Effects of carbon–dioxide-bicarbonate mixtures on oxidative phosphorylation by cauliflower mitochondria

1. Carbon dioxide-bicarbonate mixtures markedly inhibited oxidation and phosphorylation rates of mitochondria prepared from cauliflower. Inhibition occurred with succinate, malate, citrate, isocitrate and NADH as substrates. 2. Indophenol-reductase systems with malate, succinate, isocitrate and NADH as substrates were inhibited by 5% and 15% carbon dioxide. Cytochrome c oxidase was not inhibited by 15% carbon dioxide.     

Detection of IgG and IgM antibodies with ELISA technique in human trichomoniasis

The direct wet mount examination of vaginal secretion, widely applied for the diagnosis of Trichomonas vaginalis infection in woman patients, is rapid and economical, however, the sensitivity of this technique is not so high. In this study enzyme-linked immunosorbent assay (ELISA) was employed for the detection of serum anti-T. vaginalis IgG and IgM antibodies from 30 vaginal trichomoniasis patients and 30 non-infected healthy persons. The results were as follows: 1. Serum ELISA-IgG value was 0.37 +/- 0.134 (Mean +/- S.D.) in vaginal trichomoniasis patients and 0.21 +/- 0.054 in healthy controls (p less than 0.005), and the sensitivity and specificity of ELISA for serum IgG antibody were 70.0% and 96.7%, respectively. 2. Serum ELISA-IgM value was 0.33 +/- 0.177 (Mean +/- S.D.) in vaginal trichomoniasis patients and 0.11 +/- 0.051 in healthy controls (p less than 0.005), and the sensitivity and specificity of ELISA for serum IgM antibody were 70.0% and 96.7%, respectively. 3. The ELISA-IgG values showed a significant correlation with ELISA-IgM values (r = 0.77, p less than 0.005). With above results, it is assumed that ELISA is a reliable method for the diagnosis of T. vaginalis infection and simultaneous measurement of serum IgG and IgM with this technique is recommended.     

Uptake and acylation of 2-acyl-lysophospholipids by Escherichia coli.

The efficiency of extracellular 2-acyl-lysophospholipid incorporation into Escherichia coli membranes and the acyl donor utilized to acylate the 2-acyl-lysophospholipid was determined. Exogenous 2-acyl-lysophospholipids were acylated via the acyl-acyl carrier protein synthetase/2-acylglycerophosphoethanolamine acyltransferase pathway. The maximum extent of 2-acyl-lysophospholipid incorporation into the membrane was approximately 2.5% of the normal phospholipid biosynthetic rate.     

Use of lacZ fusions to measure in vivo expression of the first three genes of the Escherichia coli unc operon.

We have constructed in-frame lacZ protein fusions to the first three genes of the Escherichia coli unc operon, which codes for the subunits of the proton-translocating ATPase. We have used these constructions to measure the relative in vivo expression of these genes. The second and third genes, uncB and uncE, which code for the a and c subunits of the F0 sector, were expressed at relative levels of approximately 1:10, although the measured expression of uncB depended upon how much of the gene was fused to lacZ. These rates compared favorably with the relative numbers of a and c subunits (a1:c10) in the purified F1F0 complex. The in vivo expression of uncI, the first gene of the operon, was very low, at best 10 to 20 times less than the expression of uncB.     

The primary structure of a protein carboxyl methyltransferase from bovine brain that selectively methylates L-isoaspartyl sites

Protein L-isoaspartyl methyltransferase (PIMT) transfers the methyl group of S-adenosyl-L-methionine to free alpha-carboxyl groups of atypical L-isoaspartyl residues in proteins. The complete primary structure of the type I isoform of bovine brain PIMT was determined by sequence analysis of peptides generated by endoprotease Lys-C, trypsin, cyanogen bromide, and endoprotease Asp-N digests. The correct composition of every peptide was verified by fast atom bombardment mass spectrometry. The efficiency of sequencing by tandem mass spectrometry was examined for several peptides by comparing its speed and accuracy with automated Edman degradation. Tandem mass spectrometry was used to determine the structure of the NH2-terminal blocked peptide derived from a hydroxylamine cleavage. PIMT is 226 residues with Mr = 24,500 and contains acetyl alanine as the amino-terminal residue. The partial sequence (141 residues from 8 tryptic peptides) of a homologous human red cell PIMT (Gilbert, J. M., Fowler, A., Bleibaum, J., and Clarke, S. (1988) Biochemistry 27, 5227-5233) shows a 97% identity with the corresponding peptides of the bovine brain enzyme. The complete brain enzyme sequence reported here bears no significant homology to any other known class of methyltransferase including those which methylate the side chain gamma-carboxyl group of receptor proteins involved in bacterial chemotaxis.     

Arachidonic acid mobilization and phosphoinositide turnover by the terminal complement complex, C5b-9, in rat oligodendrocyte x C6 glioma cell hybrids

Previously, we have shown that rat oligodendrocytes release phospholipid and generate arachidonic acid (AA) and leukotriene B4 in response to sublytic C5b-9 formation. In the present study, we investigated the biochemical pathways by which C5b-9 generates AA from clone ROC-1, a fusion product of rat oligodendrocytes and C6 glioma. Cells were incubated for 24 h in the presence of [3H]AA or [3H]myoinositol. They were then sensitized with antibody against hybrid cell stroma and treated for 1 h with C9-depleted human serum (C9D-HS) or C9D-HS reconstituted with C9. Alternatively, cells were treated with C8,C9D-HS or C8,C9D-HS reconstituted with C8 or C8 plus C9 for 1 h. Qualitative and quantitative analysis of the released [3H]AA and [3H]myoinositol radiolabeled products were performed by thin layer chromatography/autoradiography and anion exchange chromatography, respectively. The major [3H]AA radiolabeled products after C5b-9 stimulation comigrated with intact phospholipid and AA standards, and the major [3H]myoinositol radiolabeled product was inositol-1-phosphate. Treatment of cells with phospholipase A2 inhibitors, mepacrine and bromophenacyl bromide, abolished AA release by C5b-9. In the absence of extracellular Ca2+, C5b-9 also failed to induce the release of AA. Interestingly, 1-(5-isoquinolinsulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinases, inhibited AA release by C5b-9, whereas AA release stimulated by the calcium ionophore A23187 was not blocked by H-7. The results suggest that AA generation by C5b-9 from the ROC-1 clone involves activation of Ca2+-dependent phospholipase A2 which is regulated by protein kinase-dependent mechanisms.     

Light Activation of Pyruvate, Orthophosphate Dikinase in Maize Mesophyll Chloroplasts: A Role for Adenylate Energy Charge

Pyruvate, orthophosphate dikinase (EC 2.7.9.1 [EC] ) was activatedin the light and inactivated following a dark treatment in intactmaize mesophyll chloroplasts. Addition of catalase (100–250units/ml) to the assay medium was necessary to obtain good activationand to keep the enzyme in an active state during illumination.Arsenate and carbonyl cyanide m-chlorophenyl-hydrazone, uncouplersof photophosphorylation, inhibited the activation. Pyruvate,which has been proposed to have a critical role in supportingthe light activation of pyruvate, orthophosphate dikinase, actuallyinhibited the activation. The pyruvate level in the chloroplastsuspension decreased when the enzyme was light-activated. Measurementsof adenylates and pyruvate in the chloroplasts indicated thatthe energy state of the chloroplasts was more important forthe light activation than was the level of pyruvate. ¹Present address: Department of Biochemistry, Faculty of Science,Saitama University, 255, Shimo-Okubo, Urawa, 338 Japan ²Present address: National Institute of Agrobiological Resources,Yatabe, Tsukuba, Ibaraki, 305 Japan (Received May 2, 1989; Accepted October 2, 1989)     

Phosphatidylethanolamine synthesis by castor bean endosperm : membrane bilayer distribution of phosphatidylethanolamine synthesized by the ethanolaminephosphotransferase and ethanolamine exchange reactions

A base exchange reaction for synthesis of phosphatidylethanolamine by the endoplasmic reticulum of castor bean (Ricinus comminus L. var Hale) endosperm has been examined. The calculated Michaelis-Menten constant of the enzyme for ethanolamine was 5 micromolar and the optimal pH was 7.8 in the presence of 2 millimolar CaCl₂. l-Serine, N-methylethanolamine and N,N-dimethylethanolamine all reduced ethanolamine incorporation, while d-serine and myo-inositol had little effect. These inhibitions of ethanolamine incorporation were found to be noncompetitive and ethanolamine also noncompetitively inhibited l-serine incorporation by exchange. The activity of the ethanolamine base exchange enzyme was affected by several detergents, with the best activity being obtained with the zwitterionic defjtergent 3-3-cholamidopropyl) dimethylammonio-2-hydroxyl-1-propanesulfonate.