Inactivation of Hydrogenase in Cell-free Extracts and Whole Cells of Chlamydomonas reinhardi by Oxygen

O₂ irreversibly inactivates hydrogenase from Chlamydomonas reinhardi. The mechanism for the inactivation involves the reaction of one molecule of hydrogenase with one molecule of O₂ (or two oxygen atoms) in the transition complex of the rate-limiting step. The second order rate constant for this reaction is 190 atmospheres⁻¹ minute⁻¹ (1.4 × 10⁵ molar⁻¹ minute⁻¹). At levels above 0.01 atmosphere O₂, the increased numbers of O₂ molecules may compete for the site of inactivation hindering the proper orientation for inactivation of any one O₂ molecule and resulting in lowered rates of inactivation.     

Oögenesis in a marine teleost,Blennius pholis L.

Summary Oögenesis in the oviparous marine teleost, Blennius pholis L., is examined. Eleven developmental stages are identified by ultrastructural observations when changes in the distributions of the organelles and inclusions are described. An exogenous source for the protein yolk precursors is indicated, but less clear is the endogenous contribution. Changes in the follicle epithelium are described together with the formation of the zona which is considered to be follicular in origin. Two types of follicle cell are distinguished and these probably function differently in the process of zona formation. The zona becomes divided into the externa and interna, the latter probably resulting from the chemical ordering by disulphide bonding of the proteinaceous material of the former.We are indebted to Professor E.W. Knight-Jones in whose department the work was carried out, and to the Natural Environment Research Council for support for one of us (S.E.S.).     

The appearance of oestrone sulphate in the peripheral plasma of the pig early in pregnancy

Detectable concentrations of oestrone sulphate were present in 50% of the plasma samples collected from pregnant animals by Day 17. No oestrone sulphate was detected in plasma from cyclic nonpregnant pigs.     

A rinsing and incubation chamber used for immunocytochemistry of vibratome sections

Summary This paper describes the construction and use of a washing/incubation chamber that may be used for peroxidase-antiperoxidase immunocytochemistry of vibratome sections. The advantages of the chamber include: all sections are processed simultaneously with exception of exposure to the primary antiserum; the exposure of the sections to DAB substrate can be precisely controlled; and also the sections are not directly manipulated during the procedure resulting in a much greater integrity of the tissue. Use of the chamber reduces the amount of labor normally required to wash the sections thorughout the procedure. Application of this system to the development and standardization of quantitative immunocytochemical techniques is also discussed.     

Glucose Metabolism in Sediments of a Eutrophic Lake: Tracer Analysis of Uptake and Product Formation

The uptake of glucose and the formation of end products from glucose catabolism have been measured for sediments of eutrophic Wintergreen Lake with a combination of tritiated and ¹⁴C-labeled tracers. Time course analyses of the loss of [³H]glucose from sediments were used to establish rate constants for glucose uptake at natural substrate concentrations. Turnover times from these analyses were about 1 min for littoral and profundal sediments. No seasonal or site differences were noted in turnover times. Time course analyses of [U-¹⁴C]glucose uptake and ¹⁴C-labeled end product formation indicated that glucose mass flow could not be calculated from end product formation since the specific activity of added [¹⁴C]glucose was significantly diluted by pools of intracellular glucose and glucose metabolites. Mass flow could only be accurately estimated by use of rates of uptake from tracer studies. Intermediate fermentation end products included acetate (71%), propionate (15%), lactate (9%), and only minor amounts of butyrates or valerates. Addition of H₂ to sediments resulted in greater production of lactate (28%) and decreased formation of acetate (50%), but did not affect glucose turnover. Depth profiles of glucose uptake indicated that rates of uptake decreased with depth over the 0- to 18-cm interval and that glucose uptake accounted for 30 to 40% of methanogenesis in profundal sediments.     

Protease priming of neutrophil superoxide production. Effects on membrane lipid order and lateral mobility.

Phagocyte superoxide (O2-) response is primed by a variety of physiologic compounds including the neutrophil secretory proteases cathepsin G and elastase. To study whether protease priming of neutrophil O2- response is related to changes in membrane physical state, we examined enzyme effects on the order and lateral mobility of lipid probes in intact neutrophil membranes. Exposure to cathepsin G (5 micrograms/ml) or elastase (10 micrograms/ml) caused a significant decrease in fluorescence anisotropy of the probe trimethylammonium diphenylhexatriene in neutrophil plasma membranes (0.279 to 0.256 for cathepsin G, 0.274 to 0.256 for elastase, p less than 0.02 for both), indicating a decrease in phospholipid chain order in the surface membrane bilayer. Cathepsin G and elastase also caused significant increases in membrane lipid lateral mobility as measured by excimer formation of the fluorescent probe 1-pyrenedecanoic acid (for cathepsin G, a 107% increase, and for elastase, a 44% increase in excimer/monomer fluorescence ratio, p less than 0.001). Enzyme effects on membrane structure were dependent on intact proteolytic activity, and were cell specific; the proteases had no effect on lipid order or lateral mobility in liposomes. In corollary studies, the possible association between the physical state of the polymorphonuclear leukocyte membrane and O2- generation was analyzed with the membrane modifying compounds, linoleic acid, ethanol, and cholesterol. Cell exposure to linoleic acid (1 microM) caused a significant decrease in lipid order and an increase in lipid lateral mobility along with increased O2- production to N-formyl-Met-Leu-Phe (fMLP) (191%) and phorbol myristate acetate (PMA) (39%), p less than 0.02 for each. 3 mM ethanol also augmented O2- response to fMLP (31%) and PMA (48%) and caused a significant decrease in lipid order, but did not affect lipid lateral mobility. Treatment with cholesteryl hemisuccinate (100 micrograms/ml) resulted in increased lipid order and decreased lipid lateral mobility, as well as decreased neutrophil superoxide response to fMLP (-61%, p less than 0.001) and PMA (-50%, p less than 0.02). We then examined whether modulation of membrane physical state may explain the mechanism of action of a known priming agent by studying the effects of low concentrations of a diacylglycerol. Cells treated with 10 microM 1-oleoyl-2-acetyl-sn-glycerol had a greater than 8-fold increase in superoxide response to fMLP (p less than 0.001) while demonstrating a significant decrease in lipid order (0.289 to 0.281, p less than 0.01) and a 50% increase in lipid lateral mobility (p less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated membrane proteinase

Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.     

Intragenic Suppressors of Folding Defects in the P22 Tailspike Protein

Within the amino acid sequences of polypeptide chains little is known of the distribution of sites and sequences critical for directing chain folding and assembly. Temperature-sensitive folding (tsf) mutations identifying such sites have been previously isolated and characterized in gene 9 of phage P22 encoding the tailspike endorhamnosidase. We report here the isolation of a set of second-site conformational suppressors which alleviate the defect in such folding mutants. The suppressors were selected for their ability to correct the defects of missense tailspike polypeptide chains, generated by growth of gene 9 amber mutants on Salmonella host strains inserting either tyrosine, serine, glutamine or leucine at the nonsense codons. Second-site suppressors were recovered for 13 of 22 starting sites. The suppressors of defects at six sites mapped within gene 9. (Suppressors for seven other sites were extragenic and distant from gene 9.) The missense polypeptide chains generated from all six suppressible sites displayed ts phenotypes. Temperature-sensitive alleles were isolated at these amber sites by pseudoreversion. The intragenic suppressors restored growth at the restrictive temperature of these presumptive tsf alleles. Characterization of protein maturation in cells infected with mutant phages carrying the intragenic suppressors indicates that the suppression is acting at the level of polypeptide chain folding and assembly.