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G C Shore

Publications and source records attributed to G C Shore.

84 records · Page 5Linked to original sources
PubMed1981

Biogenesis of the mitochondrial enzyme, carbamyl phosphate synthetase. Appearance during fetal development of rat liver an rapid repression in freshly dispersed hepatocytes.

Synthesis of carbamyl phosphate synthetase was undetectable in fetal rat liver at 16 days gestation but by 4-5 days after birth (11-12 days later), this single protein accounted for approx. 5% of total liver protein and roughly 1% of total liver protein synthesis. Likewise, translatable mRNA coding for the enzyme was absent from 16-day fetal livers but then rapidly accumulated reaching maximum levels just after birth. The in vitro primary translation product of carbamyl phosphate synthetase mRNA corresponded to a higher molecular weight biosynthetic precursor of the enzyme; peptide maps obtained from the precursor synthesized both in vivo and in vitro and from the mature enzyme made in vivo were the same. When livers of neonatal rats were perfused with collagenase and further treated to yield a preparation of freshly dispersed hepatocytes highly active in general protein synthesis, a procedure which took about 45 min to complete, biosynthesis of carbamyl phosphate synthetase was found to be completely absent in these cells. The mRNA coding for the enzyme, however, could be extracted from the dispersed hepatocytes and was actively translatable in vitro, at levels approximately 75% of those for mRNA obtained from intact liver. Repression of biogenesis of carbamyl phosphate synthetase in dispersed hepatocytes, therefore, must involve a mechanism which shifts the mRNA coding for the enzyme out of active polysomal complexes and renders it further untranslatable in vivo but not in vitro.

Animals↗
PubMed1981

Biogenesis of a 35-kilodalton protein associated with outer mitochondrial membrane in rat liver.

Biochemical analyses following subcellular fractionation of rat liver indicated that the outer mitochondrial membrane contains a number of membrane-specific proteins of which a 35-kilodalton species (OMM-35) is a prominent component. These results were confirmed and extended by electron microscope immunocytochemical tests based on the protein A-gold technique. OMM-35 is tightly bound to the outer mitochondrial membrane, e.g. it was not released by sonication in the presence of 1.5 M KCl and 0.1% sodium deoxycholate. However, it did not react with the photoaffinity probe azidopyrene, which indicates that OM-35 is located peripherally on the membrane rather than buried deep in the lipid bilayer as an intrinsic protein. Since low levels of detergent were required for OMM-35 in intact mitochondria to react with exogenous antibodies, OMM-35 is probably located on the side of the outer membrane which faces the interior of the organelle. When rat liver mRNA was translated in a messenger-dependent cell-free system derived from rabbit reticulocytes, antiserum against OMM-35 precipitated a single polypeptide product which migrated on sodium dodecyl sulfate-polyacrylamide gels with molecular weight characteristics of a protein slightly larger (by Mr = 500) than OMM-35 obtained from isolated outer mitochondrial membrane. The mRNA coding for OMM-35 was recovered exclusively from membrane-free polysomes. Thus, the route followed for synthesis and subsequent insertion of OMM-35 into the outer membrane of mitochondria is the post-translational pathway which has been previously described for proteins destined for the interior compartments of this organelle.

Animals↗
PubMed1981

Chloramphenicol inhibits hormone-dependent induction of cytoplasmic mRNA coding for the mitochondrial enzyme, carbamyl phosphate synthetase, in Rana catesbeiana tadpoles.

In tadpoles injected with thyroxine (T4), synthesis of carbamyl phosphate synthetase 1 is induced so that within 7-8 days this single polypeptide represents one of the most abundant proteins in the liver. Translational assays in vitro showed that liver RNA from control animals had very low levels of translatable mRNA coding for the enzyme whereas carbamyl phosphate synthetase mRNA activity was prominent in liver from tadpoles which had been treated with T4 for just 2 days. When the primary translation product of carbamyl phosphate synthetase mRNA was immunoprecipitated from a messenger-dependent rabbit reticulocyte cell-free system programmed with total liver mRNA, it migrated on SDS-polyacrylamide gels more slowly than the in vivo form of the enzyme and otherwise demonstrated characteristics which were very similar to the precursor for carbamyl phosphate synthetase previously described in rat liver. If tadpoles were treated for 2 days with T4 plus an inhibitor of mitochondrial protein synthesis, chloramphenicol, T4-dependent induction of both enzyme synthesis and translatable carbamyl phosphate synthetase mRNA activity were repressed by 45-65%. The two measurements, synthesis in vivo and mRNA activity in vitro, were made on the same liver and correlated closely in their response to chloramphenicol. The data suggest that a product of mitochondrial protein synthesis may be involved in mediating hormonal regulation of the nuclear gene coding for carbamyl phosphate synthetase.

Animals↗
PubMed1981

Processing of the precursor for the mitochondrial enzyme, carbamyl phosphate synthetase. Inhibition by rho-aminobenzamidine leads to very rapid degradation (clearing) of the precursor.

Pulse-chase experiments using liver explants incubated in modified Eagle's medium showed that newly synthesized precursor for carbamyl phosphate synthetase (pCPS) passes very rapidly through the cytosolic compartment en route to mitochondria (t 1/2 is approximately 2 min). Since even a small pool of precursor could not be detected in association with mitochondria, processing of pCPS must occur either coincident with or immediately following its transmembrane uptake by the organelle. Treatment of explants with the protease inhibitor, p-aminobenzamidine, however, inhibited normal processing of the precursor. But, rather than accumulate in the cell, newly synthesized pCPS was nonspecifically degraded with kinetics (t 1/2 of 2-3 min) which are consistent with the idea that the precursor was almost instantly degraded upon reaching the blocked processing enzyme in a mitochondrion. The protease inhibitor had little or no effect on synthesis of pCPS. This was determined by isolating polysomes aminobenzamidine; the two polysome preparations were about equally active in synthesizing pCPS in vitro in the presence of an inhibitor of polypeptide chain initiation, aurin tricarboxylic acid.

Amidines↗
PubMed1979

In vitro synthesis of a putative precursor to the mitochondrial enzyme, carbamyl phosphate synthetase.

A simple and rapid procedure is described for purification of carbamyl phosphate synthetase from the matrix fraction of rat liver mitochondria. Antibodies to the enzyme were raised in sheep and purified from antiserum by affinity chromatography on enzyme-bound Sepharose columns. When membrane-free polyribosomes, isolated from a cytosolic fraction of rat liver, were incubated in a messenger-dependent rabbit reticulocyte protein-synthesizing system in the presence of [35S]methionine, the purified antibody precipitated a product of translation representing 0.2% of total trichloroacetic acid-insoluble radioactivity. It demonstrated mobility characteristics in sodium dodecyl sulfate-polyacrylamide gels expected for a polypeptide of molecular mass approximately 5500 daltons larger than the mature mitochondrial form of the enzyme (160,000 daltons). Proteolysis of both the mature and presumptive in vitro precursor forms of the enzyme yielded respective sets of peptide fragments which gave similar patterns upon gel electrophoresis. Excess mitochondrial enzyme effectively competed with the in vitro product for interaction with anti-carbamyl phosphate synthetase antibody.

Animals↗
PubMed1979

Synthesis of intracellular membrane proteins in vitro. Relation between rough endoplasmic reticulum and mitochondrial outer membrane.

Hepatic rough microsomes were incubated in a messenger-dependent protein-synthesizing system from rabbit reticulocytes. Up to 30% of the total product labelled with [35S]methionine, and subsequently recovered with the microsomes, was located in an intrinsic protein fraction associated with these membranes, i.e. was retained by the membrane following extensive sonication in the presence of 1.5 M KCl, 0.1% deoxycholate, and 5 mM ethylenediaminetetra-acetate (EDTA). When products synthesized with the use of membrane-free mRNA from rough microsomes and free polysome were post-incubated with rough microsomes, ribosome-stripped rough microsomes, or outer mitochondrial membrane, low amounts of intrinsic-type polypeptide product were recovered with these membranes. Higher recovery was achieved, however, when ribosome-stripped rough microsomes were added at the beginning of polypeptide synthesis in a reticulocyte lysate supplemented with additional ribosomal-wash factors. Analysis of these products by polyacrylamide gel electrophoresis showed that a number co-migrated with intrinsic proteins located in both rough microsomes and mitochondrial outer membrane. In addition, a prominent in vitro product co-migrated with a major protein which is located in outer mitochondrial membrane fractions, but is barely detectable in rough microsomal fractions. The present experiments were unable to detect a unique set of intrinsic polypeptides which were synthesized and assembled in vitro under the direction of mRNA from free polysomes, and not from rough microsomes. The results suggest that synthesis of at least some intrinsic membrane proteins which are destined for the outer mitochondrial membrane occurs on rough ER in rat liver.

Animals↗
PubMed1977

Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria.

Low-speed centrifugation (640 g) of rat liver homogenates, prepared with a standard ionic medium, yielded a pellet from which a rapidly sedimenting fraction of rough endoplasmic reticulum (RSER) was recovered free of nuclei. This fraction contained 20-25% of cellular RNA and approximately 30% of total glucose-6-phosphatase (ER marker) activity. A major portion of total cytochrome c oxidase (mitochondrial marker) activity was also recovered in this fraction, with the remainder sedimenting between 640 and 6,000 g. Evidence is provided which indicates that RSER may be intimately associated with mitochondria. Complete dissociation of ER from mitochondria in the RSER fraction required very harsh conditions. Sucrose density gradient centrifugation analysis revealed that 95% dissociation could be achieved when the RSER fraction was first resuspended in buffer containing 500 mM KCl and 20 mM EDTA, and subjected to shearing. Excluding KCl, EDTA, or shearing from the procedure resulted in incomplete separation. Both electron microscopy and marker enzyme analysis of mitochondria purified by this procedure indicated that some structural damage and leakage of proteins from matrix and intermembrane compartments had occurred. Nevertheless, when mitochondria from RSER and postnuclear 6,000-g pellet fractions were purified in this way fromanimals injected with [35S]methionine +/- cycloheximide, mitochondria from the postnuclear 6,000-g pellet were found to incorporate approximately two times more cytoplasmically synthesized radioactive protein per milligram mitochondrial protein (or per unit cytochrome c oxidase activity) than did mitochondria from the RSER fraction. Mitochondria-RSER associations, therefore, do not appear to facilitate enhanced incorporation of mitochondrial proteins which are newly synthesized in the cytoplasm.

Animals↗
PubMed1977

Two fractions of rough endoplasmic reticulum from rat liver. II. Cytoplasmic messenger RNA's which code for albumin and mitochondrial proteins are distributed differently between the two fractions.

Subcellular fractions were obtained from rat liver homogenates under conditions which prevented degradation of polysomes (pH 8.5 and high ionic strength). Rough endoplasmic reticulum (RER) was recovered in high yields from a low-speed nuclear pellet (rapidly sedimenting endoplasmic reticulum, RSER) and from a postmitochondrial supernate (rough microsomes). The polysomal RNA content of these two fractions was very similar. When polyA+-RNA's were translated inthe mRNA-dependent wheat embryo cell-free system, both fractions yielded polypeptide products which had similar electrophoretic patterns on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Activities of messenger RNA's which code for albumin and for polypeptides destined for transport to the inner membrane and matrix of mitochondria (i.e. 'mitoplasts') were assayed by translating in the more active rabbit reticulocyte cell-free system followed by immunoprecipitation of radioactive products and coelectrophoresis with immunoprecipitated marker proteins on SDS-polyacrylamide gels. These tests indicated that albumin mRNA is about equally distributed between the two fractions of RER, or slightly enriched in the RSER fraction when activity is expressed as a percentage of total polypeptide synthesis. Activities of cytoplasmic mRNA's which code for at least some mitoplast proteins could be detected in both fractions, but all were enriched in the rough microsome fraction, not the RSER (two- to threefold when corrected for differences in total polypeptide synthesis in the lysate). Comparisons of mRNA's from free vs. membrane-bound polysomes indicated that most of the albumin mRNA activity (86-91%) and mitoplast protein mRNA activities (75%) were present in the bound fraction. Assuming that RSER and rough microsomes do not derive exclusively from different cells types, the evidence suggests that, compared to albumin and most other membrane-bound mRNA's, cytoplasmic mRNA's coding for mitoplast proteins may be preferentially segregated or compartmentalized within the cell on the microsomal class of RER.

Albumins↗