Chromatography Of Cell Extracts Research Articles

Characterization of glutamine transport in Streptococcus mutans.

Glutamine transport in glucose-energized cells of Streptococcus mutans Ingbritt exhibited Michaelis-Menten-type kinetics with a Vmax of 13.4 nmol/mg dry weight/min and a Kt of 4.1 microM. Diffusion of glutamine into de-energized cells of S. mutans displayed similar type kinetics, with a Kt of 6.8 microM but with a markedly reduced Vmax of 53.9 pmol/mg dry weight/min. Glutamine transport in S. mutans is not proton motive force-driven, as the intracellular accumulation of glutamine by energized cells far exceeded the thermodynamic limits of the proton motive force, and the dissipation of this proton motive force by gramicidin in a high K+ medium did not decrease the intracellular glutamine concentration. Glutamine transport is therefore likely to be energized by ATP hydrolysis. The activity of the transporter was maximal between pH 6.0 and 7.0 and decreased rapidly above pH 7.0. The transport of glutamine was not competitively inhibited by asparagine, glutamate or aspartate, indicating a specific glutamine transport system. Reversed-phase high-pressure liquid chromatography of cell extracts revealed that approximately 26% of the glutamine taken into the cell was converted to glutamate within 10 min. The results are consistent with transported glutamine being converted to glutamate and ammonia by the action of an intracellular glutaminase. Glutamine therefore may be an important source of nitrogen for the cell.

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line.

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with Kd of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with Kd 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with Kd 0.42 was identified as rat alpha-CGRP as was the peak with Kd 0.53. The peak with Kd 0.68 was identified as 19-37 rat alpha-CGRP and the peak with Kd 0.85 as 28-37 rat alpha-CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated alpha-CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat beta-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.

Glycogen synthase kinase-3 is rapidly inactivated in response to insulin and phosphorylates eukaryotic initiation factor eIF-2B

We have studied the control of insulin-regulated protein kinases in Chinese hamster ovary cells transfected with the human insulin receptor (CHO.T cells). Among these enzymes is one that is obtained after chromatography of cell extracts on Mono-S, whose activity is decreased (7.3 +/- 1.9-fold) within 10 min of insulin treatment. This enzyme phosphorylates glycogen synthase and the largest subunit of protein synthesis eukaryotic initiation factor (eIF)-2B (the guanine nucleotide exchange factor). The kinase appears to be glycogen synthase kinase-3 (GSK-3), on the basis of: (1) its ability to phosphorylate a peptide based on the phosphorylation sites for GSK-3 in glycogen synthase, and (2) the finding that the fractions possessing this activity contain immunoreactive GSK-3, whose peak is coincident with that of kinase activity, as judged by immunoblotting using antibodies specific for the alpha- and beta-isoforms of GSK-3. The decrease in kinase activity induced by insulin was reversed by treatment of the column fractions with protein phosphatase-2A. These data indicate that insulin rapidly causes inactivation of GSK-3 and that this is due to phosphorylation of GSK-3. The implications of these findings for the control of glycogen and protein metabolism are discussed.

The distribution and taxonomic value of fatty acids and eicosanoids in the Lipomycetaceae and Dipodascaceae.

Using radioimmunoassay, blood platelet aggregation studies and GC-MS the existence of prostaglandins in the endomycetalean yeast Dipodascopsis uninucleata was confirmed by our group. These findings triggered the search for similar eicosanoids in the rest of the Endomycetales. We commenced by scanning for the easily detectable precursors of eicosanoids, linoleic- and linolenic acid. We selected two families (i.e. Lipomycetaceae and Dipodascaceae), both producing these precursors, for further investigation. Representative strains of the two families were tested for their ability to grow in the presence of 1 mM aspirin, a specific inhibitor of prostaglandin biosynthesis. In contrast to the lipomycetaceous species the dipodascaceous species were insensitive to this drug. These results were verified when representative strains of both families were investigated for their ability to produce eicosanoids from externally fed radio-labeled arachidonic acid along an aspirin sensitive pathway. Thin layer chromatography of culture extracts, followed by autoradiography, showed that while none of the Dipodascaceae produced aspirin sensitive arachidonic acid metabolites, the members of the Lipomycetaceae tested positive for these metabolites. These findings supported the separation of the lipomycetaceous yeast Dipodascopsis from the Dipodascaceae. The findings also correlate with the delimitation of these yeasts in two families (i.e. Dipodascaceae and Lipomycetaceae). Further investigation indicated that prostaglandin production by the genus Dipodascopsis is mainly associated with ascosporogenesis. Thin layer chromatography of cell extracts from Dipodascopsis tóthii, followed by scintillation counting, indicated the presence of PGF2 alpha and PGE2 during ascosporogenesis.

Processing of pro-CGRP in a rat medullary thyroid carcinoma cell line transfected with protease inhibitors

A rat medullary thyroid carcinoma cell line, CA77, was used to study the effect of a series of biosynthesized protease inhibitors on the proteolytic cleavage of the endogenously synthesized pro-CGRP. This cell line efficiently converted the pro-CGRP to mature CGRP as assessed by chromatography of cell extracts followed by radioimmunoassay for CGRP. CA77 cells were transfected with expression vectors encoding protease inhibitors: the Arg-serpins, α 1-antitrypsin Pittsburgh (358 Met → Arg) and plasminogen activator inhibitor 1, the Kazal type serine protease inhibitor, pancreatic secretory trypsin inhibitor, and the general thiol protease inhibitor, cystatin C. Only the chromatography of cell extracts from CA77 cells transfected with a plasmid encoding cystatin C showed an apparent higher content of unprocessed pro-CGRP as compared to non-transfected cells. No effect on pro-CGRP processing could be measured in the CA77 cells transfected with plasmids encoding the three serine protease inhibitors. CA77 cells were also transfected with two constructs encoding chimeric proteins consisting of cystatin C and the precursor for neuropeptide Y. Release experiments using 8-bromo cAMP as the secretagogue showed that the chimer was co-released with CGRP. However, no effect of this chimer upon pro-CGRP processing could be detected. It is concluded that the processing of pro-CGRP in the CA77 cell line was very efficient and that four different protease inhibitors and two cystatin C/NPY chimers synthesized by this neuroendocrine cell line had only minimal effect upon the processing of CGRP.

Activation of casein kinase II in response to insulin and to epidermal growth factor.

Insulin treatment enhances casein kinase II (CKII) activity in 3T3-L1 mouse adipocytes and H4-IIE rat hepatoma cells, the magnitude of the activation varying from 30% to 150%. Activation of CKII was apparent after 5 min of exposure of 3T3-L1 cells to insulin, was maximal by 10 min, and persisted through 90 min. The insulin-stimulated activity was inhibited by low concentrations of heparin and was stimulated by spermine. Activation of CKII was effected by physiological concentrations of insulin (EC50 = 0.15 nM), suggesting that the effect is a true insulin response and not one mediated through insulin-like growth factor receptors. Epidermal growth factor (100 ng/ml for 10 min) also activated CKII in A431 human carcinoma cells, which is consistent with other observations that insulin and epidermal growth factor may have some common effects. Insulin stimulation of CKII activity was due to an increase in the maximal velocity of the kinase; the apparent Km for peptide substrate was not altered. Enhanced activity did not appear to result from increased synthesis of CKII protein, because cycloheximide did not block the effect and because an immunoblot developed with antiserum to CKII showed no effect of insulin on the cytosolic concentration of CKII. Because insulin-stimulated CKII activity was maintained after chromatography of cell extracts on Sephadex G-25, it is unlikely that the effect is mediated by a low-molecular-weight activator of the kinase. Rather, the results are consistent with the possibility that insulin activates CKII by promoting a covalent modification of the kinase.

Somatostatin production by a human medullary thyroid carcinoma cell line.

Somatostatin (SRIF, SRIF-14) is a known product of the normal and malignant parafollicular cell of the thyroid. In this report we characterize SRIF production by the TT cells, a line of transformed calcitonin-producing cells derived from a human medullary thyroid carcinoma. The cells were found to contain (5-12 ng/10(6) cells) and secrete (3-10 ng/10(6) cells X 48 h) immunoreactive SRIF. Molecular sieve chromatography of cell extracts under denaturing conditions showed a major peak with a mol wt slightly larger than 12,700, probably representing pro-SRIF and a second peak which coeluted with SRIF; in one gel chromatogram a very small peak was also noted which coeluted with SRIF-28, but represented less than 0.4% of the total immunoreactive SRIF. Short term secretion of calcitonin and SRIF was stimulated by calcium in vitro (0.5-4 mM) in a dose-related manner. mRNA isolated from the TT cells hybridized to a specific bovine fetal pancreatic SRIF DNA (BFPS-2); there was no hybridization to identical amounts of mRNA from the atT-20/D16, 3T3, or RINC5F cell lines. In vitro translation of the TT cell mRNA followed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the product revealed a single protein band of approximately 13,000 daltons. It was completely abolished when the immunoprecipitation was performed in the presence of excess unlabeled SRIF. Northern transfer of TT cell cytoplasmic RNA and hybridization with FBPS-2 cDNA showed a single hybridizing band with an apparent size of approximately 750 nucleotides. Our observations demonstrate the production of SRIF by a continuous line of human medullary thyroid carcinoma cells and provide a model for studying the biosynthesis and secretion of SRIF in the parafollicular cell.

Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12

Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme.

Uptake and aminoacylation of exogenous [formula omitted] tRNA by mouse fibroblasts

Mouse fibroblasts (L-cells) in suspension culture take up exogenous Escherichia coli tRNA in the presence of DEAE-Dextran. Tritium-labeled formylmethionine tRNA and valine tRNA are both taken up at very low levels. Tritium activity is associated solely with 4S material as judged by chromatography of cell extracts on Sephadex G-100. Further analysis of this material on a dihydroxyboryl-substituted cellulose indicates that a small portion of the tRNA taken up is acylated by the L-cells.

Terminal deoxynucleotidyl transferase in acute lymphoblastic leukemias and chronic T cell proliferations

Cells from patients with acute lymphoblastic leukemias (ALL) (28 cases), T-derived chronic lymphocytic leukemias (CLL) (6 cases), Sezary syndrome (3 cases) and thymomas (3 cases) were studied for both immunological membrane phenotype and terminal deoxynucleotidyl transferase (TdT) content (using chromatography of cell extracts on phosphocellulose). Cells from 25 cases of ALL contained TdT. T-derived ALL had relatively homogenous TdT values whereas a wider range of TdT activity was found in non-T non-B ALL. Cells from 3 ALL cases had no detectable enzyme; two were B-derived ALL, one T-derived ALL. Cells from the three thymomas and from one case each of Sezary syndrome and T-CLL had TdT activity. The significance of TdT in neoplastic disorders is discussed in the light of the known distribution of TdT in various subpopulations of normal mature T-cells and T-cell precursors.