Biomedical subjects

F Kalousek

Publications and source records attributed to F Kalousek.

At least 55 records · Page 3Linked to original sources

PubMed1985

A cDNA clone for the precursor of rat mitochondrial ornithine transcarbamylase: comparison of rat and human leader sequences and conservation of catalytic sites.

We have cloned a DNA complementary to the messenger RNA encoding the precursor of ornithine transcarbamylase from rat liver. This complementary DNA contains the entire protein coding region of 1062 nucleotides and 86 nucleotides of 5'- and 298 nucleotides of 3'-untranslated sequences. The predicted amino acid sequence has been confirmed by extensive protein sequence data. The mature rat enzyme contains the same number of amino acid residues (322) as the human enzyme and their amino acid sequences are 93% homologous. The rat and human amino-terminal leader sequences of 32 amino acids, on the other hand, are only 69% homologous. The rat leader contains no acidic and seven basic residues compared to four basic residues found in the human leader. There is complete sequence homology (residues 58-62) among the ornithine and aspartate transcarbamylases from E. coli and the rat and human ornithine transcarbamylases at the carbamyl phosphate binding site. Finally, a cysteine containing hexapeptide (residues 268-273), the putative ornithine binding site in Streptococcus faecalis, Streptococcus faecium, and bovine transcarbamylases, is completely conserved among the two E. coli and the two mammalian transcarbamylases.

Amino Acid Sequence↗

PubMed1985

A leader peptide is sufficient to direct mitochondrial import of a chimeric protein.

Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2-terminal 'leader' peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26-K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti-DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti-DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell-free system and incubated post-translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post-translational import of a chimeric precursor protein by mitochondria.

Animals↗

PubMed1985

Arginine in the leader peptide is required for both import and proteolytic cleavage of a mitochondrial precursor.

Most mitochondrial proteins are encoded in the nucleus and translated in the cytoplasm as larger precursors containing NH2-terminal "leader" peptides, which are strikingly basic in overall amino acid composition. Recent experiments indicate that these leader peptides are both necessary and sufficient to direct post-translational recognition and import of precursors by mitochondria. In this report, we demonstrate a critical role for one or more of the basic arginine residues in the leader peptide of the subunit precursor for the human mitochondrial matrix enzyme, ornithine transcarbamoylase (ornithine carbamoyltransferase, carbamoylphosphate: L-ornithine carbamoyltransferase, EC 2.1.3.3). The distal three of four basic residues, all arginines, in the leader peptide of ornithine transcarbamoylase were replaced at once with charge-neutral glycine residues. The altered ornithine transcarbamoylase precursor failed to be taken up by intact mitochondria in vitro. Moreover, it also failed to be proteolytically cleaved upon incubation with a mitochondrial matrix fraction containing the Zn2+-dependent protease, which normally cleaves the leader peptide.

Amino Acid Sequence↗

PubMed1984

RNA required for import of precursor proteins into mitochondria.

A cytoplasmic RNA moiety is necessary for posttranslational uptake of nuclear-encoded mammalian proteins destined for the mitochondrial matrix. Post-translational addition of ribonuclease to a reticulocyte lysate-programmed cell-free translation mixture inhibited subsequent import of six different mitochondrial matrix enzyme precursors into rat liver mitochondria. The required RNA is highly protected, as indicated by the high concentrations of ribonuclease necessary to produce this inhibition. The dependence of the inhibitory effect on temperature, duration of exposure to ribonuclease, and availability of divalent cations is characteristic of the nuclease susceptibility of ribonucleoproteins. The ribonuclease-sensitive component was found in a 400-kilodalton fraction which contains the mitochondrial protein precursors.

Animals↗

PubMed1984

Structure and expression of a complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase.

Most mitochondrial proteins are encoded in the nucleus and are translated on free cytoplasmic ribosomes as larger precursors containing amino-terminal "leader" sequences, which are removed after the precursors are taken up by mitochondria. We have deduced the complete primary structure of the precursor of a human mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), from the nucleotide sequence of cloned complementary DNA. The amino-terminal leader peptide of OTC is 32 amino acids in length and contains four arginines but no acidic residues. Cleavage of the leader peptide from the "mature" protein occurs between glutamine and asparagine residues. The sequence of mature human OTC resembles that of the subunits of both OTC and aspartate transcarbamylase from Escherichia coli. The biological activity of the cloned OTC complementary DNA was tested by joining it with SV40 (an animal virus) regulatory elements and transfecting cultured HeLa cells, which do not normally express OTC. Both the precursor and mature forms of the OTC subunit were identified; in stable transformants, enzymatic activity was also detected.

Amino Acid Sequence↗

PubMed1984

Newly processed ornithine transcarbamylase subunits are assembled to trimers in rat liver mitochondria.

We have characterized further the biogenesis in vitro of ornithine transcarbamylase, a homotrimeric mitochondrial matrix enzyme synthesized in the cytoplasm as a larger precursor. When cell-free translation mixtures containing the ornithine transcarbamylase precursor (40 kDa) were chromatographed on Bio-Gel P-200 columns, all of the precursor eluted as aggregates or complexes with molecular weights greater than 200 kDa. None of the precursor bound to a ligand affinity column containing delta-N-(phosphonoacetyl)-L-ornithine (delta-PALO), a transition-state analog and competitive inhibitor of carbamyl phosphate binding, which recognizes native ornithine transcarbamylase. In contrast, a significant portion of the labeled mature-sized subunits, formed when intact mitochondria processed the precursor, bound specifically to the delta-PALO column, were eluted by carbamyl phosphate, and chromatographed on a Bio-Gel P-300 column with a mobility identical to that of native, trimeric ornithine transcarbamylase. No such binding to delta-PALO was observed for the mature-sized monomer or dimer, or for the intermediate-sized ornithine transcarbamylase polypeptide. Moreover, processing by a mitochondrial matrix fraction failed to yield trimeric enzyme, despite producing ample amounts of mature-sized monomer. We conclude that delta-PALO recognizes only trimeric ornithine transcarbamylase composed of mature-sized subunits and that such trimers can be assembled in vitro by intact mitochondria following translocation and proteolytic processing.

Animals↗

PubMed1983

Biogenesis of ornithine transcarbamylase in spfash mutant mice: two cytoplasmic precursors, one mitochondrial enzyme.

Extracts of liver from hemizygous affected mice with the X-linked spfash mutation have 5 to 10 percent of normal ornithine transcarbamylase (OTC) activity, yet the homogeneous enzyme isolated from these extracts is identical to that in controls. The OTC messenger RNA from mutant livers programs the synthesis of two distinct OTC precursor polypeptides--one normal in size, the other distinctly elongated. Both precursors are imported and proteolytically processed by mitochondria, but only the normal one is assembled into active trimer. This novel phenotype may result from a mutation in the structural gene for OTC leading, primarily, to aberrant splicing of OTC messenger RNA and, secondarily, to formation of a structurally altered precursor whose posttranslational pathway is ultimately futile because its mature mitochondrial form is not capable of assembly and functional expression.

Animals↗

PubMed1983

Biosynthesis and mitochondrial processing of the beta subunit of propionyl coenzyme A carboxylase from rat liver.

Propionyl-CoA carboxylase (ADP-forming) (EC 6.4.1.3), an oligomer of nonidentical subunits (alpha 4 beta 4), has been localized to the mitochondrial matrix. As a first step in examining this enzyme's biogenesis, we have investigated in vitro the cell-free, rat liver RNA-directed synthesis of the beta subunit, and its post-translational transport and processing by rat liver mitochondria. The beta subunit is synthesized as a precursor approximately 7,500 daltons larger than its mature mitochondrial counterpart. The extension segment, comprising approximately 60 amino acids, is located at the NH2 terminus of the precursor. Intact mitochondria translocate the precursor across both mitochondrial membranes, and a protease localized to the mitochondrial matrix cleaves the precursor to a polypeptide identical in size and peptide composition to the mature beta subunit.

Animals↗

PubMed1983

Molecular cloning of the cDNA coding for rat ornithine transcarbamoylase.

Ornithine transcarbamoylase is a mitochondrial matrix enzyme composed of three identical subunits encoded on the X chromosome. The subunit is synthesized on cytoplasmic polysomes as a precursor that is cleaved during transport into mitochondria. We report here the isolation and characterization of cDNA clones containing sequences corresponding to the mRNA encoding the ornithine transcarbamoylase subunit. cDNA was synthesized using rat liver mRNA enriched by polysome immunoadsorption for the low-abundance messenger species encoding the enzyme subunit. After insertion of cDNA into plasmid pBR322 and cloning in Escherichia coli, identification of the desired plasmids was accomplished by (i) differential colony hybridization using cDNA probes synthesized from mRNA of various tissues; (ii) differential blot hybridization using cDNA probes synthesized from mRNA enriched for or depleted of the ornithine transcarbamoylase message; (iii) hybrid-selected translation assays; and (iv) most definitively, structural analysis, which matched 25 consecutive amino acid residues determined by sequential Edman analysis of the carboxyl-terminal portion of the purified enzyme subunit with coding sequence present in the insert of one of the plasmids.

Amino Acid Sequence↗

PubMed1983

Absence of cross-reacting material in isolated propionyl CoA carboxylase deficiency: nature of residual carboxylating activity.

Fibroblast extracts and fetal liver homogenates from patients with propionic acidemia due to inherited deficiency of propionyl CoA carboxylase (PCC) were analyzed for the presence of immunologically cross-reactive PCC protein. Using several rabbit antisera raised against homogeneous human liver PCC, homogeneous pig heart PCC, or the individual non-identical subunits of the human liver enzyme, we found no detectable cross-reacting material by direct or competitive immunotitration in several cell lines from patients in either major complementation group (pcc A; pcc C) with isolated PCC deficiency. In contrast, cells of a patient from the bio complementation group contained normal amounts of immunoreactive PCC. Further analysis of the pcc A and pcc C mutants revealed that their residual propionyl CoA carboxylating activity varied greatly depending on the concentration of extract or homogenate protein used in the PCC assay. When propionyl CoA carboxylation was assayed at high protein concentration in a fetal liver homogenate from a pcc C patient, the apparent PCC activity was comparable to that found in normal human fetal liver. Significantly, the specific activity in the mutant, but not in the control, extract declined steeply as protein concentration was lowered, and this loss could not be prevented by adding PCC substrates, bovine serum albumin, glycerol, or 2-mercaptoethanol. Moreover, detailed analyses of immunotitration curves of control fibroblasts extracts showed that fresh extracts contained an amount of nonimmunotitratable carboxylating activity corresponding to the residual activity present in fresh extracts of mutant cell lines. We conclude that the residual propionyl CoA carboxylating activity found in isolated PCC deficiency represents another carboxylase that can utilize propionyl CoA as a substrate rather than a mutant form of PCC with markedly different immunochemical and physicochemical properties.

Carboxy-Lyases↗

PubMed1980

Isolation and characterization of propionyl-CoA carboxylase from normal human liver. Evidence for a protomeric tetramer of nonidentical subunits.

We have purified propionyl-CoA carboxylase from normal, postmortem human liver to homogeneity. The isolation procedure, which provided an approximately 3000-fold purification and an overall yield of 26%, employed initial centrifugation of a cetyltrimethylammonium bromide-treated homogenate, followed by sequential chromatographic separations using DEAE-cellulose, Blue Sepharose, and Bio-Gel A-1.5m. The native enzyme has a molecular weight of approximately 540,000 and is composed of nonidentical subunits (alpha and beta) of Mr = 72,000 and 56,000, respectively. When studied with analytical isoelectrofocusing techniques, it focuses as a single peak at pH 5.5. Each mole of native enzyme contains 4 mol of bound biotin, virtually all of which is found with the larger (alpha) subunit. The apparent Km values for ATP, propionyl-CoA, and bicarbonate are 0.08 mM, 0.29 mM, and 3.0 mM, respectively. The enzyme also catalyzes the carboxylation of acetyl-CoA and butyryl-CoA to a limited degree, but not that of crotonyl-CoA. Propionyl-CoA carboxylase is quite stable over a temperature range from -50--37 degrees C and over a pH range from 6.2 to 8.4. It has a broad pH optimum from pH 7.2 to 8.8. Limited proteolysis with trypsin results in slow, time-dependent deactivation of the enzyme with preferential cleavage of the smaller subunit. Antiserum prepared against the native enzyme is shown to be monospecific by immunodiffusion and immunoelectrophoresis.

Acyl Coenzyme A↗

PubMed1979

In vitro synthesis of a putative precursor of mitochondrial ornithine transcarbamoylase.

Ornithine transcarbamoylase (OTCase; ornithine carbamoyltransferase; carbamoyl phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3), a major mitochondrial matrix enzyme in ureotelic animals, is synthesized on cytoplasmic ribosomes and translocated across both mitochondrial membranes to the matrix. In an attempt to identify the primary translation product (or an early intermediate) that is the substrate for this transport process, we translated rat liver polysomal RNA in vitro by using the rabbit reticulocyte lysate system. Immunoprecipitation of the [35S]methionine-labeled translation mixture was performed by using monospecific OTCase antiserum and the immunoadsorbent Staphylococcus aureus. Approximately 0.3% of total trichloroacetic acid-insoluble 35S-labeled material was specifically precipitated. Analysis of the precipitate by fluorography of a dried sodium dodecyl sulfate/polyacrylamide gel showed a single major translation product whose mobility corresponded to a polypeptide of 43,000 daltons, a value approximately 4000 daltons greater than that noted for the "mature" OTCase subunit isolated from rat liver. This translation product was not precipitated by preimmune rabbit serum, and excess unlabeled mature OTCase competed with it for interaction with OTCase antiserum. These results suggested that rat liver OTCase, like a number of other cytoplasmically synthesized organellar proteins, is initially made as a larger precursor that contains an amino acid sequence necessary to confer on OTCase its transport properties. The potential application of these findings to the study of inherited complete OTCase deficiency in humans is discussed.

Animals↗

PubMed1979

Essential arginine residues in the active sites of propionyl CoA carboxylase and beta-methylcrotonyl CoA carboxylase.

At least one arginine residue is essential for substrate binding in or near the active sites of propionyl CoA carboxylase (PCC) and beta-methylcrotonyl CoA carboxylase (beta MCC) in cultured human fibroblasts. This conclusion is based on studies of enzyme inhibition by phenylglyoxal, a reagent which specifically modifies arginine residues. Human fibroblast PCC both in extracts and in a 20-fold purified preparation was nearly completely protected from phenylglyoxal inhibition following incubation with propionyl CoA or ATP. It appears that a phosphate group from either ATP or the CoA moiety of propionyl CoA reacts with the essential arginine residue(s). beta MCC which was similarly inhibited by phenylglyoxal was protected by beta-methylcrotonyl CoA and ATP. Thus phenylglyoxal may be used to label specific arginine residues within the active sites of previously sequenced carboxylases.

Acyl Coenzyme A↗

PubMed1978

Isolation and characterization of ornithine transcarbamylase from normal human liver.

We report experiments describing the isolation and characterization of ornithine transcarbamylase from normal human liver. Our preparative procedure employs initial centrifugation and heat steps, intermediate batch-wise adsorption and desorption from ion exchange resins and column chromatographic elution from hydroxylapatite, and final purification by gel filtration chromatography and glycerol density gradient centrifugation. The enzyme, purified 580-fold in this way, is homogeneous as judged by native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Human ornithine transcarbamylase has a molecular weight of 114,000 and is a trimer of identical 38,000 molecular weight subunits. It focuses at pH 6.8 as a single band on polyacrylamide gel, has a COOH-terminal phenylalanine, an NH2-terminal glycine, an apparent Km for L-ornithine of 0.4 mM and for carbamyl phosphate of 0.16 mM, and a pH optimum of 7.7. The enzyme is quite stable over a temperature range from -50 degrees to +60 degrees C and over the pH range from 5.8 to 8.2. The quaternary structure and amino acid composition of the human enzyme are very similar to those of its bovine homologue.

Amino Acids↗

PubMed1976