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T Coolbear

Publications and source records attributed to T Coolbear.

28 records · Page 2Linked to original sources
PubMed1990

Purification and some properties of a thermostable DNA polymerase from a Thermotoga species.

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.

Anaerobiosis↗
PubMed1984

Increase of sialyltransferase activity in the serum and liver of inflamed rats.

Induction of inflammation by turpentine injection caused 1.5-2-fold increase of both sialyl- and galactosyltransferase activity in liver homogenates. The effect was apparent after 12 h of turpentine treatment. Serum sialyltransferase activity started to increase in the inflamed rats after 18 h, reaching a maximum of 4-fold at 48 h. In contrast, galactosyltransferase activity in serum showed no significant increase. The coordinated and temporal increase of sialyltransferase activity in liver and serum suggest involvement of a specific mechanism for the preferential release of this enzyme into serum.

Animals↗
PubMed1983

Key role of dolichol phosphate in glycoprotein biosynthesis.

Studies on the developmental changes in oviducts of hormone-treated chicks and embryos of sea urchins have indicated that the level of dolichol phosphate in the tissues may serve as a control for asparagine-linked glycoprotein biosynthesis. Acute-phase reactant glycoprotein biosynthesis is greatly increased in inflamed rats given a single injection of turpentine. As most of the serum glycoproteins are synthesized via the dolichol pathway, the rate of synthesis of mannosyl and glucosyl dolichol monophosphate and of glucosyl dolichol pyrophosphoryl oligosaccharide, CTP-dependent dolichol phosphokinase, and the level of dolichol phosphate were measured in the livers of inflamed rats. The activities of the glycosyltransferases were increased at least twofold as a result of inflammation. It was also observed that dexamethasone treatment reversed the inflammation-induced increase of mannosyl and glucosyl transfer to dolichol monophosphate. The endogenous level of dolichol phosphate and dolichol kinase activity were increased in microsomes 24 h after inflammation. With exogenous dolichol added to the microsome assay, increased kinase activity was observed as early as 6 h after turpentine injection. The increase of dolichol phosphate in inflammation is most likely due to both greater availability of dolichol and an increase in the level of CTP-dependent dolichol kinase. Studies with purified subcellular fractions showed that dolichol kinase activity is primarily localized in the rough endoplasmic reticular fraction. Since this is the major site of dolichol-phosphate-linked N-glycosylation reactions, a key role of dolichol phosphokinase activity in rough microsomes to initiate the first steps of N-glycoprotein synthesis seems plausible.

Animals↗
PubMed1981

Effects of turpentine-induced inflammation on the synthesis of dolichol-linked intermediates of N-glycosylation and the phosphorylation of dolichol by CTP-dependent dolichol kinase.

Inflammation was induced in rats by the subcutaneous injection of turpentine. Microsomes were prepared from the livers between 2 and 72 h after injection. Mannose and glucose incorporation into mannosyl and glucosyl dolichyl monophosphate was increased 2-fold over saline-injected controls 24 h after induction of inflammation. Synthesis of glycosylated dolichyl pyrophosphoryl oligosaccharides was also increased compared to controls. Extraction and assay of dolichol monophosphate from inflamed and control rat liver microsomes indicated that the endogenous levels of the lipid were elevated in the inflamed state. CTP-dependent phosphorylation of endogenous dolichol was also found to increase in microsomes from inflamed rats 24 h after injection of turpentine. When exogenous dolichol was added to the microsomal system an increase in phosphorylation was observed as early as 6 h after turpentine injection. Furthermore, the increase appeared to be biphasic, there being two peaks of elevated activity at 12 and 36-48 h after induction of inflammation. The earlier peak was the greater of the two. The results suggest that the increase in glycosylation of dolichol derivatives was due to greater amounts of endogenous dolichol monophosphate. The increase in dolichol monophosphate was itself due to greater availability of dolichol and an increase in the levels of CTP-dependent dolichol kinase.

Animals↗
PubMed1979

The effects of triton X-100 on the transfer of mannose, glucose and n-acetylglusomine phosphate to dolichol monophosphate by preparations of rough and smooth endoplasmic reticulum and of mitochondria of rat liver.

Triton X-100 and exogenous dolichol monophosphate have been used to investigate the nature of enzymes responsible for the transfer of mannose, glucose and N-acetylglucosamine phosphate from nucleotide donors to dolichol monophosphate in vesicles derived from rough and smooth endoplasmic reticulum and mitochondria. Mitochondria were shown to contain the highest specific activities of these enzymes. The responses of the glycosyltransferases to increasing concentrations of Triton X-100 and the effect on these responses of exogenous dolichol monophosphate suggest that the enzymes for mannose and glucose transfer are less hydrophobic, and therefore less intrinsic, in the membrane than the enzyme for N-acetylglucosamine phosphate transfer. In smooth vesicles the results are consistent with mannosyl- and glucosyl-transferases being located at both inner and outer faces of the membrane. In rough vesicles and in mitochondria mannosyl- and glucosyl-transferases were confirmed at the outer face. There is, however, only one site of N-acetylglucosamine phosphate transfer, this being more hydrophobically located in the membrane than the other sites of glycosyl transfer. Mitochondrial enzyme activity closely resembled that of rough endoplasmic reticulum in response to Triton X-100 and exogenous dolichol monophosphate, and is probably associated with the outer membrane.

Acetylglucosamine↗
PubMed1977

Separation and properties of two arylamidases from rat cardiac-muscle extracts.

Two main arylamidase activities were separated from a particle-free supernatant of rat heart by chromatography on DEAE-Sephadex. Although both enzymes hydrolysed L-leucine 4-nitroanilide, only peak-II enzyme hydrolysed L-lysine 4-nitroanilide. A third minor peak (Ia) contained an enzyme that was active mainly on the L-lysine 4-nitroanilide. The mol.wts. of the enzymes in peaks I and II were approx. 257000 and 105000 respectively. The pH optimum was approx. pH7.0 for peak-I enzyme and 7.0-8.0 for peak-II enzyme. Both enzymes were inhibited by addition of puromycin, p-hydroxymercuribenzoate, o-phenanthroline and bivalent metal ions. Addition of dithiothreitol resulted in stimulation of both activities. Dialysis against o-phenanthroline resulted in inhibition of peak-I and -II enzymes, but after dialysis against EDTA only peak-II enzyme was inhibited.

Aminopeptidases↗