield to a metabolite with the chromatographic behavior of 1,25-dihydroxyvitamin D3. The identity of this metabolite as 1,25-dihydroxyvitamin D3 was confirmed both by its potency in displacing 1,25-dihydroxyvitamin D3 in the chick cytosol receptor assay and by mass spectral analysis. These results suggest that 1,25-dihydroxyvitamin D3 may be formed in the epidermis to regulate vitamin D production by the epidermis and to provide an alternative to 1,25-dihydroxyvitamin D3 production by the kidneys.

Abstract: Reaction centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26.1 were depleted of Fe by a simple procedure involving reversible dissociation of the H subunit. The resulting intact Fe-depleted RCs contained 0.1-0.2 Fe per RC as determined from atomic absorption and electron paramagnetic resonance (EPR) spectroscopy. Fe-depleted RCs that have no metal ion occupying the Fe site differed from native RCs in the following respects: (1) the rate of electron transfer from QA- to QB exhibited nonexponential kinetics with the majority of RCs having a rate constant slower by only a factor of approximately 2, (2) the efficiency of light-induced charge separation (DQA----D+QA-) produced by a saturating flash decreased to 63%, and (3) QA appeared readily reducible to QA2-. Various divalent metal ions were subsequently incorporated into the Fe site. The electron transfer characteristics of Fe-depleted RCs reconstituted with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+ were essentially the same as those of native RCs. These results demonstrate that neither Fe2+ nor any divalent metal ion is required for rapid electron transfer from QA- to QB. However, the presence of a metal ion in the Fe site is necessary to establish the characteristic, native, electron-transfer properties of QA. The lack of a dominant role of Fe2+ or other divalent metals in the observed rate of electron transfer from QA- to QB suggests that a rate-limiting step (for example, a protonation event or a light-induced structural change) precedes electron transfer.

Abstract: The dependence on thiol pK of the second-order rate constant (kS) for reaction of thiolate anions with MMTS was shown to follow the Bronsted equation log kS = log G + beta pK with log G = 1.44 and 3.54 and beta = 0.635 and 0.309 for aryl and alkyl thiols, respectively. The reactivity toward MMTS of the protonated thiol group was found to be negligible in comparison to that of the thiolate anion. For 2-mercaptoethanol the reactivity toward MMTS of the protonated form of the thiol group was shown to be at least 5 X 10(9) smaller than that of the thiolate anion. The pH dependence of the second-order rate constant for reaction of the thiolate group of Cys-25 at the active site of papain was determined and shown to be consistent with the previously determined low pK for Cys-25 and its electrostatic interaction with His-159. The small dependence of the reactivity of Cys-25 on thiol pK (beta approximately 0.09) suggested that the charge-charge interactions that act through space to perturb the pK of the nucleophile at the active site of papain and perhaps other enzymes may serve to increase the fraction of nucleophile present in the reactive basic form without introducing the decrease in nucleophilic reactivity seen in model systems where pK's are lowered primarily by charge-dipole interactions.

Abstract: Factor XIII is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The complete amino acid sequence of the a subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gtll cDNA library prepared from human placenta mRNA was screened with an affinity-purified antibody against the a subunit of human factor XIII and then with a synthetic oligonucleotide probe that coded for a portion of the amino acid sequence present in the activation peptide of the a subunit. Six positive clones were identified and shown to code for the a subunit of factor XIII by DNA sequence analysis. A total of 3831 base pairs was determined by sequencing six overlapping cDNA clones. This DNA sequence contains a 5' noncoding region or a region coding for a portion of a pro-piece or leader sequence, the mature protein (731 amino acids), a stop codon (TGA), a 3' noncoding region (1535 nucleotides), and a poly(A) tail (10 nucleotides). When the a subunit of human factor XIII was digested with cyanogen bromide, 11 peptides were isolated by gel filtration and reverse-phase HPLC. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 363 amino acid residues were identified. These amino acid sequences were in complete agreement with those predicted from the cDNA. The a subunit of factor XIII contained the active site sequence of Tyr-Gly-Gln-Cys-Trp, which is identical with that of tissue transglutaminase.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The physical properties in water of a series of 1:1 acid-soap compounds formed from fatty acids and potassium soaps with saturated (10-18 carbons) and omega-9 monounsaturated (18 carbons) hydrocarbon chains have been studied by using differential scanning calorimetry (DSC), X-ray diffraction, and direct and polarized light microscopy. DSC showed three phase transitions corresponding to the melting of crystalline water, the melting of crystalline lipid hydrocarbon chains, and the decomposition of the 1:1 acid-soap compound into its parent fatty acid and soap. Low- and wide-angle X-ray diffraction patterns revealed spacings that corresponded (with increasing hydration) to acid-soap crystals, hexagonal type II liquid crystals, and lamellar liquid crystals. The lamellar phase swelled from bilayer repeat distances of 68 (at 45% H2O) to 303 A (at 90% H2O). Direct and polarized light micrographs demonstrated the formation of myelin figures as well as birefringent optical textures corresponding to hexagonal and lamellar mesophases. Assuming that 1:1 potassium hydrogen dioleate and water were two components, we constructed a temperature-composition phase diagram. Interpretation of the data using the Gibbs phase rule showed that, at greater than 30% water, hydrocarbon chain melting was accompanied by decomposition of the 1:1 acid-soap compound and the system changed from a two-component to a three-component system. Comparison of hydrated 1:1 fatty acid/soap systems with hydrated soap systems suggests that the reduced degree of charge repulsion between polar groups causes half-ionized fatty acids in excess water to form bilayers rather than micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: Oligopeptides have been synthesized that are structurally related to the antiviral antitumor antibiotic netropsin, but in which each of the pyrrole units is successively replaced by an imidazole moiety, as well as their di- and triimidazole-containing counterparts. These compounds bind to duplex DNA with constants in the range (1.06-1.98) X 10(6) M-1 but not to single-stranded DNA. Since they bind to T4 DNA, it is inferred that, like the parent antibiotic netropsin, they are also minor groove selective. This series of compounds exhibits a progressively decreasing preference for AT sites in binding studies with both native DNAs and synthetic oligonucleotides and a corresponding increasing acceptance of GC base pairs. Footprinting experiments utilizing a 139 base pair HindIII/NciI restriction fragment from pBR 322 DNA revealed that these lexitropsins, or information-reading oligopeptides, recognize more sites than the parent netropsin. In addition, some regions of enhanced nuclease action as the result of drug binding to the fragment were identified. The diimidazole compound in particular recognizes GC-rich sites, implying the formation of new hydrogen bonds between G-C(2)NH2 in the minor groove and the additional N3 imidazole nitrogens. It is clear however that, since the lexitropsins appear to tolerate the original (AT)4 site, an N-methylimidazole group on the ligand will permit either a GC or AT base pair in the binding sequence. Another factor that may be significant in molecular recognition is the high negative electrostatic potential of A X T regions of the minor groove, which is likely to strongly influence binding of these cationic species to DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: The chirality of biologically active S-adenosyl-L-methionine (AdoMet) is S,S, where the designations refer to the sulfur and the alpha-carbon, respectively. This paper describes a cation-exchange high-performance liquid chromatographic (HPLC) method for separating (S,S)-AdoMet from the biologically inactive (R,S)-AdoMet that results from racemization at the sulfur. This method was used to measure the rates of the degradation reactions of (S,S)-AdoMet as a function of pH. These reactions and the first-order rate constants, which were found at 37 degrees C and pH 7.5, are racemization, 1.8 X 10(-6) s-1; cleavage to homoserine lactone and 5'-(methylthio)adenosine, 4.6 X 10(-6) s-1; and hydrolysis to adenine and S-pentosylmethionine, 3 X 10(-6) s-1. Racemization showed no change in rate over the pH range from 7.5 to 1.5. The cleavage reaction persisted until the pH was lowered to 1.5, but hydrolysis ceased at pH 6. Commercial samples of nonradioactive AdoMet contained 20-30% (R,S)-AdoMet, while a sample of [methyl-3H]AdoMet had less than 1% (R,S)-AdoMet. Preparing enzyme substrates by mixing such samples will cause an underestimate of specific activity and an overestimate of the amount of product. The (R,S)-AdoMet/(S,S)-AdoMet ratio in mouse liver was 0.03, much less than the value of 0.19 calculated from the above rate constants. An enzyme extract from mouse liver did not degrade (R,S)-AdoMet, but a more thorough search may find such an activity. In any event, the cleavage and hydrolysis reactions partially balance the racemization of (S,S)-AdoMet in vivo and prevent excessive accumulation of (R,S)-AdoMet.

Abstract: The interaction of the bile salt cholate with unilamellar vesicles was studied. At low cholate content, equilibrium binding measurements with egg yolk lecithin membranes suggest that cholate binds to the outer vesicle leaflet. At increasing concentrations, further bile salt binding to the membrane is hampered. Before the onset of membrane solubilization, diphenylhexatriene fluorescence anisotropy decreases to a shallow minimum. It then increases to the initial value in the cholate concentration range of membrane solubilization. At still higher cholate concentrations, a drop in fluorescence anisotropy indicates the transformation of mixed disk micelles into spherical micelles. Perturbation of the vesicle membranes at molar ratios of bound cholate/lecithin exceeding 0.15 leads to a transient release of oligosaccharides from intravesicular space. The cholate concentrations required to induce the release depend on the size of the entrapped sugars. Cholesterol stabilizes the membrane, whereas, in spite of enhanced membrane order, sphingomyelin destabilizes the membrane against cholate. Freeze-fracture electron microscopy and phosphorus-31 nuclear magnetic resonance (31P NMR) also reflect a change in membrane structure at maximal cholate binding to the vesicles. In 31P NMR spectra, superimposed on the anisotropic line typically found in phospholipid bilayers, an isotropic peak was found. This signal is most probably due to the formation of smaller vesicles after addition of cholate. The results were discussed with respect to bile salt/membrane interactions in the liver cell. It is concluded that vesicular bile salt transport in the cytoplasm is unlikely and that cholate binding is restricted to the outer leaflet of the canalicular part of the plasma membrane.