Synthesis of the glycoprotein hormone alpha subunit and placental alkaline phosphatase by HeLa cells: effect of tunicamycin, 2-deoxyglucose, and sodium butyrate.
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Biomedical subjects
Publications and source records attributed to G S Cox.
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Differences in scores assigned by five speech-language pathologists were determined for 96 language samples utilizing assessments of total words, total sentences, words per sentence, syntax quotient (usage and endings), syntax quotient (structures), and an abstract-concrete scale. Results of the study showed statistically significant interscorer differences for words per sentence, syntax quotient (usage and endings), and the abstract-concrete scale. Although the mean differences between scorers for total words, total sentences, and syntax quotient (structures) were not statistically significant, substantial absolute differences between scorers were found on all six measurement procedures.
The glycoprotein hormone alpha subunit secreted by HeLa cells was retained by concanavalin A:Sepharose and by ricin:agarose, indicating, that the tumor protein has carbohydrate side chains containing both mannose and galactose residues. Lectin chromatography of the intracellular hormone suggests it is probably a precursor to the secreted protein, it was bound by concanavalin A but not by ricin, suggesting the presence of a high mannose core oligosaccharide but the absence of terminal sugar residues. The glycosylation inhibitors tunicamycin and 2-deoxy-D-glucose caused a reduction in alpha subunit secretion comparable to their reduction of general protein synthesis but considerably less than their inhibition of protein glycosylation. Various HeLa lines secreted alpha subunit at widely different rates, with HeLa CCL 2.2 having the highest rate of production, HeLa CCL 2.1 having the lowest, and HeLa CCL 2 being intermediate. Dose-response curves for alpha subunit from the different HeLa lines and from tunicamycin- and deoxyglucose-treated cells were sufficiently parallel to indicate similar immunological characteristics. The incorporation of radiolabeled glucosamine and N-acetylmannosamine into secreted proteins varied among the cell lines examined and was generally comparable to their hormone production rates. Concanavalin A:Sepharose chromatography of the alpha subunit secreted by HeLa CCL 2.2 and CCL 2.1 indicated that both proteins possess oligosaccharide side chains containing mannose while chromatography of these proteins on ricin:agarose suggested that less of the alpha subunit from CCL 2.1 contains galactose than that from CCL 2.2.
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In order to understand better the regulation of human placental proteins the activity of placental lactogen messenger RNA has been examined. Total RNA was extracted from normal term placentas and purified by chromatography on oligo(dT)-cellulose. The poly(A)-containing fraction stimulated amino acid incorporation 5- to 10-fold in wheat germ cell-free extracts, and immunoprecipitation of the translation products with antiserum directed against human placental lactogen (hPL) suggests that about 2% of the peptides contain hPL determinants. Analysis of the material precipitated with hPL antiserum by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed two major species, one co-migrating with hPL and the other migrating slightly slower than hPL. On DEAE-cellulose chromatography the former material eluted close to authentic hPL while the latter material eluted at higher ionic strength than hPL, indicating a difference in net charge of these two species. Tryptic peptide analysis of the large material and authentic hPL shows marked similarities in the primary structure of these two proteins. The slower migrating peptide has an apparent molecular weight about 3000 larger than hPL and thus may represent a precursor molecule. Both cell-free products could be competed out of immunoprecipitates by a large excess of authentic hPL, confirming their immunologic similarities. Centrifugation of the placental poly(A)-containing RNA through aqueous glycerol gradients indicates that the hPL mRNA sediments at about 14 S.
The synthesis and template properties of T4 vegetative DNA were studied. The DNA-containing material in lysates of cells taken 20 min past T4 infection sediments in sucrose gradients as two major components. Both fractions function as templates for amino acid incorporation in a DNA-dependent in vitro system (coupled transcription-translation). The slower sedimenting activity is not present in uninfected cells and appears in wild type T4-infected cells only after 12 min at 30 degrees, shortly after DNA synthesis starts. It is dependent for its activity on an added S-30 fraction from either uninfected or T4-infected cells and is completely inhibited by deoxyribonuclease or rifampin. On a weight basis the slower sedimenting template is about 30 to 70% as active as mature T4 DNA when supplemented with S-30 extracts from uninfected cells. The spectrum of proteins synthesized in response to the slower sedimenting template is different from that produced in response to mature T4 DNA. In contrast to mature DNA, this template is capable of directing the synthesis of material that precipitates with antiserum directed against whole T4 particles. Thus, it appears capable of directing the synthesis of mRNA for phage structural proteins, i.e. late proteins. The faster sedimenting component is about 8-fold less active for stimulating amino acid incorporation than mature DNA. Significant amounts of RNA polymerase are associated with this DNA in active transcription complexes, yet polyacrylamide gel electrophoresis of the proteins synthesized in response to this fraction show a pattern that resembles the early proteins made from mature T4 DNA in extracts from uninfected cells.
The DNA in gently lysates of T4-infected Escherichia coli cells sediments in sucrose gradients as two major components; the slower sedimenting component is designated as the S-5 fraction and the faster sedimenting component as the pad fraction. The distribution of these fractions in lysates of cells infected with T4 maturation-defective and DNA-arrest mutants was determined, and their template activities were compared in a DNA-dependent amino acid-incorporating system. The S-5 DNA template was found to be completely absent in E. coli B cells infected with a T4 maturation-defective mutant (gene 55). On the other hand, DNA sedimenting as the S-5 component is greatly increased, while that sedimenting as the pad component is virtually absent in nonpermissive cells infected with a DNA-arrest mutant (gene 46). The S-5 fractions prepared from cells infected with a DNA ligase mutant (gene 30) and a gene 30 gene 46 double mutant are reduced in their ability to stimulate amino acid incorporation compared to similar preparations from cells infected with wild type T4 or a gene 46 mutant. Moreover, the template activity of partially purified replicative DNA prepared from cells infected with phage-carrying mutations either on gene 30 or in both genes 46 and 56 (dCTPase) is lower than that of DNA obtained from cells infected with wild type phage. The polypeptide products of reaction mixtures programmed with several of the mutant DNAs were found to be qualitatively different from polypeptides synthesized in response to either mature DNA or replicative DNA prepared from cells infected with wild type phage. These data suggest that the expression of phage DNA may be significantly influenced by physical changes in the DNA arising from abnormal replication.
The transport of alpha-methylglucoside and a variety of amino acids including histidine, leucine, lysine, proline, and threonine are reduced 5- to 50-fold in Escherichia coli K1061, an unsaturated fatty acid auxotroph, when cells are grown in medium containing glucose as a carbon source and supplemented with either cis-vaccenic acid or cis-9, 10-methylenehexadecanoic acid. Cells grown in glucose medium containing oleate, palmitoleate, or cis-7-hexadecenoate do not exhibit this effect and transport alpha-methylglucoside and amino acids at rates comparable to those of a wild type strain. For cis-vaccenate- and cis-9, 10-methylenehexadecanoate-grown cells the capacity to transport amino acids and alpha-methylglucoside is normal when glycerol or succinate is the energy supply or when cyclic adenosine 3':5'-monophosphate is added to cultures growing on glucose. These results suggest that in this strain several transport systems are affected by catabolite repression, but the phenomenon is unique since repression requires not only glucose but specific unsaturated fatty acids, cis-vaccenic acid, or cis-9, 20-methylenehexadecanoic acid.
The uptake into isolated HeLa nuclei of radioactive cytosol proteins and purified Escherichia coli ribosomal protein L7 is stimulated up to 4-fold by pancreatic deoxyribonuclease (DNase I). Similar effects are not observed with pancreatic ribonuclease A or phospholipase C. The results reported suggest that there is a general stimulatory effect of DNase on protein uptake by nuclei.
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The vegetative DNA isolated from T4-infected Escherichia coli W4597 (UDPG PPase(-)) was about two to six times more active in stimulating protein synthesis in cell-free extracts than that isolated from T4-infected E. coli B06. This suggested that nonglucosylated vegetative DNA may be a better template than the glucosylated form. This view was supported by experiments measuring RNA synthesis on mature T-even DNAs with a range of glucose contents. The extent of (14)C-GTP polymerization was inversely proportional to the glucose content of the DNA. Differences were also observed in both the kind and quantity of polypeptides produced in response to these DNAs.