Characterization of a factor that stimulates hydrolysis of GTP bound to ras gene product p21 (GTPase-activating protein) and correlation of its activity to cell density

1988 ◽  
Vol 8 (10) ◽  
pp. 4169-4173
Author(s):  
M Hoshino ◽  
M Kawakita ◽  
S Hattori
Keyword(s):  
Gene Product ◽  
Cell Density ◽  
Cultured Cells ◽  
Specific Activity ◽  
Gtpase Activating Protein ◽  
Ras Gene ◽  
Cell Extracts ◽  
Mouse Tissues ◽  
Almost All ◽  
Hydrolysis Of

The postmicrosomal fraction of the extract from NIH 3T3 and BALB/c 3T3 cells stimulated the hydrolysis of GTP bound to H-ras gene product p21 by severalfold. The stimulation was observed with normal p21 but not with p21 with valine as the 12th residue. This specificity is similar to that of GTPase-activating protein (GAP) for N-ras p21 described by M. Trahey and F. McCormick (Science 238:542-545, 1987). Consistent with this specificity, analysis of p21-bound nucleotides in living cells revealed that almost all normal p21 bound GDP, whereas oncogenic mutant p21s bound both GTP and GDP. Similar activity was also found in various mouse tissues, with brain tissue showing the highest specific activity. When cell extracts were prepared from cultured cells, there was a linear relationship between GAP activity and cell density. These results suggest the factor is involved in the regulation of cell proliferation.

scholarly journals Characterization of a factor that stimulates hydrolysis of GTP bound to ras gene product p21 (GTPase-activating protein) and correlation of its activity to cell density.

1988 ◽  
Vol 8 (10) ◽  
pp. 4169-4173 ◽  
Author(s):  
M Hoshino ◽  
M Kawakita ◽  
S Hattori
Keyword(s):  
Gene Product ◽  
Cell Density ◽  
Cultured Cells ◽  
Specific Activity ◽  
Gtpase Activating Protein ◽  
Ras Gene ◽  
Cell Extracts ◽  
Mouse Tissues ◽  
Almost All ◽  
Hydrolysis Of

The postmicrosomal fraction of the extract from NIH 3T3 and BALB/c 3T3 cells stimulated the hydrolysis of GTP bound to H-ras gene product p21 by severalfold. The stimulation was observed with normal p21 but not with p21 with valine as the 12th residue. This specificity is similar to that of GTPase-activating protein (GAP) for N-ras p21 described by M. Trahey and F. McCormick (Science 238:542-545, 1987). Consistent with this specificity, analysis of p21-bound nucleotides in living cells revealed that almost all normal p21 bound GDP, whereas oncogenic mutant p21s bound both GTP and GDP. Similar activity was also found in various mouse tissues, with brain tissue showing the highest specific activity. When cell extracts were prepared from cultured cells, there was a linear relationship between GAP activity and cell density. These results suggest the factor is involved in the regulation of cell proliferation.

Mass spectroscopic analysis of phosphatidylinositol synthesis using 6-deuteriated-myo-inositol: comparison of the molecular specificities and acyl remodelling mechanisms in mouse tissues and cultured cells

2004 ◽  
Vol 32 (6) ◽  
pp. 1057-1059 ◽  
Author(s):  
A.D. Postle ◽  
H. Dombrowsky ◽  
H. Clarke ◽  
C.J. Pynn ◽  
G. Koster ◽  
...  
Keyword(s):  
Molecular Species ◽  
Cultured Cells ◽  
Ionization Mass ◽  
Membrane Composition ◽  
Mouse Tissues ◽  
Wide Range ◽  
Kinetics Of ◽  
Hydrolysis Of

Mammalian cell PtdIns (phosphatidylinositol) in vivo is enriched in the sn-1-stearoyl 2-arachidonoyl species, the physiological precursor of phosphatidylinositol 4,5-bisphosphate. Mechanisms regulating this specificity are unclear but are typically lost for cells in culture. We used ESI-MS (tandem electrospray ionization-mass spectrometry) to determine the molecular species of PtdIns synthesized by mouse tissues in vivo compared with cultured cells in vitro. After incorporation of deuteriated myo-d6-inositol over 3 h, endogenous and newly synthesized PtdIns and lysoPtdIns species were quantified from precursor scans of m/z 241− and m/z 247− respectively. PtdIns was synthesized as a wide range of species irrespective of the final membrane composition. Analyses of isotope enrichments argued against acyl remodelling as the major regulatory mechanism: composition of the lysoPtdIns pool under all conditions reflected that of either endogenous or newly synthesized PtdIns and was always at equilibrium. The kinetics of PtdIns synthesis, together with the prolonged time scale required for achieving final equilibrium compositions suggest that selective transport between membranes and/or hydrolysis of selected molecular species are the most probable mechanisms regulating compositions of PtdIns and, ultimately, phosphatidylinositol 4,5-bisphosphate.

scholarly journals Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2005 ◽  
Vol 187 (6) ◽  
pp. 2077-2083 ◽  
Author(s):  
Sherry V. Story ◽  
Claudia Shah ◽  
Francis E. Jenney ◽  
Michael W. W. Adams
Keyword(s):  
Metal Ion ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
High Specific Activity ◽  
Native Form ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts ◽  
Hydrolysis Of

ABSTRACT Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of l-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

On the Preparation of Bovine α-Thrombin

1978 ◽  
Vol 39 (01) ◽  
pp. 193-200 ◽  
Author(s):  
Erwin F Workman ◽  
Roger L Lundblad
Keyword(s):  
Immobilized Enzyme ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Guanidine Hydrochloride ◽  
Low Molecular Weight ◽  
Reversible Process ◽  
Gel Beads ◽  
Tissue Thromboplastin ◽  
C 50 ◽  
Hydrolysis Of

SummaryAn improved method for the preparation of bovine α-thrombin is described. The procedure involves the activation of partially purified prothrombin with tissue thromboplastin followed by chromatography on Sulfopropyl-Sephadex C-50. The purified enzyme is homogeneous on polyacrylamide discontinuous gel electrophoresis and has a specific activity toward fibrinogen of 2,200–2,700 N.I.H. U/mg. Its stability on storage in liquid media is dependent on both ionic strenght and temperature. Increasing ionic strength and decreasing temperature result in optimal stability. The denaturation of α-thrombin by guanidine hydrochloride was found to be a partially reversible process with the renatured species possessing properties similar to “aged” thrombin. In addition, the catalytic properties of a-thrombin covalently attached to agarose gel beads were also examined. The activity of the immobilized enzyme toward fibrinogen was affected to a much greater extent than was the hydrolysis of low molecular weight, synthetic substrates.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

IN VITRO METHODS FOR THE STUDY OF THE ADENOHYPOPHYSIAL FUNCTIONS TO SECRETE GONADOTROPHIN

1971 ◽  
Vol 68 (1_Suppl) ◽  
pp. S27-S40 ◽  
Author(s):  
T. Kobayashi ◽  
T. Kigawa ◽  
M. Mizuno ◽  
T. Watanabe
Keyword(s):  
Cell Culture ◽  
Organ Culture ◽  
Cultured Cells ◽  
Cell Sheet ◽  
Short Term ◽  
Hypothalamic Extract ◽  
Numerous Cell ◽  
Single Cell Culture ◽  
Almost All

ABSTRACT There are several in vitro methods to analyse the function of the adenohypophysis or the mechanisms of its regulation. The present paper deals with single cell culture, organ culture and short term incubation techniques by which the morphology and gonadotrophin-secreting function of the adenohypophysis were studied. In trypsin-dispersed cell culture, the adenohypophysial cells showed extensive propagation to form numerous cell colonies and finally develop into a confluent monolayer cell sheet covering completely the surface of culture vessels. Almost all of the cultured cells, however, became chromophobic, at least at the end of the first week of cultivation, when gonadotrophin was detectable neither in the culture medium nor in the cells themselves. After the addition of the hypothalamic extract, gonadotrophin became detectable again, and basophilic or PAS-positive granules also reappeared within the cells, suggesting that the gonadotrophs were stimulated by the extract to produce gonadotrophin. In organ culture and short term incubation, the incorporation of [3H] leucine into the adenohypophysial cells in relation to the addition of hypothalamic extract was examined. It was obvious that the ability to incorporate [3H] leucine into the gonadotrophs in vitro was highly dependent upon the presence of the hypothalamic extract.

scholarly journals Catalytic Diversity of GH30 Xylanases

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4528
Author(s):  
Katarína Šuchová ◽  
Vladimír Puchart ◽  
Nikolaj Spodsberg ◽  
Kristian B. R. Mørkeberg Krogh ◽  
Peter Biely
Keyword(s):  
Methyl Ester ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Current Knowledge ◽  
Carboxyl Group ◽  
Weak Effect ◽  
Bacterial Enzymes ◽  
Catalytic Activities ◽  
Almost All ◽  
Free Carboxyl

Catalytic properties of GH30 xylanases belonging to subfamilies 7 and 8 were compared on glucuronoxylan, modified glucuronoxylans, arabinoxylan, rhodymenan, and xylotetraose. Most of the tested bacterial GH30-8 enzymes are specific glucuronoxylanases (EC 3.2.1.136) requiring for action the presence of free carboxyl group of MeGlcA side residues. These enzymes were not active on arabinoxylan, rhodymenan and xylotetraose, and conversion of MeGlcA to its methyl ester or its reduction to MeGlc led to a remarkable drop in their specific activity. However, some GH30-8 members are nonspecific xylanases effectively hydrolyzing all tested substrates. In terms of catalytic activities, the GH30-7 subfamily is much more diverse. In addition to specific glucuronoxylanases, the GH30-7 subfamily contains nonspecific endoxylanases and predominantly exo-acting enzymes. The activity of GH30-7 specific glucuronoxylanases also depend on the presence of the MeGlcA carboxyl, but not so strictly as in bacterial enzymes. The modification of the carboxyl group of glucuronoxylan had only weak effect on the action of predominantly exo-acting enzymes, as well as nonspecific xylanases. Rhodymenan and xylotetraose were the best substrates for exo-acting enzymes, while arabinoxylan represented hardly degradable substrate for almost all tested GH30-7 enzymes. The results expand current knowledge on the catalytic properties of this relatively novel group of xylanases.

Transcription of c-onc genes c-rasKi and c-fms during mouse development

1983 ◽  
Vol 3 (6) ◽  
pp. 1062-1069
Author(s):  
R Müller ◽  
D J Slamon ◽  
E D Adamson ◽  
J M Tremblay ◽  
D Müller ◽  
...  
Keyword(s):  
Gene Product ◽  
Time Course ◽  
Specific Pattern ◽  
Yolk Sac ◽  
Transport Processes ◽  
Mouse Development ◽  
Mrna Species ◽  
Mouse Tissues ◽  
Sarcoma Virus ◽  
Significant Expression

We investigated the expression of cellular sequences c-rasKi and c-fms, which are homologous to the oncogenes of Kirsten rat sarcoma virus and the McDonough strain of feline sarcoma virus, during murine development and in a variety of mouse tissues. The c-rasKi gene was found to be transcribed into two mRNA species of approximately 2.0 and 4.4 kilobases, whereas a single c-fms-related transcript of approximately 3.7 kilobases was identified. The c-rasKi gene appeared to be expressed ubiquitously, since similar levels of transcripts were observed in embryos, fetuses, extraembryonal structures, and a variety of postnatal tissues. In contrast, significant expression of c-fms was found to be confined to the placenta and extraembryonal membranes (i.e., combined yolk sac and amnion). The concentration of c-fms transcripts in the placenta increased approximately 15-fold (relative to day-7 to day-9 conceptuses) during development before reaching a plateau at day 14 to 15 of gestation. The time course of cfms expression in the extraembryonal membranes appeared to parallel the stage-specific pattern observed in the placenta. The level of c-fms transcripts in the extraembryonal tissues reached a level which was approximately 20- to 50-fold greater than that in the fetus. These findings suggest that the c-fms gene product may play a role in differentiation of extraembryonal structures or in transport processes occurring in these tissues. Our results indicate that the c-onc genes analyzed in the present study exert essentially different functions during mouse development.

scholarly journals A Monoacylglycerol Lipase from Mycobacterium smegmatis Involved in Bacterial Cell Interaction

2010 ◽  
Vol 192 (18) ◽  
pp. 4776-4785 ◽  
Author(s):  
Rabeb Dhouib ◽  
Françoise Laval ◽  
Frédéric Carrière ◽  
Mamadou Daffé ◽  
Stéphane Canaan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Wild Type Strain ◽  
Specific Activity ◽  
Cell Interaction ◽  
Monoacylglycerol Lipase ◽  
Homologous Proteins ◽  
Dependent Activity ◽  
Hydrolysis Of

ABSTRACT MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg−1. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.

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