Journal of applied biochemistry最新文献

Electron microscopic examination of the effect of linoleic acid hydroperoxide on cultured smooth muscle cells from human umbilical artery revealed that incubation of the cells with 5.0 nmol/ml (in terms of malondialdehyde) of the hydroperoxide for 3 h caused a decrease in the electron density of the mitochondrial matrix and that dilatation of the rough-surfaced endoplasmic reticulum occurred when the cells were incubated with 10 nmol/ml of the hydroperoxide. These concentrations are higher by one order of magnitude than those required for the same effects on cultured endothelial cells from human umbilical vein. A similar difference in susceptibility to injury by linoleic acid hydroperoxide was found between cultured endothelial and smooth muscle cells from fetal calf aorta. The effects of the hydroperoxide on the respiration of these cells paralleled the observed morphological changes.

Micrococcus lysodeikticus (M. luteus) ATPase digested in a controlled manner with trypsin behaves like the native protein when chromatographed on alkyl agarose supports. The enzyme immobilized on the supports through noncovalent interaction is able to hydrolyze ATP with a specific activity similar to that of native membrane-bound ATPase. However, the response of M. lysodeikticus ATPase to the interaction with the hydrophobic columns can be modified by changing the protein-ligand ratio. These results support the notion that the catalytic site of M. lysodeikticus ATPase is not involved in the interaction with alkyl agarose, but rather that binding of the ATPase to the hydrophobic columns takes place through polypeptide or protein domains other than those which mediate binding to the native membranes, since they are very easily modified by trypsin. It is proposed that the alpha subunit plays a role in the interaction of the bacterial ATPase with hydrophobic ligands. These results are discussed in relation to the topography of the enzyme as established previously.

Enzymes can lose activity through covalent and noncovalent structure alterations. In the former, protease attack and modification by small active molecules such as oxygen are important. Conformational stability can be measured by Tm, the midpoint temperature of the thermal denaturation curve, and turnover in vivo of a number of enzymes correlates with Tm. Measurement of Tm and delta Cp leads to evaluation of delta H, T delta S, and delta G for the unfolding process. The importance of d(delta G)/dT is emphasized since it can be used to evaluate the temperature of maximum stability. There is no simple relationship between amino acid sequence and delta Gmax, nor can the effect of mutation be accurately forecast. Reversibility of folding is an important factor in stability. Tm correlates with [G]1/2, the midpoint guanidine unfolding concentration, and is the most useful predictive quantity for enzyme stability.

Tryptophanase purified from Escherichia coli B/1t7-A is inactivated by mild ozonization following pseudo-first-order kinetics. Previous data from the authors suggest that one out of two tryptophan residues (Trp's) in the enzyme subunit is preferentially oxidized concomitant with the ozone inactivation and has a direct interaction with the coenzyme, pyridoxal phosphate [PLP (M. Tokushige, Y. Fukuda, and Y. Watanabe, 1979, Biochem. Biophys. Res. Commun. 86, 976-981)]. To determine which Trp is more susceptible to ozonization and interacts with PLP, the native and ozonized enzyme proteins were cleaved by trypsin and the two Trp-containing peptides were analyzed by reverse-phase HPLC equipped with a dual-monitoring system consisting of an uv and a fluorescence monitor connected in tandem for selective detection of Trp-containing peptides. This device facilitated rapid detection and quantitation of the Trp-containing peptides which decreased upon ozonization. The results showed that Trp preferentially oxidized upon ozonization and involved in the interaction with PLP was the one in peptide T-15 rather than that in T-23, which Kagamiyama et al. originally designated (H. Kagamiyama, H. Wada, H. Matsubara, and E. E. Snell, 1972, J. Biol. Chem. 247, 1576-1585).

During the refolding and oxidation of reductively denatured ribonuclease A in solution, there is a marked lag in appearance of enzymatic activity as compared to the oxidation of sulfhydryl groups, whether such oxidation is spontaneous or is catalyzed by sulfhydryl oxidase. However, if ribonuclease is covalently attached to a derivatized glass surface, a lag period is not observed during the reformation of native structure from the completely reduced, denatured state. These results suggest that, in solution, intermolecular interactions alter the pathway of polypeptide chain folding and disulfide bond formation, leading to nonnative disulfides which do not rapidly interchange to form native pairings. The isolation of refolding polypeptide chains by covalent immobilization prevents such interactions. Presumably, such intermolecular interactions would be similarly prevented by "isolation" of nascent polypeptide chains during protein synthesis on ribosomes.

The activity of alanine aminotransferase (ALT) phenotypes was determined in 148 hemolysates of the Serbian population. The highest activity was obtained for phenotype ALT 1 (0.614 U/g Hb), intermediate for ALT 2-1 (0.475 U/g Hb), and the lowest for ALT 2 (0.395 U/g Hb). To explain the differences in catalytic activity between the ALT phenotypes, some kinetic characteristics were investigated. No difference in heat stability and calculated activation energies for ALT phenotypes could be detected. Addition of pyridoxal 5'-phosphate to the reaction system did not increase the catalytic activity. For the catalytic activity of all three phenotypes, a broad pH optimum in the range 7.1 to 7.6 was found. The Tris/HCl buffer concentration of 140 mmol/liter was optimal. The Michaelis-Menten constants for L-alanine as substrate were 2.462 mmol/liter for ALT 1, 1.965 mmol/liter for ALT 2-1, and 2.698 mmol/liter for ALT 2. For another substrate, 2-oxoglutarate, Km values were 0.299, 0.208, and 0.202 mmol/liter, respectively.

Two cellobiohydrolases and two endoglucanases were purified from a culture filtrate of the fungus Trichoderma reesei QM 9414 by simple and straightforward purification techniques. Molecular weights, isoelectric points, amino acid compositions, and carbohydrate contents are reported.

The effect of bile acids on the glycoprotein constituent of gastric mucus was investigated. Ghosh-Lai rat stomachs were instilled in vivo for 1 h with buffered saline (control) followed by various bile acids in saline at pH 2.0-7.0. Following quantitative recovery, the instillates were used for the isolation of mucus glycoprotein. The results of analyses revealed that while taurocholic acid exerted essentially identical depletory effects throughout the entire pH range tested, the cholic acid had little depletory effect on the mucus below pH 5.0, and glycocholic acid below pH 4.0. The extent of mucus glycoprotein depletion by bile acids under optimal pH conditions was clearly dependent upon the bile acid concentration as well as its type. The maximum depletory effect with the lowest concentration of bile acid was achieved with taurocholate followed by glycocholate and cholate. Comparison of the depletory effects of mono-, di-, and trihydroxy conjugated and unconjugated bile acids revealed that, within each group, the highest degree of mucus glycoprotein depletion occurred with taurine conjugates. The number of hydroxyl groups on the bile acid molecule appears to exert a less evident effect on the depletory capacity. The results suggest that the in vivo depletion of gastric mucus of its glycoprotein constituent by bile acids depends on the pH of luminal exposure and on the bile acid composition.

125Iodine-labeled human immunoglobulin G-encapsulated dipalmitoyl phosphatidylcholine liposomes were prepared with or without asialoganglioside. Distribution of radioactivity following the oral administration of these liposomes in rats was checked in liver and in blood of the portal vein and heart. Gel filtration of plasma from the portal vein showed the presence of intact liposomes and protein. In contrast, neither liposomes nor protein were detected in cardiac blood but only lower molecular weight materials. The presence of liposomes in portal blood was further suggested from experiments using 6-carboxyfluorescein-entrapped liposomes. The level of liposomes in portal venous blood plasma was found to be lower in asialoganglioside liposomes. But this level could be increased substantially, almost to that in liposomes without asialoganglioside, by injection (iv) of asialofetuin.

gamma-Glutamyl transpeptidase has been purified by chromatographic methods from endometrium of rat deciduoma. The membrane-bound enzyme, digested with papain, eluted as a single fraction during chromatography and the final preparation had a specific activity of 104.5 U/mg protein. The molecular weight of the native enzyme was approximately 70,000 and the protein could be resolved into a heavy and a light fraction on sodium dodecyl sulfate-acrylamide gel electrophoresis with molecular weights of 45,000 and 23,000, respectively. The enzyme had a pH optimum of 8.2 and Km's for donor (p-nitroanilide) and acceptor (glycylglycine) were 1 and 7.6 mM, respectively. The enzyme was inhibited by L-serine in the presence of borate with a Ki of 22 microM. The decidual enzyme was not heat stable and its electrophoretic mobility was decreased after neuraminidase treatment.