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P P Cohen

Publications and source records attributed to P P Cohen.

At least 19 recordsLinked to original sources
PubMed1981

Synthesis, intracellular transport, and processing of the precursors for mitochondrial ornithine transcarbamylase and carbamoyl-phosphate synthetase I in isolated hepatocytes.

The synthesis and intracellular transport of the mitochondrial matrix enzymes ornithine transcarbamylase (carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3.) and carbamoyl-phosphate synthetase (ammonia) I [carbon-dioxide:ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] were studied in isolated rat hepatocytes. In pulse experiments at 37 degrees C, the larger precursors of the two enzymes appeared in the cytosol of the liver cells, where radioactivity levels of the precursors reached a plateau in 10-20 min after the pulse. The pulse-labeled mature enzymes appeared in the particulate fraction (containing mitochondria) after a time lag and increased almost linearly with time up to 40 min. The specific radioactivities of the precursors in the cytosol were much higher than those of the mature enzymes in the particulate fraction. In pulse--chase experiments, the labeled precursors disappeared from the cytosol with estimated half-lives of about 1-2 min. These results indicate that ornithine transcarbamylase and carbamoyl-phosphate synthetase I are initially synthesized as larger precursors and exist in a cytosolic pool from which they are transported into mitochondria and processed there to the mature enzymes concomitantly with or immediately after transport. Although the rates of synthesis, transport, and processing were decreased about 3-fold at 25 degrees C (as compared to incubation at 37 degrees C), the pool size of the precursors in the cytosol were somewhat larger at this temperature.

Animals↗
PubMed1980

Ornithine transcarbamylases. Ordering of S-cyano peptides and location of characteristically reactive cysteinyl residues within the sequence.

The peptides produced by cleavage of the S-cyano derivatives of the ornithine transcarbamylases (ornithine carbamoyltransferase, 2.1.3.3) from bovine liver, Streptococcus faecalis ATCC 11,420, and Streptococcus faecium ATCC 8043 have been isolated and aligned. Their amino acid compositions and COOH-terminal sequences are reported. The COOH-terminal peptide (51 residues in the bovine enzyme; 66 residues in the bacterial enzymes) contains the essential sulfhydryl group.

Amino Acid Sequence↗
PubMed1980

The essential sulfhydryl group of ornithine transcarbamylases. Reaction with anionic, aromatic disulfides and properties of its cyano derivative.

The essential sulfhydryl group of the ornithine transcarbamylases (ornithine carbamoyltrasferase, 2.1.3.3) from bovine liver and Streptococcus faecalis reacts slowly with aromatic disulfides at alkaline pH. But at pH 4.5, the apparent second-order rate constant for the reaction of this group in the S. faecalis enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) (Nbs2) is 40-fold that for the reaction of 2-mercaptoethanol. This enhanced reactivity at acid pH, because it occurs only with anionic, aromatic disulfides and results in rates greater than those for low molecular weight thiols, must be due to a specific interaction with these reagents. The Nbs derivatives of both enzymes are inactive; the cyano derivatives prepared from them by cyanolysis are active but with greatly increased Kmorn. The slow rates of cyanolysis and thiolysis suggest that access to the Nbs residue is limited. Also, the red shift of the spectrum of the Nbs residue in the enzymes from that of Nbs2 implies that its microenvironment is different from that of the bulk medium. Deprotonation of a residue in the S. faecalis enzyme causes a further red shift. Since even the small, uncharged cyano group interferes with the binding of ornithine to both enzymes, the essential sulfhydryl group may actually be a part of the binding site for ornithine.

Animals↗
PubMed1980

The essential sulfhydryl group of ornithine transcarbamylases. pH dependence of the spectra of its 2-mercuri-4-nitrophenol derivative.

The essential sulfhydryl group of the ornithine transcarbamylases (ornithine carbamoyltransferase, 2.1.3.3) from bovine liver and Streptococcus faecalis reacts preferentially with 2-chloromercuri-4-nitrophenol. The spectra of this derivative between pH 4.4 AND 8.8 HAVE BEEN RESOLVED INto the spectrum of the nitrophenolate ion (III) and two species of phenol (I and II). The lambda max of I and II (both enzymes) and III (bovine) are red shifted from those of the comparable species in the same derivative of 2-mercaptoethanol. Deprotonation of a residue on the enzyme must be responsible for the transition from I to II. The pK values of the phenolic group are 7.1 (mercaptoethanol), 7.7 (bovine), and 8.8 (S. faecalis). The red shift in the lambda max of III and the modest increase in the pK of the phenolic group are consistent with a relatively hydrophobic environment for the nitrophenolate ion in the bovine enzyme. Since deprotonation of the residue in the bovine enzyme perturbs the pK of the phenolic group only slightly, its effect may be indirect. Interaction with a neighboring carboxyl group (pK 5.3) would account for the large increase in the pK of the phenolic group in the S. faecalis enzyme, which is not accompanied by an appreciable shift in the lambda max. Carbamyl-P increases the pK of the phenolic group in both enzymes, a result consistent with its binding site being close to the essential sulfhydryl group.

Animals↗
PubMed1980

Characterization of a protease apparently involved in processing of pre-ornithine transcarbamylase of rat liver.

The precursor of rat liver ornithine transcarbamylase (ornithine carbamoyltransferase; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) (pre-ornithine transcarbamylase), which was synthesized in a reticulocyte lysate cell-free system, was converted to an apparently mature form of the enzyme by isolated rat liver mitochondria. The proteolytic processing involved two steps: (i) conversion of pre-ornithine transcarbamylase (39,400 daltons) to a product of about 37,000 daltons and (ii) further conversion to the apparently mature form of the enzyme (36,00 daltons). When mitochondria were subfractionated by digitonin treatment followed by sonication of a mitoplast fraction, the proteolytic activity catalyzing the first step was recovered mainly in a matrix fraction. Some activity was found in an intermembrane space fraction. The enzyme activity in the matrix fraction has an optimal pH at about 7.5. The activity was inhibited almost completely by 2 mM leupeptin and partly by 2 mM antipain but not significantly by other microbial protease inhibitors or serine protease inhibitors. It was inhibited strongly by 2 mM EDTA, 2 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate, 2 mM p-chloromercuriphenylsulfonate, and 2 mM Hg(CH3COO)2 but not by N-ethylmaleimide or iodoacetamide. These results suggest that pre-ornithine transcarbamylase is first transported into the mitochondrial matrix and converted there to the mature form of the enzyme by a novel neutral protease(s).

Animals↗
PubMed1980

Processing of a putative precursor of rat liver ornithine transcarbamylase, a mitochondrial matrix enzyme.

A putative precursor of rat liver ornithine transcarbamylase [EC 2.1.3.3] which was about 3,400 daltons larger than the subunit of the mature enzyme (36,000 daltons) was synthesized in a rabbit reticulocyte cell-free system and immunoprecipitated using an antibody against the bovine enzyme and fixed Staphylococcus aureus cells. The mature enzyme of rat liver competed effectively with the putative precursor for interaction with the antibody. Digestion of the putative precursor by S. aureus protease gave a pattern of peptide fragments similar to that of the mature enzyme. A rat liver mitochondrial preparation converted the putative precursor to a polypeptide which comigrated with the mature subunit on sodium dodecyl sulfate/polyacrylamide gels. The "processed" product was recovered in sedimented mitochondria and was no longer susceptible to externally added proteases. These results indicate that the enzyme is synthesized as a larger precursor which may be imported into mitochondria in association with post-translational proteolytic processing to the mature form of the enzyme.

Animals↗
PubMed1979

Cell-free synthesis and processing of a putative precursor for mitochondrial carbamyl phosphate synthetase I of rat liver.

Total RNA or poly(A)(+) RNA of rat liver was translated in a rabbit reticulocyte or wheat germ protein-synthesizing system and the carbamyl phosphate synthetase I [carbamoyl-phosphate synthetase (ammonia); carbon dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16] synthesized was isolated by indirect immunoprecipitation by using antibody purified on enzyme-bound Sepharose and Staphylococcus aureus cells. The in vitro product moved on sodium dodecyl sulfate/polyacrylamide gels as a polypeptide that was about 5000 daltons larger than the subunit of the mature enzyme (160,000 daltons). The same polypeptide was also obtained by direct immunoprecipitation or by a double-antibody precipitation method. The mature enzyme competed effectively with the in vitro product for interaction with anti-carbamyl phosphate synthetase I antibody. Digestion of the in vitro product by S. aureus protease gave a pattern of peptide fragments similar to that of the mature enzyme. A mitochondrial membrane preparation from rat liver converted the in vitro product into a polypeptide that comigrated with the mature subunit on sodium dodecyl sulfate gel electrophoresis. Similar proteolytic activity was not detected in either a cytosol or a microsomal fraction of rat liver. These results indicate that the enzyme is synthesized as a larger precursor which is converted to the mature form of enzyme by posttranslational processing.

Animals↗
PubMed1979

Cell-free translation and thyroxine induction of carbamyl phosphate synthetase I messenger RNA in tadpole liver.

Total RNA of tadpole and frog (Rana catesbeiana) liver was isolated by either 7 or 8 M guanidine . HCl extraction and translated in a cell-free protein-synthesizing system derived from rabbit reticulocytes. The identity of carbamyl phosphate synthetase I[carbamoyl-phosphate synthase (ammonia); ATP:carbamate phosphotransferase (dephosphorylating), EC 2.7.2.5] synthesized in vitro with the purified enzyme was established as follows: (i) immunoprecipitation by a specific antibody; (i) comigration with purified carrier enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis; (iii) copurification with carrier enzyme by affinity chromatography on Cibacron Blue F3GA-coupled agarose; and (iv) formation of identical proteolytic cleavage products. Inclusion of protease inhibitors in the system resulted in no apparent change in the polypeptide molecular weight. These results indicate that carbamyl phosphate synthetase I is synthesized as a polypeptide that is indistinguishable from the mature enzyme by the analytical methods used and that it is not grossly modified during its transport into mitochondria. The level of translatable mRNA for carbamyl phosphate synthetase-I in tadpole liver was increased about 2-fold 1 day after thyroxine treatment and did not change significantly through 4 subsequent days of treatment. Thus the thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver is at least partly due to an increase of translatable mRNA for this enzyme.

Animals↗
PubMed1978

Preparation of crystalline carbamyl phosphate synthetase-I from frog liver.

Ammonia- and N-acetylglutamate-dependent carbamyl phosphate synthetase-I (EC 2.7.2.5), the mitchondrial enzyme involved in the initial step of urea biosynthesis, was purified to homogeneity from frog liver and crystallized. The purification involved extraction of a particulate fraction with cetyltrimethylammonium bromide in the presence of the protease inhibitors antipain, leupeptin, chymostatin, and pepstatin; acetone precipitation; and affinity chromatography with Cibacron blue F3GA-coupled agarose. The enzyme was adsorbed to the gel at pH 8.3 in the presence of 5 mM MgCl2 and eluted with magnesoum-free buffer. The enzyme crystallized as either elongated, thin, rectangular plates or as clusters of small crystals from 37 to 40% saturated ammonium sulfate. The enzyme moved as a single polypeptide band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis with a molecular weight of 160,000. In the absence of protease inhibitors, proteolysis of the enzyme occurred with the formation of an enzymatically active fragment with a subunit molecular weight of 139,000.

Animals↗
PubMed1978

Antipain inhibits thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver.

The increased activity of carbamyl phosphate synthetase I [carbamoyl-phosphate synthase (ammonia); ATP: carbamate phosphotransferase (diphosphorylating), EC 2.7.2.5] in tadpole liver observed during thyroxine-induced metamorphosis was markedly inhibited by intraperitoneal injection of the microbial protease inhibitor antipain (0.1 micrometermol/g of body weight, twice daily). A somewhat less than maximal inhibition was seen when antipain was given only during the first 2 days of thyroxine treatment. On the other hand, little inhibition was observed when the inhibitor was given after the third or fourth day of thyroxine treatment. Antipain also inhibited thyroxine-induced increases of ornithine transcarbamylase (EC 2.1.3.3), arginase (EC 3.5.3.1), and succinate-cytochrome c reductase (EC 1.3.99.1) activities. Among other microbial protease inhibitors tested, chymostatin was nearly as effective as antipain, leupeptin was less effective, and pepstatin was ineffective. Analysis of the total liver protein and of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed that the inhibition was due to decreased amount of the enzyme protein. Antipain had no significant effect on leucine incorporation into total protein of tadpole liver. These results indicate the involvement of a proteolytic step in the pretranscriptional events in thyroxine-stimulated enzyme induction.

Animals↗