Purification of Vibrio fischeri nitrite reductase and its characterization as a hexaheme c-type cytochrome

Dissimilatory nitrite reductase was isolated from anaerobically nitrate-grown Vibrio fischeri cells and purified to electrophoretic homogeneity. The enzyme catalyzes the six-electron reduction of nitrite to ammonia. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under either nonreducing or reducing conditions, the purified nitrite reductase migrated as a single protein band of Mr 57,000. Gel filtration chromatography revealed a native molecular weight of 58,000, indicating the enzyme as isolated to be present in the monomeric form. Purified nitrite reductase exhibited typical c-type cytochrome absorption spectra with the reduced alpha-band at 552.5 nm. Heme content analysis using the purified preparation indicated the enzyme to contain 5.5 heme c groups per molecule. Iron analysis showed the presence of 5.62 g iron atoms per mole of enzyme and no nonheme irons were detected. These results clearly indicate that, similar to the dissimilatory nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes, and Escherichia coli, the V. fischeri nitrite reductase is a hexaheme c-type cytochrome. Amino acid composition of V. fischeri also revealed close similarities to those of the other three hexaheme nitrite reductases previously studied. Based on this information, it is concluded that the four ammonia-forming, dissimilatory nitrite reductases isolated to date represent a homologous group of proteins with the distinct property of being hexaheme c-type cytochromes.     

Change in binding affinities of 3Y1 secreted fibronectin upon desulfation of tyrosine-O-sulfate

Desulfated form of the 3Y1 secreted fibronectin was prepared by treatment with arylsulfatases. Under optimal conditions, the degrees of tyrosine-desulfation of [35S]sulfate-labeled fibronectin by arylsulfatases from Helix pomatia (Type H-1), Patalle vulgata (Type V) and Abalone entrails (Type VIII) were determined to be 55.7%, 54.9% and 76.4%. Upon desulfation of [3H]leucine-labeled fibronectin by Type H-1 or Type V arylsulfatase, gelatin-binding affinity remained unchanged; while heparin-binding affinity increased nearly 50%. Treatment with Type VIII arylsulfatase caused a considerable decrease in gelatin-binding and a slight decrease in heparin-binding affinities. Nevertheless, desulfation by all three enzymes consistently resulted in a dramatic decrease of fibrin-binding affinity, ranging from 42.1% to 64.4%.     

Atrial natriuretic factor R1 receptor from bovine adrenal zona glomerulosa: purification, characterization, and modulation by amiloride

The atrial natriuretic factor (ANF) R1 receptor from bovine adrenal zona glomerulosa was solubilized with Triton X-100 and purified 13,000-fold, to apparent homogeneity, by sequential affinity chromatography on ANF-agarose and steric exclusion high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the purified receptor preparation in the absence or presence of dithiothreitol revealed a single protein band of Mr 130,000. Affinity cross-linking of 125I-ANF to the purified receptor resulted in the labeling of the Mr 130,000 band. The purified receptor bound ANF with a specific activity of 6.8 nmol/mg of protein, corresponding to a stoichiometry of 0.9 mol of ANF bound/mol of Mr 130,000 polypeptide. Starting with 500 g of adrenal zona glomerulosa tissue, we obtained more than 500 pmol of purified receptor with an overall yield of 9%. The purified receptor showed a typical ANF-R1 pharmacological specificity similar to that of the membrane-bound receptor. The homogeneous Mr 130,000 receptor protein displayed high guanylate cyclase activity [1.4 mumol of cyclic GMP formed min-1 (mg of protein)-1] which was not stimulated by ANF. This finding supports the notion that the ANF binding and the guanylate cyclase activities are intrinsic components of the same polypeptide. Finally, the purified ANF-R1 receptor retained its sensitivity to modulation by amiloride, suggesting the presence of an allosteric binding site for amiloride on the receptor protein.     

Actions of 17 beta-estradiol on gonadotropin release induced by drugs that activate intracellular signal transduction mechanisms in rat anterior pituitary cells

We studied the effects of 17 beta-estradiol (E2) on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release induced by drugs that activate different intracellular signal transduction mechanisms in rat anterior pituitary cells. Cells were pretreated with E2 (6 x 10(-10) M) or diluent for 24 h. Then, both E2- and diluent-pretreated cells were incubated for 4 h with E2 or diluent, respectively, with or without drugs, and in the presence or absence of gonadotropin-releasing hormone (GnRH). Media were assayed for LH and FSH by radioimmunoassays. E2 treatment had no effect on basal FSH release, but occasionally stimulated basal LH release. Phospholipase C (PLC), L-alpha-1,2-dioctanoyl glycerol (C8), veratridine, 8-bromo-cyclic adenosine 3',5'-monophosphate (8-Br-cAMP), melittin (a phospholipase A2 [PLA2] activator), arachidonic acid, PLA2, and GnRH all stimulated LH and FSH release in both E2- and diluent-treated cells. E2 treatment increased both LH and FSH release induced by GnRH, PLC, C8, veratridine, and 8-Br-cAMP, but not by melittin, arachidonic acid, and PLA2. Neither C8, PLA2, nor arachidonic acid in combination with a maximal dose of GnRH had additive effects on either LH or FSH release, whereas melittin increased the maximal response to GnRH in both E2- and diluent-treated cells. The effects of veratridine and 8-Br-cAMP depended on dose of GnRH and presence or absence of E2. These results suggest that E2 augments stimulus-coupled gonadotropin release by interacting with the Ca2+-, and/or diacylglycerol-, and cAMP-activated pathways, but not with the arachidonic acid-activated pathway.     

Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support

A method allowing initial sequencing yields of 60-85% to be consistently obtained from samples prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer is described in detail. Conducting electrophoresis at a pH near neutrality is the single most important of the modifications made to earlier procedures, but pre-electrophoresis in the presence of glutathione or sodium thioglycolate and use of Immobilon polyvinylidene difluoride membranes all contribute to the success of the technique. When tryptophan was the NH2 terminus of a protein, the phenylthiohydantoin (PTH)-derivative recovered appeared to be an irreversible oxidation product if pre-electrophoresis was not performed. Following pre-electrophoresis, the PTH-derivative recovered co-migrated with that of unmodified tryptophan, and the recovery was higher. Recovery of methionine as its PTH-derivative was not affected by pre-electrophoresis suggesting that thioglycolate in the electrophoresis buffer during sample separation prevented or reversed oxidation of methionine sulfur but did not protect tryptophan.     

Selective inhibition of glycoprotein-processing enzymes. Differential inhibition of glucosidases I and II in cell culture

In this study, we compared the effects of 2,6-dideoxy-2,6-imino-7-O-(beta-D-glucopyranosyl)-D-glycero-L-gulohep titol (MDL) to those of the glucosidase I inhibitor, castanospermine, on the purified processing enzymes glucosidases I and II. WE also compared the effects of these two inhibitors on glycoprotein processing in cell culture using influenza virus-infected Madin-Darby canine kidney cells as a model system. With the purified processing enzymes, castanospermine was a better inhibitor of glucosidase I than of glucosidase II, whereas MDL is more effective against glucosidase II than glucosidase I. In cell culture at the appropriate dose, MDL also preferentially affected glucosidase II. Thus, at 250 micrograms/ml MDL, the major [3H]glucose-labeled (or [3H]mannose-labeled) glycopeptide from the viral hemagglutinin was susceptible to endoglucosaminidase H, and the oligosaccharide liberated by this treatment was characterized as a Glc2Man7-9GlcNAc on the basis of size, resistance to digestion by glucosidase I (but sensitivity to glucosidase II), methylation analysis, and Smith degradation studies. These data indicate that at appropriate concentrations of MDL (250 micrograms/ml), one can selectively inhibit glucosidase II in Madin-Darby canine kidney cells. However, at higher concentrations of inhibitor (500 micrograms/ml), both enzymes are apparently affected. Since MDL did not greatly inhibit the synthesis of lipid-linked saccharides or the synthesis of protein or RNA, it should be a useful tool for studies on the biosynthesis and role of N-linked oligosaccharides in glycoprotein function.     

Mononuclear phagocyte activation: activation-associated antigens

Mononuclear phagocyte activation is characterized by alterations in cellular metabolism and plasma membrane composition. In rodent and human systems, antibodies (conventional heteroantibodies or monoclonal reagents) that identify plasma membrane antigens selectively expressed by activated macrophages and monocytes have been generated. Among these activation-associated determinants is Mo3e (p50,80), a protease-sensitive antigen that is expressed by human monocytes activated in culture by exposure to bacterial lipopolysaccharide, muramyl dipeptide, or phorbol myristate acetate (PMA) (as well as other biologically active phorbol compounds). Mo3e is also expressed by the monoblastic cell line U-937 after culture in medium containing PMA and other pharmacological activators of protein kinase C (4 beta-phorbol-12,13-dibutyrate, 4 beta-phorbol-12,13-didecanoate, mezerein, and cell-permeable 1,2-diacylglycerol). The human promyelocytic cell line HL-60 becomes Mo3e positive after exposure in vitro to certain inducers of monocytic differentiation (PMA, dibutyryl cyclic AMP, and cholera toxin plus 3-isobutyl-1-methylxanthine). The surface expression of Mo3e is blocked by inhibitors of protein synthesis, N-linked glycosylation, and protein kinase activation, as well as by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and calcium antagonists. These data suggest the involvement of glycoprotein synthesis, protein kinase activation, and calcium ions in the stimulated expression of Mo3e by activated human mononuclear phagocytes. Anti-Mo3e antibody blocks the human monocyte response to migration inhibitory factor (MIF), which indicates an association between the expression of Mo3e antigen and responsiveness to MIF.     

Normal human neutrophils are a source of a specific interleukin 1 inhibitor

In the course of our study on neutrophil production of an interleukin 1 (IL-1)-like factor, we found that the addition of polymorphonuclear neutrophils (PMN) to monocytes cultured in the presence of zymosan resulted in decreased IL 1 activity of the resultant supernatant, suggesting that PMN may contain an inhibitor of IL 1. The objective of this investigation was to study this IL 1 inhibitor which normal human PMN contain. The inhibitor is constitutively present in the PMN because 0 hr PMN lysates and unstimulated PMN supernatants also show inhibitory activity. The PMN inhibitor inhibits IL 1 (crude and partially purified) in a dose-response manner and does not affect basal [3H]thymidine incorporation in the presence or absence of PHA-P. The PMN inhibitor does not have any effect on interleukin 2 (IL 2)-induced proliferation of the IL 2-dependent CTLL cells. The inhibitor can be generated in the absence of serum and is not produced as a result of proteolytic activity from PMN enzymes. The inhibitor is heat-labile and is most stable at neutral pH. Gel filtration studies on Sephadex G-200 indicate that the inhibitor is heterogeneous in size. Two inhibitory peaks, at 45,000 to 70,000 m.w. and at greater than 160,000 m.w., were observed. When zymosan-stimulated PMN supernatant was chromatographed, there was separation of inhibitory factor from a 17,000 m.w. proliferating factor. Presence of this PMN inhibitor may be important in negative regulation of IL 1.