PubMed Health⌕ Search

Biomedical subjects

V Manne

Publications and source records attributed to V Manne.

30 records · Page 2Linked to original sources
PubMed1986

Purification and properties of synephrinase from Arthrobacter synephrinum.

Synephrinase, an enzyme catalyzing the conversion of (-)-synephrine into p-hydroxyphenylacetaldehyde and methylamine, was purified to apparent homogeneity from the cell-free extracts of Arthrobacter synephrinum grown on (+/-)-synephrine as the sole source of carbon and nitrogen. A 40-fold purification was sufficient to produce synephrinase that is apparently homogeneous as judged by native polyacrylamide gel electrophoresis and has a specific activity of 1.8 mumol product formed/min/mg protein. Thus, the enzyme is a relatively abundant enzyme, perhaps comprising as much as 2.5% of the total protein. The enzyme essentially required a sulfhydryl compound for its activity. Metal ions like Mg2+, Ca2+, and Mn2+ stimulated the enzyme activity. Metal chelating agents, thiol reagents, denaturing agents, and metal ions like Zn2+, Hg2+, Ag1+, and Cu2+ inhibited synephrinase activity. Apart from (-)-synephrine, the enzyme acted upon (+/-)-octopamine and beta-methoxysynephrine. Molecular oxygen was not utilized during the course of the reaction. The molecular mass of the enzyme as determined by Sephadex G-200 chromatography, was around 156,000. The enzyme was made up of four identical subunits with a molecular mass of 42,000.

Arthrobacter↗
PubMed1986

Reversal of transformed phenotype by monoclonal antibodies against Ha-ras p21 proteins.

The transforming activities of p21 ras proteins have been determined by micro-injection of these proteins into NIH3T3 cells. In order to facilitate functional studies on the effect of ras proteins on malignant transformation and normal cellular growth, analysis has been made with three monoclonal antibodies (YA6-172, Y13-238 and Y13-259) as originally reported by Furth et al. (J virol 43 (1982) 294). Purified immunoglobulin of Y13-259 has the highest titer of binding to bacterially synthesized p21 ras proteins. Experimental analyses indicate that only Y13-259 antibody will neutralize the transforming activity of the co-injected bacterially synthesized ras protein and the neutralization effect was blocked by co-injection of excess ras protein. In addition, micro-injection of Y13-259 immunoglobulin into transformed NIH3T3 cells (obtained by DNA transfection of NIH3T3 cells with molecularly cloned ras gene) reversed their transformed phenotypes. These results indicate that both bacterially synthesized p21 ras proteins and the natural ras proteins produced in NIH3T3 cells were neutralized by Y13-259 antibody.

Animals↗
PubMed1985

Effect of divalent metal ions and glycerol on the GTPase activity of H-ras proteins.

The product of the protooncogenic ras gene (p21N ras) exhibits a weak GTPase activity. A significant increase in the GTPase activity associated with p21N ras protein was obtained by using glycerol in the assay mixture. Of the several metal ions tested, only Mg++ and Mn++ are effective divalent cations that support the GTPase activity of p21N ras protein. p21N ras protein exhibits higher GTPase activity and yields higher [3H] GDP binding in the presence of MnCl2 than with MgCl2. Optimal GTPase and [3H] GDP binding are obtained at micromolar concentrations of MgCl2 or MnCl2. Concentrations in the millimolar range of either MgCl2 or MnCl2 are inhibitory to the GTPase activity, whereas [3H] GDP binding was not affected.

Cations, Divalent↗
PubMed1985

Bacterial gene expression and biotechnology.

This brief review article has been intended to give a few up-to-date examples of the dramatic impact that our knowledge of gene expression (especially bacterial gene expression) has had in the area of biotechnology. This area is in a state of such rapid growth that it has only been possible to present a limited overview of the subject matter. We have tried to illustrate our points with examples from work in which we have had some direct involvement. It should be apparent that continued increase in our understanding of gene expression should provide additional opportunities for expanded application of the new methodology.

Bacteria↗
PubMed1985

Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity.

Several ras genes have been expressed at high levels in Escherichia coli and the resultant ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. We were able to detect a weak GTPase activity associated with the purified proteins. The normal cellular ras protein (p21N) exhibits approximately equal to 10 times higher GTPase activity than the "activated" proteins. Even though the turnover rate of the reaction is very low (0.02 mol of GTP hydrolyzed per mol of p21N protein per minute), the reaction appears to be catalytic; one molecule of p21N hydrolyzes more than one molecule of GTP. The GTPase and the GDP binding activities both have been recovered from a Mr 23,000 protein eluted following NaDodSO4/polyacrylamide gel electrophoresis, suggesting that these two activities are associated with the same protein. Mg2+ ions and dithiothreitol are required for GTPase activity and the optimal pH is between 7 and 8. Guanidine X HCl, which is required for solubilizing bacterially expressed ras protein, is strongly inhibitory to GTPase activity at concentrations higher than 0.5 M.

Amino Acid Sequence↗
PubMed1985

Effects of phospholipids and ADP-ribosylation on GTP hydrolysis by Escherichia coli-synthesized Ha-ras-encoded p21.

The Ha-ras protooncogene product p21, which may be involved in control of cellular growth, is a membrane protein that binds guanine nucleotides and hydrolyzes GTP. p21 GTPase activity is stimulated by lysophosphatidylcholine; a delay in activation was observed unless p21 was incubated with the phospholipid prior to assay. Maximal activation by the phospholipid was observed over a narrow concentration range; the presence in the assay mixture of lysophosphatidylcholine at concentrations above this optimum markedly inhibited p21 GTPase. GTP hydrolysis was also stimulated, but to a lesser degree, by phosphatidylcholine. Phosphatidylinositol and phosphatidylserine did not significantly enhance GTPase activity. The stimulatory effect of phospholipid was mimicked, in part, by nonionic detergents. p21 may be related to other GTPases, the regulatory guanine nucleotide-binding G proteins of the hormone-sensitive adenylate cyclase complex and transducin of the retinal light-activated phosphodiesterase system. The G proteins and transducin are heterotrimers; the alpha subunits possess GTPase activity and the beta gamma subunit complex along with agonist-receptor complex or light-activated rhodopsin enhance GTP hydrolysis. p21 GTPase activity was slightly stimulated by rhodopsin, but, in contrast to the GTPase activity of transducin, stimulation was not light-dependent. GTP hydrolysis was enhanced somewhat by beta gamma subunit complex in the absence, but not in the presence, of rhodopsin. Like the G proteins and transducin, activity of p21 was altered by ADP-ribosylation. Modification of p21 catalyzed by an NAD: arginine ADP-ribosyltransferase purified from turkey erythrocytes decreased both GTPase activity and guanine nucleotide binding activity.

Adenosine Diphosphate Ribose↗
PubMed1984

Mechanism of translational control by partial phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

Catalysis of ternary complex formation by the GDP exchange factor (GEF), in the presence of Mg2+, is blocked by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF-2). We proposed earlier that this phosphorylation interferes with the interaction between eIF-2 and GEF (then termed ESP). If so, inhibition should be related to the extent of phosphorylation. However, work in other laboratories indicated that in fully inhibited, heme-deficient lysates only 20-40% of the eIF-2 is phosphorylated. To understand the nature of the molecular lesion in eIF-2-alpha phosphorylation we used a system of pure components in which the rate of exchange of eIF-2-bound [3H]GDP with unlabeled GDP (via the reaction eIF-2-GDP + GEF in equilibrium eIF-2-GEF + GDP) was measured by using mixtures of eIF-2(alpha P) X [eH]GDP and eIF-2 X [3H]GDP in different proportions at constant concentration of eIF-2 X GEF. If, for example, the ratio of eIF-2 X GEF to total (phosphorylated and unphosphorylated) eIF-2 X [3H]GDP was 0.25, the exchange was found to be maximally inhibited when the proportion of eIF-2(alpha P) X [3H]GDP in hte mixture reached 25%. This suggests that the reaction stops because the available GEF is trapped in an inactive complex with eIF-2(alpha P). In the absence of free GEF, eIF-2 would not be able to recycle and initiation would come to a standstill when the available eIF-2 is tied up as eIF-2 X GDP. The trapping of GEF by eIF-s(alpha P) is strongly supported by the following observation. Incubation of eIF-2 X GEF with excess [3H]GDP leads to the formation of eIF-2 X [3H] GDP and free GEF and, if eIF-2(alpha 32P) X GDP is also present, all of the GEF is converted to eIF-2(alpha 32P) X GEF. This suggests that, whereas the equilibrium of the reaction eIF-2 X GEF + GDP in equilibrium eIF-2 X GDP + GEF favors the formation of free GEF, the equilibrium of the reaction eIF-2(alpha P) X GDP + GEF in equilibrium eIF-2(alpha P) X GEF + GDP is in favor of the association of GEF to eIF-2(alpha P).

Animals↗
PubMed1984

Guanosine nucleotide binding by highly purified Ha-ras-encoded p21 protein produced in Escherichia coli.

High-level expression of the p21 protein product of the BALB murine sarcoma virus v-ras gene (similar to the product of the Harvey murine sarcoma virus v-Ha-ras gene) has been reported recently, and highly purified preparations of this protein have been obtained. We used a nitrocellulose filter assay for measuring the binding of GDP and GTP to the purified protein. Previously p21 antibodies had been used to precipitate p21-guanosine nucleotide complexes from crude extracts containing the protein. Using the filter assay, we find that the v-Ha-ras gene product binds [3H]GDP stoichiometrically. The binding is time-dependent and is faster at 30 degrees C than at 0 degrees C. Optimum binding is obtained in the presence of dithiothreitol and magnesium ions and at pH 7.4. In terms of its GDP binding activity, p21 is heat stable and pronase sensitive. The dissociation constants (Kd) of p21 for [3H]GDP and [3H]GTP, determined by Scatchard analysis, are 6 X 10(-8) M and 2.5 X 10(-8) M, respectively.

Cloning, Molecular↗
PubMed1983

Polypeptide chain initiation in eukaryotes: reversibility of the ternary complex-forming reaction.

In the last step of polypeptide chain initiation in eukaryotes, the interaction of the 40S preinitiation complex eIF-2.GTP.Met-tRNAi.40S [the complex between the 40S ribosomal subunit and the ternary complex containing equimolar amounts of eukaryotic initiation factor 2 (eIF-2), GTP, and eukaryotic initiator methionyl tRNA (Met-tRNAi)] with a 60S ribosomal subunit in the presence of mRNA, cap binding protein (with "capped" messengers), ATP, and the initiation factors eIF-3, eIF-4a, -4b, -4c, and eIF-5, results in the formation of an 80S initiation complex (Met-tRNAi.80S.mRNA) with concomitant hydrolysis of GTP and liberation of eIF-2 for recycling in subsequent initiation events. However, at physiological Mg2+ concentrations, GDP is known to have approximately equal to 100-fold greater affinity than GTP for eIF-2 and eIF-2 is believed to be released in the form of an eIF-2.GDP complex. Previously, we have shown that initiation factor SP (for eIF-2-stimulating protein) promotes the exchange of eIF-2-bound GDP for GTP and catalyzes ternary complex formation in the presence of Met-tRNAi. Binding of GDP by eIF-2 is indeed so tight that, as we now show, homogeneous preparations of eIF-2 contain upward of 0.5 mol of GDP/mol of eIF-2. We further show that, in the presence of Mg2+ and catalytic amounts of SP, ternary complex formation conforms to the overall reversible reaction eIF-2.GDP + GTP + Met-tRNAi in equilibrium eIF-2.GTP.Met-tRNAi + GDP.

Eukaryotic Initiation Factor-2↗
PubMed1982

A bacteriophage T4 DNA packaging related DNA-dependent ATPase-endonuclease.

A new bacteriophage T4-induced DNA-dependent ATPase-endonuclease was purified to essential homogeneity from an extract of late infected Escherichia coli. Both DNA-dependent ATPase and endonuclease activities co-chromatograph, co-sediment, and have been renatured from a single 43-kilodalton protein eluted following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that both activities are exerted by one multifunctional protein. Duplex, single-stranded, and supercoiled DNAs are all effective activators of the high specific activity ATPase which produces ADP and inorganic PO4. The enzyme displays a broad specificity towards the nucleoside and deoxynucleoside triphosphates, and the ATPase activity is strongly inhibited by DNA-intercalating compounds. The endonuclease appears to be most active on supercoiled DNA, producing double-stranded breaks in duplex DNA, and does not require nucleoside triphosphates. An antiserum against the purified enzyme immunoprecipitated it, inhibited its ATPase activity, and also precipitated from extracts a T4-induced protein of Mr = 43,000. This antigen was not found in uninfected E. coli, or following a gene 55am mutant (late protein synthesis defective) infection, and was not detected following infection with T4 amber mutants of any early capsid protein gene which blocks T4 head protein cleavage in vivo. In a pulse-chase experiment, the radioactive antigen was not found following a pulse of radioactive amino acids, but appeared after a chase with excess nonradioactive amino acids. The enzyme-related antigen is apparently produced by cleavage of a precursor by the T4 head assembly proteinase which processes a number of prohead proteins. These processing reactions are dependent in vivo upon assembly of the prohead and are required for its maturation. The evidence suggests that this enzyme functions in head assembly and DNA packaging, and originates as the cleavage product of a prohead precursor protein.

DNA, Viral↗
PubMed1982

Heat-stable inhibitor of translation in reticulocyte lysates.

Inhibition of translation in hemin-containing reticulocyte lysates by catalytic subunit (cS) preparations of cAMP-dependent protein kinase from bovine heart, reported earlier by our group, is due to a highly active heat-stable protein contaminant (HS). The specific activity for translational inhibition goes up by a factor of 10 when cS is heated for 10 min at 80 degrees C, which completely destroys histone phosphorylation activity. HS has been purified to homogeneity from bovine heart. It consists of a single polypeptide chain (Mr approximately 68,000). HS inhibits translation with biphasic kinetics similar to those of hemin deficiency and induces pronounced phosphorylation of the alpha subunit of the eukaryotic initiation factor eIF-2. The inhibition is relieved by eIF-2 or GTP but not by high concentrations of double-stranded RNA, thus ruling out involvement of the double-stranded RNA-activated inhibitor. Judged by poly(U) translation, HS has no effect on chain elongation. When added to crude preparations of the proinhibitor form (proHCI) of the heme-controlled translational inhibitor (HCI), HS appears to produce an increase of the HCI-to proHCI ratio. The mode of action of HS is as yet unknown.

Animals↗