Search PubMed⌕ Search

Biomedical subjects

S H Pai

Publications and source records attributed to S H Pai.

At least 37 records · Page 2Linked to original sources
PubMed1984

Purification of Eco R1 endonuclease on heparin Sepharose-4B column chromatography.

Restriction endonucleases play a very important role in genetic engineering and DNA mapping. Among hundreds of restriction endonucleases, the Eco R1 enzyme is the most useful and widely investigated enzyme. After sonication and ultracentrifugation, crude extracts of E. coli RY 13 were purified by employing the polyethyleneimine precipitate, ammonium sulfate precipitate and heparin Sepharose-4B affinity column chromatography. The Eco R1 enzyme were purified at about 42 folds and the specific activity was about 100,000 U/mg of protein. The whole purification procedure was finished within two days. The recovery was about 42%. The enzyme was sufficiently concentrated for direct specific DNA hydrolysis.

Chromatography, Affinity↗
PubMed1982

Kinetic mechanism of threonyl-tRNA synthetase from human placenta.

Using purified enzyme and homologous tRNA, we have investigated the order of substrate binding and product release for the human placental threonyl-tRNA synthetase by isotope exchange, initial velocity, dead-end inhibition and product inhibition studies. The kinetic patterns obtained from these studies are consistent with a unique Bi Uni Uni Bi ping-pong mechanism. The order of addition of the first two substrates ATP and threonine is random, while the release of products follows an obligatory sequence, with AMP as the last product to dissociate from the enzyme.

Adenosine Triphosphate↗
PubMed1982

Purification and subunit structure studies of human placental threonyl-tRNA synthetase.

Human threonyl-tRNA synthetase has been purified from full-term placenta over 5000-fold with a 13% overall yield by a combination of affinity chromatographic methods and conventional procedures. The product was apparently homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the esterification of approximately 3500 nmol L-threonine to tRNA per min per mg of protein at 37 degrees, corresponding to a molecular activity of approximately 700/min. The apparent molecular weight of the native enzyme ranged from 210000 to 220000 by gel-filtration on Sephadex G-200, and was either approximately 110000 or 228000 by sucrose gradient centrifugation for different preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single band of molecular weight 85000 or 115000 for different preparations. These results suggest that human placental threonyl-tRNA synthetase has a subunit structure of the type alpha 2 with a subunit molecular weight of about 100000. However, some other molecular forms with lower specific activity were also found to exist under certain conditions.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Protein kinases of Dictyostelium discoideum, strain AX-2.

In cell homogenates of Dictyostelium discoideum, strain AX-2, four major soluble protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) and one membrane-associated protein kinase activity were identified. The enzymes showed high affinity for casein. One of the enzymes was purified by affinity chromatography on casein-coated Sepharose. The soluble high molecular weight enzymes phosphorylated histones, whereas the low molecular weight enzymes did not. The same protein kinase species were present in vegetative and aggregation-competent cells. Their specific activity, however, changed during the development to aggregation competence. None of the enzymes was stimulated by cyclic AMP or cyclic GMP, regardless of their origin from vegetative or aggregation-competent cells.

Caseins↗
PubMed1976

Multiple molecular forms of cysteinyl-tRNA synthetase from rat liver: purification and subunit structure.

Cysteinyl-tRNA synthetase (L-cysteine:tRNACys ligase (AMP-forming), EC 6.1.1.16) has been purified from rat liver in 23% overall yield. The enzyme was resolved by hydroxyapatite chromatography into three active forms (Fractions CRS-1, CRS-2 and CRS-3). The total activity ratio was about 0.7:2:1. The fractions CRS-2 and CRS-3 contained no other detectable aminoacyl-tRNA synthetase activity. CRS-2 was homogeneous by polyacrylamide gel electrophoresis, CRS-3 gave two active bands with mobilities corresponding to those of CRS-1 and CRS-2. The molecular weight of CRS-2 was about 240 000 by electrophoretic mobilities on the gels of various porosity, and 115 000-140 000 by sucrose gradient centrifugation. By gel-filtration, CRS-1, CRS-2 and CRS-3 exhibited apparent molecular weights of 122 000, 235 000 and 300 000, respectively. By sodium dodecyl sulfate gel electrophoresis, both CRS-2 and CRS-3 gave a single major band of 120 000 daltons. Stoichiometric study of cysteinyl adenylate formation indicated that CRS-2 has two active sites per molecule. These results are consistent with a dimeric structure of the type alpha2 for the major form of rat liver cysteinyl-tRNA synthetase, composed of two probably identical subunits of about 120 000 daltons. Available evidence also suggests that CRS-1 and CRS-3 are alpha and alpha3 (or alpha4), respectively.

Amino Acyl-tRNA Synthetases↗
PubMed1975

Protein kinase of bacteriophage T7. 1. Purification.

A protein kinase, ATP:protein phosphotransferase (EC 2.7.1.37) was detected in Escherichia coli after infection with bacteriophage T7. The enzyme was purified from the ribosomal wash fraction by conventional methods, affinity chromatography on Cibacron blue and on lysozyme coupled to Sepharose, and by cellogel electrophoresis. An approximately 5000-fold purification was achieved.

Chromatography, Affinity↗
PubMed1975

Protein kinase of bacteriophage T7. 2. Properties, enzyme synthesis in vitro and regulation of enzyme synthesis and activity in vivo.

Protein kinase, which was isolated from cells infected with T7, is indeed a viral gene product. This is shown by DNA-dependent synthesis in vitro. The protein kinase transfers phosphate from ATP to seryl or threonyl residues in protein. The enzyme has only a relative requirement for magnesium ions, but is only active at low ionic strength. The best substrate is lysozyme. T7 protein kinase activity is not stimulated by cyclic 3':5'-AMP and/or cyclic 3':5'-GMP. The T7 protein kinase carries -- SH groups essential for activity. There is indication that the enzyme phosphorylates itself and causes self inactivation, which may explain the fast disappearance of enzyme activity in vivo. Bacteriophage T3 also induces a protein kinase which is similar to the T7-induced enzyme in all respects tested.

Ammonium Chloride↗
PubMed1975

Transcriptional units for ribosomal proteins of Escherichia coli.

Transcriptional units for ribosomal proteins in Escherichia coli were measured using the ultraviolet sensitivities of the rates of synthesis of individual ribosomal proteins. The ultraviolet sensitivities of gene transcriptions are proportional to the distances from the promoters. The longest transcriptional units for ribosomal proteins are 3.6 x 10(6) of DNA molecular weight corresponding to 1.8 x 10(6) of RNA or to 180 000 of protein. The length would cover 10--12 genes of ribosomal proteins (of an average Mr of 15000-18000).

Bacterial Proteins↗
PubMed1974

Protein kinase induction in Escherichia coli by bacteriophage T7.

After bacteriophage T7 infection, a protein kinase (EC 2.7.1.37; ATP:protein phosphotransferase) activity can be demonstrated in E. coli in vivo by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Cell-free extracts catalyzed the transfer of the terminal phosphoryl group of [(gamma)-(32)P]ATP to endogenous protein acceptor or to added histone. The bond between phosphate and protein shows the characteristics of serine phosphate: it is stable in 1 N HCl (100 degrees ) and cleaved by 1 N KOH (37 degrees ) and by alkaline phosphatase treatment. Moreover, after partial acid hydrolysis, radiophosphate migrates with marker O-phosphoserine on polyethyleneimine-cellulose thin-layer chromatograms. Enzyme activity in uninfected cells is negligible. Ultraviolet irradiation of the phage genome prevents the appearance of the protein kinase; irradiation of the host genome does not. The enzyme activity occurs 4 min after infection and its gene maps in the early region (promoter proximal to gene 1). Ribosomal proteins are phosphorylated in vivo and are substrates in vitro. Enzyme activity in vitro is not changed by addition of cyclic AMP or cyclic GMP.

Adenosine Triphosphate↗
PubMed1974

Translational control induced by bacteriophage T7.

Phage T7 discontinues host gene expression by translational and transcriptional control mechanisms. Translational control is exerted by the T7 translational-repressor. This protein inhibits the synthesis of beta-galactosidase (EC 3.2.1.23) in vivo and in vitro and the synthesis of the T3 enzyme S-adenosylmethioninehydrolase (EC 3.3.1.-). The translational-repressor does not interfere with T7-specific enzyme synthesis. The T7 translational-repressor purifies with the initiation factors. The repressor interacts with the initiation of translation of host enzymes. The translational-repressor gene is close to the promotor for RNA polymerase of Escherichia coli.

Chromosome Mapping↗