Hydrogen peroxide involvement in the rhodanese inactivation by dithiothreitol.

The conditions required to obtain rhodanese inactivation in the presence of dithiothreitol indicate the involvement of hydrogen peroxide produced by metal-ion catalyzed oxidation of dithiothreitol. Inhibition of dithiothreitol oxidation by a chelating agent, or by removal of hydrogen peroxide by catalase prevents the enzyme inactivation. The inactivated enzyme contains a disulfide bond resulting from the oxidation of the catalytic sulfhydryl group and another sulfhydryl group close to it. This disulfide might be formed via a sulfenic intermediate.     

Isolation and characterization of androgen-dependent non-histone chromosomal protein from dorsolateral prostate of rats

Rat prostate contains a unique androgen-dependent non-histone protein (Matuo et al. (1)). The non-histone protein was isolated in homogeneous form by extraction of nuclei from the dorsolateral prostate with 0.35 M NaCl in the presence of 1 mM PMSF and chromatography on a CM-Sepharose column. The final fraction was greater than 98% pure as judged by electrophoreses in SDS- and acid/urea-gels. The purified protein had a molecular weight of approximately 20,000, and an isoelectric point of approximately 11.5. Its absorption peak was at 276 nm and A(1%, 276nm)=9.3. The protein is characterized by the absence of cysteine, histidine and tryptophan, and by the high content of methionine, tyrosine and phenylalanine.     

Synthesis of a photoaffinity probe for the beta-adrenergic receptor.

The synthesis and characterization of a beta-adrenergic photo-affinity label, N-(-2-hydroxy-3-naphthoxypropyl)-N′ (-2-nitro-5-azidophenyl ethylenediamine, (NAP-propranolol) is described. The inhibition constants (Ki) for the NAP-propranolol inhibition of ³H-dihydroalprenolol binding and the inhibition of (?)-isoproterenol-stimulated adenylate cyclase in turkey erythrocytes are 100 nM and 19 nM respectively.     

Organ specific alcohol metabolism: placental chi-ADH

Human placenta contains a single detectable isozyme of alcohol dehydrogenase that has been isolated and characterized. It migrates toward the anode on starch gel electrophoresis and can be stained with pentanol but not ethanol as substrate. Its kinetic and molecular characteristics are identical with those of the recently discovered chi-ADH (Class III) isozyme from human liver. Placental ADH is present in the cytosol of this organ in small amounts, 6 mg/kg fresh tissue. It oxidizes ethanol very slowly--even at ethanol concentrations that would reflect intoxication when found in serum. Thus, placental alcohol dehydrogenase cannot play a significant role in the ethanol metabolism of pregnant women.     

Studies on mechanism of double hydroxylation. I. Evidence for participation of NADH-cytochrome c reductase in the reaction of benzoate 1,2-dioxygenase (benzoate hydroxylase).

Benzoate 1,2-dioxygenase system which catalyzed double hydroxylation of benzoate was obtained from $\text{Pseudomonas arvilla}$ and was shown to consist of two protein components (component A and B). Component A which was purified and was shown to be homogeneous upon sodium dodecyl sulfate disc gel electrophoresis retained high activity of NADH-cytochrome $\text{c}$ reductase. Both of benzoate 1,2-dioxygenase activity and NADH-cytochrome $\text{c}$ reductase activity were simultaneously induced by benzoate. Dichlorophenolindophenol which could serve as an electron acceptor of the NADH-cytochrome $\text{c}$ reductase inhibited the activity of benzoate 1,2-dioxygenase. These results suggest the possibility that NADH-cytochrome $\text{c}$ reductase activity is required for benzoate 1,2-dioxygenase.     

The mechanism of replication of phi x174 DNA. XVI. Evidence that the phi x174 viral strand is synthesized discontinuously

Chymostatin is a naturally occurring inhibitor of serine proteases that have chymotryptic-like specificity. This tetrapeptide inhibitor is produced by various species of Streptomyces bacteria. Chymostatin reacts with the serine enzyme Streptomyces griseus protease A in the crystalline state to produce an adduct, the structure of which is in agreement with hemiacetal formation between the C-terminal l-phenylalaninal residue of the inhibitor and the O^γ atom of the active Ser195 residue of S. griseus protease A. The 2.8 Å difference electron density map of the complex is also consistent with the novel structural features previously deduced spectroscopically for chymostatin; i.e. an essential (for inhibition) aldehyde function in the C-terminal l-phenylalaninal residue, an unusual arnino acid, 2-(2-iminohexahydro-(4 S)-pyrimidyl)-(S)-glycine as the third residue from the C terminus and an N-terminal amino group blocked by a (1S)-carboxyphenylethyl-carbamoyl group. There is no significant movement of the active site residues of S. griseus protease A upon complexation with chymostatin.     

Tonofilament protein, a keratin from rat epidermis

Tonofilament protein from newborn rat epidermis was found to be antigenically related to mouse keratins. Tonofilament protein, like keratins, could be extracted with 8 M urea in Tris buffer. The yield was enhanced by the presence of dithiothreitol. A higher molecular weight (64,000 dalton) protein antigenically cross-reacting with tonofilament protein but with a slightly different amino acid composition was also extracted.     

Human alpha-1-proteinase inhibitor mechanism of action: evidence for activation by limited proteolysis.

Human plasma alpha-1-proteinase inhibitor (α₁-antitrypsin) has been re-isolated from its complex with porcine trypsin. The re-isolated protein (α₁-PI^*) was found to be non-inhibitory and 8,000 lower in molecular weight than the native inhibitor. Sequence analysis of α₁-PI^* showed that an amino terminal peptide had been lost, apparently the result of cleavage at a Lys-Thr bond. These data indicate that limited proteolysis is the first step in the inhibitory mechanism.     

Purification and properties of a membrane-bound aldehyde dehydrogenase from rat liver microsomes

A membrane-bound aldehyde dehydrogenase was solubilized from rat liver microsomes and purified about 150-fold by chromatography on ω-aminohexyl- and 5′-AMP-Sepharose columns with a recovery of about 40%. The purified enzyme was homogeneous upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its monomeric molecular weight was estimated to be 51,000. In aqueous solution, it existed as large, polymeric aggregates. Its activity towards straight-chain aliphatic aldehydes increased as their carbon chain length was increased at least up to dodecanal, whereas aldehyde dehydrogenase in the cytosolic fraction of rat liver was most active with hexanal as substrate.     

Studies on the oligomeric structure of yeast phosphofructo-kinase by means of cross-linking diimidoesters.

Intracellular cross-linking of yeast phosphofructokinase with a series of diimidoesters of different chain length resulted in the appearance of tetramers as largest cross-linked product of the enzyme subunits. The native enzyme is evidently composed of eight subunits being arranged in two tetramers α₄β₄. In the tetramers the monomers are probably assembled in tetrahedral geometry.     

Characterization of the deoxyribonuclease determined by lambda reverse as exonuclease VIII of Escherichia coli.

Gottesman et al. (1974) detected a new DNAase in Escherichia coli infected with λ reverse, a recombination-proficient substitution mutant of phage λ which is deleted for the λ recombination genes. We have purified this enzyme, using the procedure developed for the purification of exonuclease VIII (Kushner et al., 1974), a DNAase produced by E. coli K-12 strains carrying sbcA^? mutations. The λ reverse exonuclease (Exoλrev) is identical to exonuclease VIII by several criteria. The two enzymes elute at similar salt concentrations from DEAE-cellulose and DNA-cellulose; sediment at the same velocity in glycerol gradients, corresponding to a molecular weight of about 1.4 × 10⁵; migrate at the same R_F in sodium dodecyl sulfate/polyacrylamide gels, indicating a polypeptide molecular weight of 1.4 × 10⁵; exhibit maximum activity at 20 mm-Mg²⁺ and pH 8 to 9; and are much more active on double-stranded DNA than on heat-denatured DNA. Both enzymes are rendered sedimentable by antiserum against Exoλrev. This evidence supports the hypothesis that the non-λ DNA substitution in λ reverse includes recE, the structural gene for exonuclease VIII.     

DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid.

Treatments in vivo of Escherichia coli with oxolinic acid, a potent inhibitor of DNA gyrase and DNA synthesis, lead to DNA cleavage when extracted chromosomes are incubated with sodium dodecyl sulfate. This DNA breakage has properties similar to those obtained in vitro with DNA gyrase reaction mixtures designed to assay production of supertwists: it is oxolinic acid-dependent, sodium dodecyl sulfate-activated, and at saturating drug concentrations produces double-strand DNA cleavage with a concommitant tight association of protein and DNA. In addition, identical treatments performed on a nalA mutant strain exhibit no DNA cleavage. Thus the DNA cleavage sites probably correspond to chromosomal DNA gyrase sites. Sedimentation measurements of the DNA cleavage products indicate that there are approximately 45 DNA breaks per chromosome. This value is similar to the number of domains of supercoiling found in isolated Escherichia coli chromosomes, suggesting one gyrase site per domain. At low oxolinic acid concentrations single-strand cleavages predominate after sodium dodecyl sulfate treatment, and the inhibition of DNA synthesis parallels the number of sites that obtain a single-strand scission. Double-strand breaks arise from the accumulation of single-strand cleavages in accordance with a model where each cleavage site contains two independent drug targets, one on each DNA strand. Since the nicking-closing subunit of gyrase is the target of oxolinic acid in vitro, we suggest that each gyrase site contains two nicking-closing subunits, one on each DNA strand, and that DNA synthesis requires both to be functional.     

Identification of a major extracellular non-collagenous glycoprotein synthesised by human skin fibroblasts in culture

The major protein released into the medium by human skin fibroblasts in culture has been shown to be a fucosylated glycoprotein (designated MFGP). Analysis by gel filtration chromatography and polyacrylamide gel electrophoresis demonstrated that under reducing conditions MFGP has a molecular weight of approx. 250,000, but occurs as a disulphide-linked aggregate in the medium. Three lines of evidence are presented to establish that MFGP is a non-collagenous molecule.     

Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoretic procedure

A sodium dodecyl sulfate-gel electrophoretic procedure which allows the separation of isolated cytochrome c oxidase from different mammalian sources into 13 different polypeptides is described. Application of the silver-staining procedure results in the same protein pattern as obtained by Coomassie blue staining. From the correlation of the gel bands with 12 isolated polypeptides from which the complete amino acid sequence is known, it is concluded that mammalian cytochrome c oxidase consists of 13 different polypeptides which can all be separated by the described procedure.     

A micromethod for complete removal of dodecyl sulfate from proteins by ion-pair extraction

Methods are presented for the complete removal of dodecyl sulfate from proteins. Themethods utilize the extraction of dodecyl sulfate anions as ion pairs with triethylammonium or tributylammonium cations into an organic solvent. The protein is insoluble in the organic solvent and is recovered as a precipitate. The methods are applicable to microgram as well as milligram amounts of protein. In all cases studied, the recovery of protein ranges from 70 to 100%. The recovered protein is suitable for N-terminal Edman degradation, tryptic peptide mapping, and amino acid analysis and can be renatured to regain enzymatic activity and antigenicity.     

Separation of type III collagen from type I collagen and pepsin by differential denaturation and renaturation.

Types I and III collagens were solubilized from fetal human skin by limited digestion with pepsin and precipitated by dialysis against 0.02 M Na₂HPO₄. Heat denaturation of the collagens in 2 M guanidine-HCl, pH 7.5, resulted in the precipitation of the contaminant pepsin which could be removed by centrifugation. Renaturation of the denatured collagens by dialysis against deionized water at 22° for 2 hours selectively precipitated the type III collagen fibrils. Type I collagen remained in solution. The simplicity and high recovery (77%) make this a suitable approach for the rapid estimation of type III collagen in small tissue samples.