Monoclonal antibodies to cytochrome c from Paracoccus denitrificans: effects on electron transport reactions

The effect of a monoclonal antibody to a soluble cytochrome c from Paracoccus denitrificans was tested on the membrane-bound electron-transport system of this bacterium. This antibody (F3-10.2) and one previously described (F3-29.4) (Kuo, L.M., Davies, H.C. and Smith, L. (1984) Biochim. Biophys. Acta 766, 472-482) were deduced to bind to the cytochrome c in the area including amino acid residue number 23 on a loop on the side of the heme crevice. In contrast to the observations with the previously tested antibody, the present data show the second antibody to block completely the reaction of the cytochrome c with cytochrome c oxidase but not that with cytochrome c reductase. Neither antibody has an appreciable inhibitory effect on the NADH oxidase of the isolated detergent-treated membranes. The two antibodies bind in different ways, giving insight into the interaction of a soluble protein with membrane-bound enzymes. The data indicate that the reaction sites on the cytochrome c for the oxidase and reductase moieties of P. denitrificans are different. They also argue against the need for a dissociable cytochrome c comparable to that which functions on the mitochondrial inner membrane.     

Plants have isoforms for acyl carrier protein that are expressed differently in different tissues

Two closely related isoforms of acyl carrier protein (I and II) have been purified from spinach leaves. Differences in the N-terminal amino acid sequence and amino acid composition indicate that these proteins are coded by different genes. The two spinach leaf isoforms have been resolved and characterized by ion-exchange high-performance liquid chromatography, by thin layer isoelectric focusing, and by differences in mobility upon polyacrylamide gel electrophoresis. Both isoforms are effectively bound by antibodies raised to acyl carrier protein I. However, in competition experiments isoform II is only about 40% effective in blocking isoform I binding to antibody. Therefore, the isoforms are immunologically related but hold only some antigenic sites in common. Immunoblot analysis ("Western blotting") of crude spinach leaf tissue extracts probed with antibody to acyl carrier protein I reveals both isoforms. In addition, both forms of acyl carrier protein are present in dark-grown leaf tissue and in isolated chloroplasts. However, in spinach seeds and roots only acyl carrier protein II can be detected. Similar results are observed with extracts of castor oil plant leaf and seed. Therefore, the expression of the two acyl carrier protein isoforms is tissue specific.     

Substrate specificity of phospholipid/Ca2+-dependent protein kinase as probed with synthetic peptide fragments of the bovine myelin basic protein

The substrate specificity of phospholipid/Ca2+-dependent protein kinase (protein kinase C) was studied using synthetic peptides, in particular those corresponding to the amino acid sequence around serine 115 in bovine myelin basic protein (MBP). It was found that MBP (104-118) and MBP (104-123) were substrates for the enzyme, with apparent Km values of 14 and 10 microM, respectively. Neither MBP (111-118) nor MBP (111-123) were phosphorylated, indicating that an additional segment of sequence extending toward the N terminus, but not toward the C terminus, was essential for the substrate activity of the peptides. Of the alanine-substituted analogs examined, [Ala 105] MBP (104-118) was comparable to the parent peptide, whereas [Ala 107] MBP (104-118) and [Ala 113] MBP-(104-118) were much poorer substrates. These findings indicated that lysine 105 was not essential, but both arginine 107 and arginine 113 were important specificity determinants. Initial studies revealed that [Ala 113] MBP (104-118) inhibited phosphorylation by the enzyme of the parent peptide and, to a lesser extent, the intact MBP(1-170). Serine 115 was the only site phosphorylated in the analog peptides [Ala 105] MBP (104-118) and [Ala 107]MBP (104-118). In the parent peptide, serine 115 was the initial site of phosphorylation but after prolonged phosphorylation other sites became phosphorylated (serine 110 and/or serine 112), further supporting the concept that arginine residues act as essential substrate specificity determinants for phospholipid/Ca2+-dependent protein kinase.     

The fermentation process for l-phenylalanine production using an auxotrophic regulatory mutant of Escherichia coli

Summary A process for l-phenylalanine production was studied using a tyrosine auxotrophic regulatory mutant of Escherichia coli, resistant to both -2-thienyl-dl-alanine and p-fluoro-dl-phenylalanine. Fermentations were carried out in a 30-1 fermentor with intermittent feeding of glucose plus phosphate. The mutant accumulated l-phenylalanine in the fermentation broth up to 15 g/l at pH 7.0 and 33°C. Column chromatography on a strong cation exchanger was employed as the most effective step in the purification of l-phenyl-alanine from the broth. This step brought about 4-fold concentration of the product with 96% recovery.     

Correlation of 3,4-dihydroxybutyl 1-phosphonate resistance with a defect in cardiolipin synthesis in Escherichia coli.

Escherichia coli treated for 1 h with 100 microM rac-3,4-dihydroxybutyl 1-phosphonate (DBP), a glycerol-3-phosphate analog, die when sorted at 5 degrees C, whereas the viability of untreated cells is relatively unaffected. This observation formed the basis of a selection procedure that was used to isolate mutants that are partially resistant to DBP. One such mutant, strain 6204, is constitutive for DBP transport, exhibits a particularly high degree of cold resistance, has the same doubling time as the parent, and is similar to the parent strain in terms of incorporation of DBP into the lipid fraction. Glycerol-3-phosphate and phosphatidylglycerol phosphate synthetases obtained from strain 6204 and its parent were identical in terms of DBP recognition. The parent strain is killed when incubated in the presence of a combination of 70 microM rac-DBP and 0.25% deoxycholate, whereas strain 6204 continues to grow, albeit more slowly, in the presence of this combination. Strain 6204 can be distinguished from the parent strain on agar plates (low phosphate minimal medium with glucuronate as the sole carbon source) containing 15 microM rac-DBP. The insertion of Tn10 near the 6204 mutation has facilitated genetic manipulations. All phenotypic effects attributed to strain 6204 appear to be due to a single mutation. Genetic analysis indicates that Tn10, inserted near the gene responsible for DBP resistance, maps in the vicinity of 27 min. Three-factor crosses reveal a gene order of hemA-Dbpr-Tn10(zch)-trp. The only gene for phosphoglyceride metabolism known to map in this region is the gene associated with cardiolipin synthetase, cls. Genetic results suggest that the mutation responsible for DBP resistance maps in or very near cls. Analysis of the lipids isolated from untreated strain 6204 (and from each of the transductants prepared by P1 vir-mediated transfer of DBP resistance of wild-type strains) reveals that cardiolipin synthesis is defective. These results strongly suggest that the mutation responsible for DBP resistance has its primary effect on cardiolipin synthesis. To further test this hypothesis, strains with an authentic cls mutation were constructed and examined for resistance to DBP. These strains had growth properties that were identical with those of strain 6204. Wild-type strains and mutants defective in cardiolipin synthesis were treated with DBP and 20 mM magnesium or calcium chloride. Simultaneous treatment of either cell type with DBP and divalent cation not only failed to stimulate growth but, quite the contrary, had a marked synergistic growth inhibitory effect.     

Kinetics of the interaction of hemin liposomes with heme binding proteins

As a model for the transport of hemin across biological membranes, sonicated phosphatidylcholine liposomes with incorporated hemin were characterized. The interaction of the hemin liposomes with the heme binding proteins albumin, apomyoglobin, and hemopexin was examined as a function of liposome charge and cholesterol content. In all cases, there was an almost complete transfer of hemin from liposome to protein; a rapid phase and a slow phase were observed for the transfer. For negatively charged liposomes (with 11% dicetyl phosphate), the rapid and slow phases showed observed rates of transfer of ca. 2 and 0.01 s-1, respectively, for all three proteins. The presence of cholesterol in the liposomes decreased the observed rates by a factor of 2, and positively charged liposomes (with 11% stearylamine) showed about one-fifth the observed rates of negatively charged liposomes. The observed rates were independent of protein concentration, indicating that the rate-determining step is hemin efflux from the lipid bilayer. The hemin interaction with the phospholipid bilayer is suggested to be primarily hydrophobic with some electrostatic character. The two phases are suggested to arise from two different populations of hemin within the liposomes and are interpreted as arising from two different orientations of hemin within the bilayer.     

Characterization of the subunit structure of gonadotropin receptor in luteinized rat ovary

Gonadotropin receptors with specificity, high affinity and low capacity for luteinizing hormone and human chorionic gonadotropin (hCG) have been identified in rat luteal cells. To investigate the nature of the receptor, we have employed disuccinimidyl suberate, a cross-linker noncleavable by reducing agents, and dithiobis(succinimidyl propionate), a cleavable cross-linker, to covalently cross-link the 125I-hCG . receptor complex. The molecular weight of 125I-hCG-linked receptor complex and the receptor subunit structure were determined by electrophoresis in either 10 or 4.5% acrylamide in the presence of 0.1% sodium dodecyl sulfate with or without reducing agents. Autoradiographic analysis of the 125I-hCG-linked receptor separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing condition revealed a single labeled band corresponding to Mr = 305,000 +/- 15,000. However, electrophoresis performed in the presence of 50 mM dithiothreitol and 2% beta-mercaptoethanol resulted in the appearance of four labeled bands corresponding to Mr = 105,000 +/- 4,000, 96,000 +/- 5,000, 74,000 +/- 4,000, and 62,000 +/- 4,000 concomitant with the loss of the labeled band in the Mr = 305,000 region. Further experiments demonstrated that these four labeled bands were derived from the same molecular species. In addition, the 125I-hCG-linked receptor in the absence of reducing agent was not dissociated into subunits even by treatment with strong denaturing agent (8 M urea). The appearance of the cross-linked 125I-hCG . receptor was effectively inhibited by the unlabeled beta-subunit of hCG, intact hCG, and luteinizing hormone and partially inhibited by the alpha-subunit of hCG but not by choleratoxin, gonadotropin-releasing hormone, insulin or bovine serum albumin. These data suggest that 1) the hCG/luteinizing hormone receptor is an oligomeric complex linked by disulfide bonds and 2) that under reducing conditions, the oligomeric receptor dissociates into four nonidentical subunits.     

Phospholipid-Sensitive Ca2+-Dependent Protein Kinase Preferentially Phosphorylates Serine-115 of Bovine Myelin Basic Protein

Phospholipid-sensitive Ca2+ -dependent protein kinase (PL-Ca-PK) and cyclic AMP-dependent protein kinase (A-PK) both preferentially phosphorylated serine residues of bovine myelin basic protein (MBP). Tryptic peptide maps of MBP phosphorylated by PL-Ca-PK or A-PK, however, revealed different phosphopeptides, suggesting a difference in the intramolecular substrate specificity for the two enzymes. Serine-115 of MBP, in the sequence (-Arg-Phe-Ser(115)-Trp-), was found to be a preferred and probably major phosphorylation site for PL-Ca-PK. Because serine-115 of bovine MBP corresponds to serine-113 of rabbit MBP, an in vivo phosphorylation site reported by Martenson et al. (1983), and PL-Ca-PK is present at a very high level in brain and myelin, it is suggested that the enzyme may be responsible for the in vivo phosphorylation of this and other sites in MBP.     

Influence of pulmonary inflations on discharge patterns of phrenic motoneurons

The purpose of this study was to assess the influence of pulmonary inflations on activities of single phrenic motoneurons. Studies were performed in decerebrate and paralyzed cats; activities of phrenic nerve and single phrenic motoneurons were recorded. Animals were ventilated with a servo-respirator which produced alterations in tracheal pressure in parallel with changes in integrated activity of the phrenic nerve. At end-tidal fractional concentrations of CO2 of 0.05, phrenic motoneurons were distributed into "early" and "late" populations, depending on time of onset of activity. During the late stages of neural inspiration, differences in levels of integrated activity of the phrenic nerve became evident between cycles with and without lung inflations. At a time approximating 90% of the inspiratory duration during inflations, integrated phrenic activity was higher for cycles with inflation. Concomitantly, with lung inflations, the discharge frequencies of early phrenic motoneurons were lower, and late motoneurons began to discharge sooner than when inflations were withheld. Similar results were obtained in hypercapnia. We conclude that reflexes activated by pulmonary inflations may produce augmentation, as well as inhibition of phrenic motoneuronal activities. Factors responsible for eliciting these reflex augmentations and inhibitions are discussed.     

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.