Free-radical chain oxidation of 2-nitropropane initiated and propagated by superoxide.

The superoxide radical O2.-, whether produced by the xanthine/xanthine oxidase reaction or infused as KO2, solubilized by a crown ether in dry dimethyl sulphoxide, initiated a free-radical chain oxidation of anionic 2-nitropropane. Superoxide dismutase, but not catalase, inhibited oxidation of the nitroalkane. Xanthine oxidase suffered a syncatalytic inactivation, during the co-oxidation of 2-nitropropane, which was reversed by dialysis. Cyanide exacerbated this syncatalytic inactivation and rendered it irreversible. The frequently observed oxidations of nitroalkanes by flavoenzymes now need to be re-examined to clarify the extent to which O2.--initiated free-radical chain oxidation contributed to the overall nitroalkane oxidation.     

Purification and characterization of protease So, a cytoplasmic serine protease in Escherichia coli

A new cytoplasmic endoprotease, named protease So, was purified to homogeneity from Escherichia coli by conventional procedures with casein as the substrate. Its molecular weight was 140,000 when determined by gel filtration on Sephadex G-200 and 77,000 when estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thus, it appears to be composed of two identical subunits. Protease So had an isoelectric point of 6.4 and a K(m) of 1.4 muM for casein. In addition to casein, it hydrolyzed globin, glucagon, and denatured bovine serum albumin to acid-soluble peptides but did not degrade insulin, native bovine serum albumin, or the "auto alpha" fragment of beta-galactosidase. A variety of commonly used peptide substrates for endoproteases were not hydrolyzed by protease So. It had a broad pH optimum of 6.5 to 8.0. This enzyme is a serine protease, since it was inhibited by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. Although it was not inhibited by chelating agents, divalent cations (e.g., Mg(2+)) stabilized its activity. Protease So was sensitive to inhibition by N-tosyl-l-phenylalanine chloromethyl ketone but not by N-tosyl-l-lysine chloromethyl ketone. Neither ATP nor 5'-diphosphate-guanosine-3'-diphosphate affected the rate of casein hydrolysis. Protease So was distinct from the other soluble endoproteases in E. coli (including proteases Do, Re, Mi, Fa, La, Ci, and Pi) in its physical and chemical properties and also differed from the membrane-associated proteases, protease IV and V, and from two amino acid esterases, originally named protease I and II. The physiological function of protease So is presently unknown.     

Metabolic heterogeneity of the proximal and distal kidney tubules

Proximal and distal tubule suspensions were prepared from kidneys of Sprague-Dawley rats by an isolation procedure on a Percoll^R gradient. The marker enzymes alkaline phosphatase (brush border) and hexokinase (cytoplasmic) as well as p-aminohippurate transport capacity, gluconeogenic activity and electron microscopy were used to characterize the two kidney tubule suspensions. The results of this study indicate that cytochrome P-450 is localized to the proximal tubular cells and that the O-deethylation of 7- ethoxycoumarin was higher in the proximal than distal fraction. Both proximal and distal tubules showed glucuronidation and deacetylation capacities and a relatively equal distribution of non-protein sulfhydryls. These studies demonstrate metabolic heterogeneity of the nephron, the proximal tubule being the main site of renal xenobiotic metabolism. Understanding of metabolic heterogeneity of proximal and distal kidney tubules should provide important information regarding cell specific mechanisms of nephrotoxicity.     

Nitrate reductase-deficient mutants in barley : immunoelectrophoretic characterization

Nitrate reductase-deficient barley (Hordeum vulgare L.) mutants were assayed for the presence of a functional molybdenum cofactor determined from the activity of the molybdoenzyme, xanthine dehydrogenase, and for nitrate reductase-associated activities. Rocket immunoelectrophoresis was used to detect nitrate reductase cross-reacting material in the mutants. The cross-reacting material levels of the mutants ranged from 8 to 136% of the wild type and were correlated with their nitrate reductase-associated activities, except for nar 1c, which lacked all associated nitrate reductase activities but had 38% of the wild-type cross-reacting material. The cross-reacting material of two nar 1 mutants, as well as nar 2a, Xno 18, Xno 19, and Xno 29, exhibited rocket immunoprecipitates that were similar to the wild-type enzyme indicating structural homology between the mutant and wild-type nitrate reductase proteins. The cross-reacting materials of the seven remaining nar 1 alleles formed rockets only in the presence of purified wild-type nitrate reductase, suggesting structural modifications of the mutant cross-reacting materials. All nar 1 alleles and Xno 29 had xanthine dehydrogenase activity indicating the presence of functional molybdenum cofactors. These results suggest that nar 1 is the structural gene for nitrate reductase. Mutants nar 2a, Xno 18, and Xno 19 lacked xanthine dehydrogenase activity and are considered to be molybdenum cofactor deficient mutants. Cross-reacting material was not detected in uninduced wild-type or mutant extracts, suggesting that nitrate reductase is synthesized de novo in response to nitrate.     

THE NACREOUS WALLS OF SIEVE ELEMENTS IN SEAGRASSES

The nacreous walls of sieve elements occur in seagrasses in all three genera of the family Zosteraceae and the genus Halodule of the family Cymodoceaceae but are absent from another eight seagrass genera belonging to the families Hydrocharitaceae, Cymodoceaceae, and Posidoniaceae. They occur in leaf blades, leaf sheaths, rhizomes, and erect stems but are not present in root tissues. The nacreous wall is uneven along the inner limits reflecting irregular thickness. The wall consists of hemicellulose or pectin and cellulose, but no protein, lignin, or lipid. Ultrastructurally, the wall contains parallel microfibrils or loose fibrils embedded in an amorphous matrix. Open pores occur in sieve plates and branching plasmodesmata are present in enlarged sieve areas. Mitochondria, endoplasmic reticulum, and plastids are also present in these sieve elements.     

Phospholipid-sensitive Ca2+-dependent protein kinase and its substrates in human neutrophils

Phospholipid-sensitive Ca²⁺-dependent protein kinase (PL-Ca-PK) was found to be present at a high level in human neutrophils, with its activity localized in the particulate fraction. In contrast, cyclic AMP-dependent protein kinase (A-PK) and cyclic GMP-dependent protein kinase (G-PK), present at lower levels compared to PL-Ca-PK, were localized in the cytosolic fraction. Phosphorylation of several endogenous proteins (mol. wts. 89,000, 38,000, 34,000, 17,000 and 15,000), also localized in the particulate fraction, was stimulated specifically by a combination of phosphatidylserine and Ca²⁺, whereas no substrate proteins were observed for the calmodulin-sensitive Ca²⁺-dependent protein kinase system under the same incubation conditions. Although no substrate proteins for G-PK were detected, one substrate (mol. wt. 19,000) for A-PK was observed. Phosphorylation of substrates for PL-Ca-PK, but not that for A-PK and for enzymes independent of Ca²⁺ or cyclic AMP, was inhibited by a variety of agents, including trifluoperazine, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide], adriamycin, palmitoylcarnitine, and melittin. The present findings suggest that the $\text{phospholipid}\text{Ca}{}^{\mathrm{2+}}\text{-stimulated}$ protein phosphorylation system may be important in the membrane associated functions of human neutrophils.     

Expression of creatine kinase isoenzyme during oogenesis and embryogenesis in the mouse

Creatine kinase activity was discovered in the growing mouse oocyte and in the preimplantation embryo. Changes in the enzyme activity during the growth and maturation of the egg and during the development of the embryo up to the blastocyst stage were determined. Close similarity of the protein to the brain-type isoenzyme of creatine kinase was established immunochemically. The kinetic parameters of the brain-type isoenzyme (M. R. Iyengar, C. E. Fluellen, and C. W. L. Iyengar, 1982, J. Muscle Cell Motil. 3, 231–246) and the pattern of development-associated changes in activity suggest a possible role for creatine kinase in maintaining the reported high ATP/ADP ratio (L. Ginsberg and N. Hillman, 1975, J. Reprod. Fertil. 43, 83–90), which is essential for the biosynthetic activities of the embryo.     

Apparent Mutagenic Effect of Induction of Lambda Prophage Inserted Between lysA and thyA

Induction of a heat-inducible abnormal lambda prophage inserted between lysA and thyA in Escherichia coli resulted in a number of auxotrophic mutants in the surviving cured-cell populations. These mutants could not be accounted for by deletions arising on formation of lambda hybrid particles carrying regions adjacent to the insertion site. The properties of these mutants, which were almost all spontaneously revertable, have been described and mapped by F′ episome complementation. Tentatively, it was suggested that induction of the lambda lysogen leads to a mutagenic state.