ISSN:0008-4018    

DOI:10.1139/o80-001

出版商:NRC Research Press    

年代:1980

数据来源: NRC

摘要:

The influence of the chain length inn-alkyl β-D-xylopyranosides and of the para substituents in aryl β-D-xylopyranosides on the kinetic parameters (Ka,V) for hydrolysis of these substrates by a β-D-xylosidase fromPenicillium wortmanni, has been investigated. Binding of the corresponding 1-thio derivatives as competitive inhibitors (Ki) was studied for comparative purposes. For then-alkyl substrates, a slight dependency ofVon the chain length is noted, whereas the aryl β-D-xylopyranosides show nearly constantVvalues. The influence of the aglycon onKaandKivalues is complex; for then-alkyl derivatives the contribution of the hydrophobicity of the aliphatic chain seems predominant, although steric factors cannot be neglected. These results, together with previous observations, can tentatively be interpreted in terms of a double-displacement mechanism.

ISSN:0008-4018    

DOI:10.1139/o80-002

出版商:NRC Research Press    

年代:1980

数据来源: NRC

摘要:

Gladiolic acid (GA, 4-methoxy-5-methyl-o-phthalaldehyde-3-carboxylic acid), an antifungal aromatic ortho dialdehyde produced byPenicillium gladioliwas found to be a potent inhibitor of electron transport and oxidative phosphorylation reactions in sweet potato and mung bean mitochondria. Similar results were also found with the naturally occurring ortho dialdehydes, cyclopaldic acid, quadrilineatin, and flavipin as well as the synthetic dialdehydes, 3-formyl opianic acid ando-phthalaldehyde. Because of their highly reactive ortho-diformyl grouping, GA and structurally related dialdehydes apparently act as multisite inhibitors affecting electron transport and oxidative phosphorylation (at each coupling site). Gladiolic acid has no uncoupling effect like 2,4-dinitrophenol and does not have the same point of interaction in the energy transfer process as oligomycin. Several "partial" reactions of phosphorylation (Mg+2–DNP-stimulated ATPase; ATP–Piexchange) were strongly inhibited by the various dialdehydes. Flavipin and quadrilineatin are potent inhibitors (80% at a concentration of 25 μM) of site III phosphorylation. Gladiolic acid and related ortho dialdehydes inactivate the catalytic activity of native cytochromec in vitro. Lysyl ϵ-NH2rich cytochromecmay be a major site of GA action in the intact mitochondrion. In view of the high chemical reactivity of the ortho-diformyl group, it is suggested that mitochondrial function may be affected by aromatic ortho dialdehydes through a combination of reactions involving cross-linking of amino groups on membrane polypeptides and monofunctional reaction with free amino groups important for enzyme function, including ϵ-NH2groups on cytochromec. Cross-linking in mitochondrial membrane systems might affect function by interfering with molecular motion in the operation of the terminal portion of the electron-transport chain. The primary toxicological mode of action of GA and related dialdehydes appears to be due to inhibition of mitochondrial function.

ISSN:0008-4018    

DOI:10.1139/o80-003

出版商:NRC Research Press    

年代:1980

数据来源: NRC

摘要:

The 105 000gsupernatant from human placental homogenates, prepared in the presence of sodium taurocholate and Cutscum, contained β-glucosidase activity towards estrone glucoside as well as towards 4-methylumbelliferyl glucoside (4-MU-glucoside) and glucocerebroside. After partial purification, the estrone glucosidase was found to be active only after the addition of negatively charged phospholipid, whereas the other β-glucosidases did not exhibit this requirement. The estrone glucosidase was separated from the 4-MU-glucosidase by chromatography on Sephadex G-200 with 0.1% sodium taurocholate in the eluting buffer. The estrone glucosidase was mainly contained in material with a pi of 4.7, while the 4-MU-glucosidase was distributed in fractions with pi values of 4.7 and 6.2 to 6.4. The partially purified estrone glucosidase had a pH optimum of 5.8, as distinct from that of 6.4 found for the 4-MU-glucosidase, and differed markedly from the 4-MU-glucosidase in its response to treatment with heat, sulfhydryl reagents, and detergents. Its sensitivity to changes in pH differed from those reported for glucocerebrosidase.

ISSN:0008-4018    

DOI:10.1139/o80-004

出版商:NRC Research Press    

年代:1980

数据来源: NRC

摘要:

The relationship between plasma membrane and whole cell protein phosphorylation and chick embryo fibroblast proliferation was studied using anin vitrolabeling technique employing [γ-32P]ATP and isolated plasma membranes or whole cell homogenates. Cultures containing proliferating cells in log phase or containing cells stimulated to proliferate by the addition of serum were compared with cultures in which proliferation was inhibited due to cell density. Cell proliferation was found to be associated with increased plasma membrane basal protein kinase activity, but decreased membrane and whole cell cyclic AMP-dependent kinase activity. The phosphorylation of a number of membrane and whole cell polypeptides differed between proliferating and density-inhibited cells, suggesting that these phosphoproteins may be involved in the regulatory events associated with cell proliferation. Of interest, however, was the fact that the phosphoprotein differences found with serum-stimulated and sparse, rapidly dividing cells were generally not the same. These observations suggest that the protein phosphorylation events associated with rapid proliferation of sparse, log-phase cells may differ from those involved in serum-activated proliferation of quiescent cells.

ISSN:0008-4018    

DOI:10.1139/o80-005

出版商:NRC Research Press    

年代:1980

数据来源: NRC

摘要:

Enzyme I of the phosphoenolpyruvate – sugar phosphotransferase system (PTS) has been purified to homogeneity fromEscherichia coli. A merodiploid strain P650 which had an extra copy of the gene for enzyme I resulting in a twofold increase in the amount of activity was used. The enzyme is a dimer of 67 000 ± 5000 molecular weight subunits. At low protein concentration and 4 °C the monomer predominates, while at room temperature the dimer predominates. At higher protein concentrations (2 to 10 mg) this reversible temperature-dependent association-dissociation is not found. Enzyme I has a pH optimum of pH 7.2, aKmfor HPr of 9 ± 3 μM, aKmfor phosphoenolpyruvate of 0.18 ± 0.04 mM, and kinetics that are consistent with a bi bi Ping-Pong mechanism. No allosteric regulation of kinetic activity has been found. The amino acid composition has been determined and the ϵ1%280 nmis 4.4. Evidence suggests that the phosphorylated form of enzyme I is more stable.

ISSN:0008-4018    

DOI:10.1139/o80-006

出版商:NRC Research Press    

年代:1980

数据来源: NRC

摘要:

A variety of pyridinium, quinolinium, and benzoquinolinium cations have been investigated as potential substrates for milk xanthine oxidase at pH 9.9 and (or) pH 10.6. Steady-state kinetic parameters (kc,Kmand (or)kc/Km) have been evaluated for all substrates which are enzymically oxidized. SimpleN-alkyl pyridinium cations are neither substrates nor inhibitors, althoughN-aryl pyridinium cations are slowly oxidized to the 4-pyridinones.N-Methylpyridinium cations bearing 3-CONH2, 3-CONHCH3, 3-COCH3, 3-CO2−or 3-CN substituents are readily oxidized at C-6 and this suggests an important hydrogen-bonding interaction between an enzyme donor and the C-3 carbonyl substituent. A variety ofN-methylquinolinium cations bearing C-6 substituents are enzymically oxidized at C-2. Analogous substituent effects onkc/Kmfor these 6-substituted 1-methylquinolinium cations and the corresponding 1-(substituted phenyl)-pyridinium cations is suggestive of the relative productive binding orientations of these two classes of substrate in the active site.N-Methyl benzoquinolinium and 1,10-phenanthrolinium cations are the best cationic substrates found to date, and suggest a relatively large active-site region for the reducing substrate, and important hydrophobic interactions between enzyme and substrate. The overall enzymic specificity observed for these cationic substrates allows a mapping of the general features of the reducing substrate binding site of this enzyme

ISSN:0008-4018    

DOI:10.1139/o80-007

出版商:NRC Research Press    

年代:1980

数据来源: NRC

摘要:

Membrane-bound galactosyltransferase is solubilized and activated by exogenous lysolecithin or Triton X-100. A study on the effect of different phospholipids on the lysolecithin-solubilized enzyme showed that two charged phospholipids (i.e., phosphatidylinositol and phosphatidyl-serine) inhibited the membrane-bound enzyme in the presence of a wide range of lysolecithin concentration (up to 6 μmol/mg protein). In contrast, these phospholipids produced a biphasic effect on the enzyme solubilized with Triton X-100. In lower concentration of Triton (up to 2 μmol/mg protein), the charged phospholipids somewhat reduced the enzyme activity but a reversal of this effect was observed when Triton concentration was gradually raised (from 2 to 8 μmol/mg protein). This biphasic effect of the phospholipids was also demonstrated on purified membrane-bound galactosyltransferase in presence of low and high concentration of Triton. Electron microscopic evidence suggested that an increased concentration of phosphatidylinositol prevented membrane solubilization by lysolecithin or retained the membrane vesicular organization concurrent with a restraining effect on the enzyme. The results lend support to the hypothesis that the phospholipid microenvironment of the membrane may exert a control on the membrane-bound glycosyltransferases.

ISSN:0008-4018    

DOI:10.1139/o80-008

出版商:NRC Research Press    

年代:1980

数据来源: NRC

摘要:

A ribonuclease activity associated with influenza virus has been purified to homogeneity. This preparation is free of DNAase, phosphodiesterase, and phosphatase activities. The purified enzyme has a pH optima of 7.0 at 37 °C, and moves as a single band on sodium dodecyl sulfate – polyacrylamide gel with an estimated molecular weight of 84 000.

ISSN:0008-4018    

DOI:10.1139/o80-009

出版商:NRC Research Press    

年代:1980

数据来源: NRC

摘要:

Derivative melting profiles of calf thymus mononucleosomes have been examined for changes resulting from variations in solvent pH and ionic strength, histone H1 content, and DNA size. Samples of mononucleosomes were found to rearrange during freeze-drying to form an altered monomer and a series of noncovalent multimers. The derivative melting profiles of these particles differ significantly from those for the untreated monomer and dimer. The noncovalent dimer exhibited a new melting transition at 66 °C involving approximately 18 base pairs of DNA normally associated with the highest melting transition. Mononucleosomes were reconstituted from 6 Mguanidine hydrochloride to give particles with physical properties including melting profile which were virtually indistinguishable from those of the starting material. This result confirms the notion that no structural domains exist in the histone core that can be irreversibly denatured by noncovalent perturbations.

ISSN:0008-4018    

DOI:10.1139/o80-010

出版商:NRC Research Press    

年代:1980

数据来源: NRC