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Biomedical subjects

I Boime

Publications and source records attributed to I Boime.

At least 127 records · Page 7Linked to original sources
PubMed1980

Isolation of mRNA from bovine pituitary. The cell-free synthesis of the alpha and beta subunits of luteinizing hormone.

RNA derived from bovine steer pituitary was translated in wheat germ cell-free extracts containing [35S]methionine. Antisera generated against purified denatured alpha and beta subunits of lutropin were used to demonstrate the synthesis of both proteins in vitro. The immunoprecipitated products of the cell-free system were resolved on sodium dodecyl sulfate/polyacrylamide gels and it was observed that the molecular weight of the immunoprecipitated alpha subunit protein was approximately 14,000, while that of the beta protein was estimated to be 16,000. Since the molecular weights of authentic alpha and beta subunits are 10,600 and 14,000 respectively, the cell-free products presumably represented their pre-protein forms. The ratio of the immunoprecipitated subunit pre-proteins was dependent on the magnesium concentration in the translation mixtures; at 2.1 mM, translation of lutropin alpha and beta mRNAs was comparable. RNA isolated from cow pituitary tissue directed the synthesis of fivefold less of the alpha and beta immunoprecipitated proteins than did steer RNA. Since the blood levels of gonadal steroids are higher in the cow, the results supported the hypothesis that lutropin alpha and beta mRNA biosynthesis is repressed by these steroids. The data also suggest that synthesis of lutropin alpha and beta subunits is coordinately expressed in certain physiological situations.

Animals↗
PubMed1980

Cytological localization of placental lactogen messenger ribonucleic acid in syncytiotrophoblast layers of human placenta.

Previous histochemical studies identified human placental lactogen (hPL) in the syncytial layer rather than in the cytotrophoblast cells of human placenta. However, these studies failed to rule out the possibility that de novo synthesis occurred in a cell type distinct from that in which the highest steady state level is accumulated after intercellular transport. To address this point, localization of placental mRNA in trophoblasts was attempted by the technique of in situ hybridizaton. Term placental sections were incubated with [3H]cDNA transcribed from sucrose gradient-purified hPL mRNA. Slides were stained, fixed for autoradiography, and examined under the light microscope. The syncytial layer was covered with silver grains. The amount of this radioactivity was greatly reduced when placental fragments were incubated with globin [3H]cDNA or if hPL [3H]cDNA was hybridized to sections of human tonsil tissue. In addition, the amount of silver grains was greatly reduced when the sections were prehybridized with labeled probe. These results suggest that hPL is synthesized in the syncytial layer of placental villi and that the appearance of hPL in this region was not the result of intercellular transport.

Female↗
PubMed1979

Processing in vitro of placental peptide hormones by smooth microsomes.

Rough and smooth microsomes were prepared from ascites tumor cells, rat liver, and bovine adrenal cortex. Proteolytic removal of the signal peptide in pre-placental lactogen and asparagine-linked glycosylation of the alpha subunit of chorionic gonadotropin by these fractions were examined in mRNA-dependent lysates from ascites cells. Both processing steps were performed by smooth microsomes, which was unexpected because it has been presumed that only rough microsomes contain components for ribosomal binding. Thus smooth microsomes are apparently capable of interacting with polysomes bearing secretory nascent chains, and cleavage and asparagine-linked glycosylation activities are present in both rough and smooth endoplasmic reticulum.

Adrenal Cortex↗
PubMed1979

Glycosylation of human chorionic gonadotropin in mRNA-dependent cell-free extracts: post-translational processing of an asparagine-linked mannose-rich oligosaccharide.

Placental RNA has previously been shown to direct the synthesis of an asparagine-linked mannose-rich glycosylated form of the alpha subunit of human chorionic gonadotropin (hCG-alpha) in lysates derived from mouse ascites tumor cells. Glycosylation was dependent on the presence of homologous microsomal membranes, and the glycosylated protein was sequestered into the microsomal vesicles. Here we show that when Triton X-100 is added after 60 min of translation and the incubation is continued, there is a shift of this glycosylated form to new discrete lower molecular weight proteins. The formation of these new proteins was not the apparent result of proteolysis because (i) treatment of the fully glycosylated protein or the proteins formed in the presence of Triton with endoglycosidase H resulted in the formation of a single protein migrating at the same rate on sodium dodecyl sulfate gels; (ii) the migration of nonglycosylated hCG-alpha synthesized in the presence of membranes isolated from tunicamycin-pretreated ascites tumor cells was not changed upon Triton addition; and (iii) the Triton-induced change was inhibited by mannonolactone, yeast mannan, and purified mannose oligosaccharides. It was also shown that little processing of the mannose-rich glycoprotein occurred in the presence of microsomal membranes alone. However, addition of the ribosome-free supernatant fraction to the glycoprotein resulted in processing. These data suggest that processing of the oligosaccharide core is a compartmentalized process in which removal of sugar, presumably mannose, requires a transfer of the glycoprotein from the endoplasmic reticulum to another component of the secretory cascade.

Animals↗
PubMed1979

Characterization of an endopeptidase involved in pre-protein processing.

Proteolytic removal of the pre-segment from growing nascent chains of pre-human placental lactogen (hPL) occurred during in vitro translation of placental mRNA if crude membranes derived from ascites lysates, dog pancreas, or rat liver rough endoplasmic reticulum were added to the translation mixtures. The cotranslational proteolytic event was inhibited by the peptide protease inhibitor, chymostatin, but not by leupeptin, antipain, or elastatinal. The proteases involved in cleavage were solubilized with detergent and converted completed pre-hPL to hPL (post-translational processing). Direct assay of the solubilized membranes, with synthetic fluorogenic aminocoumarin peptide substrates, revealed no significant tryptic or elastase-like activity, but activity against a chymotrypsin substrate [(succinyl-Ala-Ala-Phe)-7-amino-4-methyl-coumarin] was found. This activity was dependent upon both an endopeptidase and an aminopeptidase. Although bestatin inhibited the aminopeptidase activity, it had no effect on the endopeptidase or on post-translational cleavage. Although this endopeptidase cleaved on the COOH side of an alanine residue, it was not inhibited by elastatinal. However, it was inhibited by high levels of chymostatin and by some serine protease inhibitors.

Animals↗
PubMed1978

Processing of placental peptide hormones synthesized in lysates containing membranes derived from tunicamycin-treated ascites tumor cells.

To examine the relationship between pre-protein cleavage and nascent chain glycosylation placental mRNA was translated in a reconstituted ascites cell-free system containing microsomal membranes prepared from tunicamycin-treated or untreated ascites tumor cells. In the absence of membranes, first trimester RNA directed the synthesis of the pre-form of the alpha subunit of human chorionic gonadotropin, whereas, in the presence of normal membranes, first trimester RNA directed the synthesis of a glycosylated form of the alpha subunit. Cell-free lysates containing membranes derived from tunicamycin-treated cells synthesized an alpha subunit protein with little, if any, carbohydrate. This protein was apparently sequestered into membranes since it was resistant to the action of trypsin which was added after translation. The pre-peptide of the alpha subunit protein was removed by treated membranes as determined by amino acid sequence analyses. The non-glycosylated protein pre-placental lactogen was also cleaved to its mature form by tunicamycin membranes. These data strongly suggest that, in vitro, glycosylation is not obligatory for pre-protein cleavage and sequestration of these placental protein hormones.

Animals↗
PubMed1978

Conversion of rat pre-proalbumin to proalbumin in vitro by ascites membranes. Demonstration by NH2-TERMINAL SEQUENCE ANALYSIS.

Rat liver poly(A)-containing RNA was translated in an ascites cell-free system. Labeled protein precipitable by antibody directed against rat serum albumin was identified as pre-proalbumin based on its size and partial NH2-terminal sequence. However, when an ascites membrane fraction was added to the translation reaction, the albumin antibody-precipitable material was smaller than pre-proalbumin. Partial NH2-terminal sequence analysis of this protein revealed that it was proalbumin. Conversion of pre-proalbumin to proalbumin by the ascites membrane fraction was complete and precise--i.e. no serum albumin was observed. Reconstitution in vitro of the processing of pre-proalbumin to its stable intracellular form, proalbumin, provides a method for studying the initial proteloytic event involved in secretion of rat serum albumin.

Amino Acid Sequence↗
PubMed1978

Isolation and partial purification of catfish pancreatic islet messenger RNA.

Poly(a)-rich mRNA has been isolated from catfish pancreatic islet total nucleic acid. Cell-free translation of the mRNA by wheat germ extracts yielded a protein of 11 000-12 000 molecular weight, estimated by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. This peptide is larger than catfish proinsulin, but contains tryptic peptides of proinsulin. Its synthesis comprises up to 23% of the cell-free product, depending on the conditions of cell-free synthesis. Synthesis is inhibited by 7-methylguanosine 5'-monophosphate suggesting the presence of a 7-methylguanosine cap on the 5' end of catfish proinsulin mRNA. Sucrose gradient centrifugation of the islet poly(A)-rich mRNA yielded 8S and 12S peaks. These fractions were translated with wheat germ extracts and it was determined that over 60% of the islet mRNA-dependent protein from the 8S fraction was preproinsulin. The 8S mRNA fraction was electrophoresed on 3% agarose-6 M urea gels and demonstrated to be several bands, ranging from 100 000-200 000 molecular weight.

Animals↗
PubMed1978

mRNA-dependent synthesis of a glycosylated subunit of human chorionic gonadotropin in cell-free extracts derived from ascites tumor cells.

Protein synthesized in ascites cell-free extracts in response to first trimester placental mRNA was immunoprecipitated with antisera directed against the alpha subunit of human chorionic gonadotropin. The immunoprecipitable proteins were resolved on sodium dodecyl sulfate/polyacrylamide slab gels. In membrane-depleted extracts placental mRNA directed the synthesis of the "preprotein" form of the alpha subunit. However, when membranes were added to the cell-free extracts a protein migrating more slowly than pre-alpha subunit was observed. This protein was specifically adsorbed to a concanavalin A column, and its migration on sodium dodecyl sulfate gels was enhanced after treatment with alpha-mannosidase (EC 3.2.1.24) or endo-beta-N-acetylglucosaminidase C(II) or H. Similar results were obtained when mRNA was translated in lysates derived from first trimester placenta instead of in ascites cell extracts. Kinetic studies of the glycosylation reaction revealed that sugar attachment can occur just prior to release of the protein. These data show that the apoprotein of the alpha subunit can be glycosylated in vitro. The data also suggest that the glycosylated protein contains a sugar core consisting of di-N-acetylchitobiose and at least four mannose residues, some of which are alpha-linked. In addition, it appears that this carbohydrate unit is present in homologous placental membranes as well as in the ascites tumor membranes.

Acetylglucosamine↗
PubMed1978

Cell-free translation of messenger RNA extracted from a human insulinoma.

Total nucleic acid has been extracted from a human pancreatic insulinoma. Purification of the messenger RNA (mRNA) fraction by oligo-dT cellulose chromatography yielded 200 micrograms poly(A)-rich mRNA. This mRNA produced a 2-fold stimulation of protein synthesis in a wheat germ cell-free system. Analysis of the translation products by gel filtration chromatography (Biogel P-30) revealed nothing smaller than an acid-alcohol-soluble protein larger than bovine proinsulin. In contrast, insulinoma slices incubated with labeled amino acids synthesized smaller proteins which comigrated with bovine proinsulin and insulin. [3H]Leucine-labeled cell-free proteins were electrophoresed on NaDodSO4-urea polyacrylamide slab gels. In the presence of insulinoma mRNA, discrete proteins of 25,000 and 11,500 mol wt were synthesized. The 11,500 mol wt protein was specifically immunoprecipitated with antiinsulin serum. Thus, cell-free translation of human insulinoma mRNA yields an immunoreactive insulin larger than proinsulin, which is the same size as fish and rat preproinsulins recently described.

Adenoma, Islet Cell↗
PubMed1977

Membrane-dependent cleavage of the human placental lactogen precursor to its native form in ascites cell-free extracts.

Messenger RNA derived from term placenta directs the synthesis of a precursor to human placental lactogen (prelactogen, molecular weight 25000) in an ascites cell-free system containing ribosome free supernatant and preincubated purified ribosomes. The processing of prelactogen to native lactogen (molecular weight 22200) was only observed when a microsomal membrane preparation was added prior to the synthesis of complete protein, i.e. before release. Placental mRNA directed the synthesis of prelactogen in asystem containing free polysomes, whereas in a comparable system containing membrane-bound polysomes both prelactogen and lactogen were synthesized. The prelactogen synthesized in the latter system could be cleaved by the addition of membranes at the start of incubation. Preprotein cleavage activity was inhibited 100% by 0.04% Tritonx-100, while protein synthesis was inhibited only about 30%. Using Triton to block cleavage specifically at intervals after mRNA and membrane additions, it was determined that the overall cleavage reaction required about 15 min. When the ascites system was incubated with charged initiator [35S]methionyl-tRNAfMet prelactogen was formed. The labeled prelactogen was processed when membranes were added after 60 min of incubation. The results indicate that prelactogen is the primary gene product, and cleavage activity is apparently associated only with the membrane-bound ribosomal fraction of the cell.

Animals↗