Characterization of two high molecular weight proteins immunoprecipitated with an antibody against rat liver cytochrome c oxidase

Isolated rat hepatocytes were labelled with [35S]methionine, dissolved in Triton X-100-containing buffer, and incubated with antibodies against rat liver cytochrome c oxidase. After separation by dodecyl sulfate-gel electrophoresis the fluorogram of immunoprecipitated proteins showed two labelled bands with apparent molecular weights of 52000 and 182000. The immunological relationship of the two proteins to cytochrome c oxidase was demonstrated by immunocompetition with the isolated enzyme and with purified subunits IV-VIII. Although the precursor nature of the two described proteins for cytoplasmically synthesized subunits of cytochrome c oxidase cannot be excluded, the following observations do not support this assumption: 1) The amount of incorporated radioactivity is too high; 2) they are exclusively located with the microsomal fraction; 3) the turnover is rather slow, compared to that of known precursor proteins.     

Differences in the products of mitochondrial protein synthesis in vivo in human and mouse cells and their potential use as markers for the mitochondrial genome in human–mouse cell hybrids

The proteins synthesized in the mitochondria of mouse and human cells grown in tissue culture were examined by electrophoresis in polyacrylamide gels. The proteins were labelled by incubating the cells in the presence of [(35)S]methionine and an inhibitor of cytoplasmic protein synthesis (emetine or cycloheximide). A detailed comparison between the labelled products of mouse and human mitochondrial protein synthesis was made possible by developing radioautograms after exposure to slab-electrophoresis gels. Patterns obtained for different cell types of the same species were extremely similar, whereas reproducible differences were observed on comparison of the profiles obtained for mouse and human cells. Four human-mouse somatic cell hybrids were examined, and in each one only components corresponding to mouse mitochondrially synthesized proteins were detected.     

Mitochondrial translation of subunits of the rotenone-sensitive NADH:ubiquinone reductase in Neurospora crassa.

The rotenone sensitive NADH:ubiquinone was isolated from mitochondria of Neurospora crassa as a monodisperse preparation with the apparent mol. wt. in Triton solution of 0.9 X 10(6). The enzyme is composed of at least 22 subunits with apparent mol. wts. in SDS between 70 and 11 kd. Six of the subunits with the mol. wts. 70, 48, 37, 25, 22 and 18 kd were radioactively labelled in the enzyme isolated from cells which had incorporated [35S]methionine in the presence of cycloheximide. These subunits are synthesized in the mitochondria. Eleven subunits were radioactively labelled in the enzyme from cells which had incorporated [35S]methionine in the presence of chloramphenicol. These subunits are synthesized in the cytoplasm. The site of translation of the other subunits could not be established by the pulse-labelling technique. The assignment of the mitochondrially synthesized subunits to unidentified reading frames on the mitochondrial DNA is discussed.     

ADP-ribosylation of proteins associated with heterogeneous nuclear RNA in rat liver nuclei

Purified rat liver nuclei were incubated in vitro in the presence of [adenylate-32P]nicotinamide adenine dinucleotide. The label was rapidly incorporated into trichloroacetic acid-insoluble material and also detected in particles carrying heterogeneous nuclear RNA. The particles were isolated by density gradient centrifugation, and their size determined to be 30-40 S from parallel experiments using nuclei labelled with [3H]uridine 5'-triphosphate under similar conditions. Treatment of the 30-40 S-particles with enzymes of different specificities showed that the label was tightly bound to proteins, not incorporated into nuclei acids and not utilized in phosphorylation of proteins. The label was detached by phosphodiesterase I from snake venom and identified as ADP-ribose and adenosine 5'-phosphate present at a ratio of 7.5 to 1 using thin layer chromatography on poly(ethyleneimine)-cellulose. Radioactively labelled (ADP-ribosylated) proteins were visualized by autoradiography following SDS-polyacrylamide gel electrophoresis. They included several major species of the ribonucleoprotein with molecular weights of 36000, 39000 and 42000, and a limited number of high molecular weight polypeptides.     

Lipophilic proteins encoded by mitochondrial and nuclear genes in Neurospora crassa.

Mitochondrial proteins soluble in neutral chloroform-methanol (2:1) were separated from lipids by ether precipitation and resolved by Sephadex G-200 filtration in the presence of dodecylsulfate into two major fractions eluting in the excluded region (peak I) and in a region of an apparent molecular weight 8000 (peak II). Residual phospholipids are found only in peak II. Peak I consists of several aggregated small polypeptides of molecular weights around 8000, which can be disaggregated by mild oxidation with performic acid. Cycloheximide stimulates almost two-fold incorporation of radioactive phenylalanine into peak I proteins but inhibits labelling of peak II proteins by 95%. Chloramphenicol and ethidium bromide inhibit the synthesis of peak I proteins by 70% and 95% respectively, but do not affect labelling of peak II proteins. At least 30% of the translation products of mitochondrial DNA in vitro behave like peak I proteins: they are soluble in organic solvents, they aggregate in dodecylsulfate buffer after removal of phospholipids and they contain species of molecular weights around 8000 that disaggregate upon oxidation. The data strongly suggest that the proteins of peak I are encoded by mitochondrial genes and synthesized on mitochondrial ribosomes, whereas the proteins of peak II are encoded by nuclear genes and synthesized on cytoplasmic ribosomes. Both groups of lipophilic proteins are very similar in their molecular weights, but the mitochondrially coded peak I proteins have the unique property of forming large heat-stable aggregates in the presence of dodecylsulfate.     

Synthesis and processing of sphingolipid activator protein-2 (SAP-2) in cultured human fibroblasts

Sphingolipid activator proteins (SAP) are relatively small molecular weight proteins that stimulate the enzymatic hydrolysis of sphingolipids in the presence of specific lysosomal hydrolases. SAP-2 has previously been demonstrated to activate the hydrolysis of glucosylceramide, galactosylceramide, and, possibly, sphingomyelin. Using monospecific rabbit antibodies against human spleen SAP-2, the synthesis and processing of SAP-2 were studied in cultured human fibroblasts. When [35S]methionine was presented in the medium to control human cells for 4 h, five major areas of radiolabeling were found. These had apparent molecular weights of 73,000, 68,000, 50,000, 12,000, and 9,000. Further studies indicated that the major extracellular product in normal cells given NH4Cl along with the [35S]methionine and in medium from cultures from patients with I cell disease had an apparent molecular weight of 73,000. The Mr = 68,000 and 73,000 species can be converted to a species with an apparent molecular weight of 50,000 by the action of endoglycosidase F. After labeling cells for 1 h followed by a 1-h chase, the Mr = 12,000 and 9,000 species appear. Treatment of the immunoprecipitated mixture with endoglycosidase F resulted in conversion of these species to one band with an apparent molecular weight of 7,600. These studies indicate that this relatively low molecular weight protein is rapidly synthesized from a relatively large molecular weight highly glycosylated precursor.     

Biogenesis of the mitochondrial ATPase from sea urchin embryos

The mitochondrial rutamycin-sensitive ATPase from sea urchin eggs was purified to homogeneity. The subunit structure of the enzyme was characterized by SDS-gel electrophoresis. Eight polypeptides were identified with molecular weights of 55,000, 52,000, 39,000, 31,000, 28,000, 23,000, 17,000 and 10,000. Developing sea urchin embryos were incubated with [2H]leucine in the presence of emetine preferentially to label mitochondrially made proteins. Under these conditions sea urchin mitochondria synthesize eight different polypeptides. Two of these proteins, with molecular weights of 31,000 and 23,000, co-purify with the ATPase. Antibody directed against the pure rutamycin-sensitive ATPase precipitated only these two proteins. Therefore, two of the eight sea urchin ATPase subunits appear to be made by mitochondria.     

Structural glycoproteins from rabbit aortic media.

Rabbit aortic intima-media fragments were incubated with [14C]mannose and [3H]fucose for 6 h to detect glycoproteins synthesized in situ. The radioactively labelled and the non-labelled samples were extracted with 0.2 mM-CaCl2/0.5 mM-dithiothreitol/0.5 mM-ATP and chloroform/methanol/water (4:4:1, by vol.). The delipidated residue was extracted with 5 M-guanidinium chloride/0.05 M-dithiothreitol/0.1 M-Tris/0.4% Na2EDTA, pH 7.5, before (extract 1) and after hydrolysis with collagenase (extract 2). The proteins in extracts 1 and 2 were S-carboxamidomethylated and separated by molecular-sieve chromatography, polyacrylamide-gel electrophoresis and isoelectric focusing in sucrose gradients in urea. The apparent molecular weights of glycoproteins were 36 000 (glycoprotein I) from extract 1, 50 000 (glycoprotein II) and 130 000 (glycoprotein III) from extract 2. The molecular weights of the non-labelled and radioactively labelled glycoproteins were identical. Glycoproteins I, II and III contain large amounts of polar amino acids and methionine. They contain neither hydroxyproline nor 3-methylhistidine. A hydroxyproline-containing component of 160 000-apparent-mol.wt. relatively rich in polar amino acids and labelled with incorporated sugars was isolated from extract 1. The incorporation in vitro of radioactive sugars into glycoproteins I, II, III and collagenous glycoproteins indicates that they are synthesized in the surviving aorta by the smooth-muscle cells.     

Characterization of [3H]palmitate- and [3H]ethanolamine-labelled proteins in the multicellular parasitic trematode Schistosoma mansoni.

Biosynthetic labelling experiments with cercariae and schistosomula of the multicellular parasitic trematode Schistosoma mansoni were performed to determine whether [3H]palmitate or [3H]ethanolamine was incorporated into proteins. Parasites incorporated [3H]palmitate into numerous proteins, as judged by SDS/polyacrylamide-gel electrophoresis and fluorography. The radiolabel was resistant to extraction with chloroform, but sensitive to alkaline hydrolysis, indicating the presence of an ester bond. Further investigation of the major 22 kDa [3H]palmitate-labelled species showed that the label could be recovered in a Pronase fragment which bound detergent and had an apparent molecular mass of 1200 Da as determined by gel filtration on Sephadex LH-20. Schistosomula incubated with [3H]ethanolamine for up to 24 h incorporated this precursor into several proteins; labelled Pronase fragments recovered from the three most intensely labelled proteins were hydrophilic and had a molecular mass of approx. 200 Da. Furthermore, reductive methylation of such fragments showed that the [3H]ethanolamine bears a free amino group, indicating the lack of an amide linkage. We also evaluated the effect of phosphatidylinositol-specific phospholipase C from Staphylococcus aureus: [3H]palmitate-labelled proteins of schistosomula and surface-iodinated proteins were resistant to hydrolysis with this enzyme. In conclusion, [3H]palmitate and [3H]ethanolamine are incorporated into distinct proteins of cercariae and schistosomula which do not bear glycophospholipid anchors. The [3H]ethanolamine-labelled proteins represent a novel variety of protein modification.     

Nucleoside transport. Photoaffinity labelling of high-affinity nitrobenzylthioinosine binding sites in rat and guinea pig lung

Binding of the potent nucleoside transport inhibitor [3H]nitrobenzylthioinosine to rat and guinea pig lung membranes was investigated. Reversible high-affinity binding was found in both species (apparent KD approximately 0.3nM). Binding was inhibited by nitrobenzylthioguanosine, adenosine and uridine. Dipyridamole was also an effective inhibitor of [3H]nitrobenzylthioinosine binding to guinea pig membranes. In contrast, rat membranes were relatively insensitive to dipyridamole. Exposure of site-bound [3H]nitrobenzylthioinosine to high intensity U.V. light resulted in the photoaffinity labelling of lung proteins with apparent molecular weights similar to that of the human erythrocyte nucleoside transporter (45,000-65,000).     

Secreted proteins of quiescent,serum-stimulated and over-confluent mouse embryo fibroblasts

Quiescent and proliferating cultures of Swiss mouse embryo fibroblasts were pulse labelled with [¹⁴C]-amino acids and the newly synthesized proteins that were secreted into the medium were resolved by electrophoresis on Polyacrylafde gradient gels. Conditioned media obtained from quiescent cultures that were stimulated to grow by the addition of 20% fetal calf serum showed the presence of two unique polypeptides of molecular weights 48000 and 26000. A polypeptide of molecular weight 45000 was present in increased amounts in serum-stimulated cells than in quiescent cells. This protein was also superinduced in quiescent cells by cycloheximide treatment. Mouse embryo fibroblasts grown under over-crowded conditions secreted two proteins of molecular weights 35000 and 11000. The 35 K polypeptide was shown to be related to the major excreted protein of transformed cells, since it was immunoprecipitated by an antiserum to major excreted protein. These results indicate that the 48 K and 26 K proteins may be proliferation specific proteins, while the 35 K protein present in the conditioned media of over-confluent cells may be a marker of morphological transformation.     

Schwann Cell Surface Proteins and Glycoproteins

Abstract: To identify surface sialoglycoproteins of rat Schwann cells and to compare molecular weights of these sialoglycoproteins with those present in rat peripheral nervous system myelin, we prepared Schwann cells from sciatic nerves of 1–3-day-old rats and cultured them in monolayer. Surface sialoglycoproteins of the cultured cells were tritium-labeled by the periodateborohydride procedure and compared with sialoglycoproteins of adult rat peripheral nervous system myelin by fluorography following polyacrylamide slab gel electrophoresis in sodium dodecyl sulfate. Three radioactive bands with apparent molecular weights of 114,000–132,000, 105,000–115,000, and 44,000–56,000 were observed in both the Schwann cell and myelin preparations. Bands of similar apparent molecular weights were noted in Schwann cells metabolically radiolabeled with d -[1,6-³H]glucosamine. A band co-migrating with myelin P₀ glycoprotein was the most intensely radiolabeled of all peptides in periodate-B³H₄^?treated myelin, but was present in only trace amounts in periodate-B³H₄^? or d -[1,6-³H]glucosamine radiolabeled Schwann cells. Many presumably non-myelin glycoproteins were identified in the cultured Schwann cells by the periodate-borohydride procedure and by incubation of the cells with d -[1,6-³H]glucosamine. An immunoprecipitation technique was used to detect radiolabeled peptides in a nonionic detergent extract of freshly prepared, surface-radioiodinated Schwann cells that were bound by a rabbit anti-Schwann cell serum preabsorbed with rat fibroblasts. Many radioactive peptides were detected in the immunoprecipitate, but the two most intensely radiolabeled had apparent molecular weights of 105,000–115,000 and 95,000–106,000. This study has identified a number of glycoproteins synthesized by cultured rat Schwann cells which resemble in apparent molecular weight the glycoproteins expressed in rat peripheral nervous system myelin and has defined Schwann cell surface proteins recognized by a specific anti-rat Schwann cell antiserum.     

Phosphorylation of both 47 and 49kDa proteins accompanies superoxide release by neutrophils

Neutrophils stimulated with activators of protein kinase C (i.e., 4 beta-phorbol 12-myristate 13-acetate; sn-1,2-dioctanoylglycerol) exhibit a dramatic, dose-dependent incorporation of 32P[Pi] into two proteins with molecular weights of ca. 47 and 49kDa. Proteins of the same molecular weights are also labelled when the cells are stimulated with a chemotactic peptide. However, with the latter stimulus, labelling of the 47kDa species is transient whereas that of the 49kDa entity persists. Labelling of both proteins always accompanied the release of O2-stimulated by these agents. The kinetics of labelling are compatible with the involvement of both phosphoproteins in the stimulation of these cells.     

HeLa cell plasma membranes : IV. Iodination of membrane proteins in synchronized cells

Intact HeLa cells and isolated HeLa cell plasma membranes were subjected to lactoperoxidase-catalysed iodination. The ¹²⁵I-labelled proteins were separated by SDS-polyacrylamide gel electrophoresis. Six protein species with apparent molecular weights from 32 000 to 200 000 were accessible to labelling from the outer cell surface, while most of the proteins present in the plasma membrane were labelled when isolated plasma membranes were iodinated. Iodination of synchronized intact cells revealed that the labelling obtained was cell cycle dependent with maximal labelling at mitosis. No changes in the distribution of radioactivity among the labelled proteins were observed when cells from different phases were iodinated.     

Glucosamine-labelled envelope proteins of Escherichia coli K-12 I. Electrophoretic studies and partial fractionation of the phenol-soluble proteins

Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-^{1 4}C]glucosamine and L-[4,5-³H]leucine were obtained by electrophoretic separation. Envelope were isolated from cells labeleed with D-[1-^{1 4}C]glucosamine—HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. Te glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-^{1 4}C₂]pimelic acid, while ortho[^{3 2}P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAS-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.     

Glucosamine-labelled envelope proteins of Escherichia coli K-12 II. Location in inner and outer membranes

Hydrophobic envelope proteins were extracted by phenol from a glucosamine- and leucine-requiring mutant of Escherichia coli K-12 (E-110). Three protein fractions labelled with D-[1-^{1 4}C]glucosamine and L-[4,5-³H]leucine were obtained by electrophoretic separation. Envelope were isolated from cells labeleed with D-[1-^{1 4}C]glucosamine—HCL and acid hydrolyzed. At least 68% of the radioactivity was recovered as glucosamine and glucose with no random distribution of label. Fingerprinting of pronase digests of glucosamine-labelled proteins showed four radioactive spots associated with peptides. Te glycoproteins were pronase- and trypsin-sensitive and had apparent molecular weights of 11 000 (fast mobility), 35 000 (intermediate mobility) and 62 000 (slow mobility) as estimated by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. The two heavier fractions were labelled with meso-diamino[1,7-^{1 4}C₂]pimelic acid, while ortho[^{3 2}P]phosphate was not incorporated into any fraction. The glucosamine radioactivity of the fast fraction underwent rapid changes upon a chase with non-radioactive glucosamine. Using a Sephadex LH-20 column, the radioactive proteins were separated from the phenol and subsequently fractionated on a DEAS-cellulose column. The DEAE-cellulose fractions were distinct from each other in the number and composition of protein bands, when analyzed by sodium dodecyl sulfate-polyacrylamide disc electrophoresis. Radioactive bands with intermediate and fast electrophoretic mobilities were found in separate DEAE-cellulose fractions.     

Chloroplast ribosomal proteins of Chlamydomonas synthesized in the cytoplasm are made as precursors

Polyadenylated RNA from Chlamydomonas was translated in a cell-free rabbit reticulocyte system that employed [35S]methionine. Antibodies made to four chloroplast ribosomal proteins synthesized in the cytoplasm and imported into the organelle were used for indirect immunoprecipitation of the labeled translation products, which were subsequently visualized on fluorographs of SDS gels. The cytoplasmically synthesized chloroplast ribosomal proteins were first seen as precursors with apparent molecular weights of 1,000 to 6,000 greater than their respective mature forms. Processing of the ribosomal protein precursors to mature proteins was affected by adding a postribosomal supernatant that had been extracted from cells of Chlamydomonas. In contrast to the chloroplast ribosomal proteins synthesized in the cytoplasm, two such proteins made within the chloroplast were found to be synthesized in mature form in cell-free wheat germ translation systems programmed with nonpolyadenylated RNA.     

Characterization of the intracellular processing and secretion of hepatic lipase in FU5AH rat hepatoma cells

The processing and secretion of newly synthesized hepatic lipase was characterized in FU5AH rat hepatoma cells. Pulse-chase experiments revealed two immunoreactive species with apparent molecular weights of 55,400 and 57,600. The 55.4 kDa species was detectable only in cell extracts, whereas the 57.6 kDa species was present in both cell extracts and media. Following a 5 min pulse with L-[35S]methionine and a 10 min chase, these two species represented only 0.003% of the total labelled protein. Quantitation of the 55.4 kDa and 57.6 kDa species in a chase time course taken together with their respective sensitivity and resistance to digestion with endo-beta-N-acetylglucosaminidase H indicates that the 55.4 kDa species is a high mannose precursor to the mature 57.6 kDa enzyme which contains only complex N-linked oligosaccharides. From a time course of endo-beta-N-acetylglucosaminidase H digestion, it was determined that hepatic lipase contains a minimum of two N-linked oligosaccharides. Treatment of the 55.4 kDa species with endo-beta-N-acetylglucosaminidase H yields a protein with a kDa value similar to that observed after treatment of the mature secreted enzyme with endo-beta-N-acetylglucosaminidase F or trifluoromethanesulfonic acid. Therefore, processing of N-linked oligosaccharides is probably the only post-translational modification responsible for the observed change in the apparent molecular weight of hepatic lipase. The half-residence times of hepatic lipase in the endoplasmic reticulum-cis Golgi region and in the cell were estimated at 34 min and 57 min, respectively. Newly synthesized hepatic lipase in Fu5AH cells is secreted constitutively and is not stored in an intracellular pool. Finally, little of the newly synthesized enzyme is degraded during the course of a 1 h chase.     

Multiple forms of human dopamine beta-hydroxylase in SH-SY5Y neuroblastoma cells

Dopamine beta-hydroxylase exists as three forms in human neuroblastoma (SH-SY5Y) cells. The membrane-bound form of the hydroxylase contains three different species with apparent relative molecular weights of 73,000, 77,000, and 82,000. The intracellular soluble form of dopamine beta-hydroxylase was present as a single species with an apparent molecular weight of 73,000. Pulse-chase experiments showed that membranous dopamine beta-hydroxylase contains two subunit forms of 73,000 and 77,000 after short chase times. The soluble hydroxylase was synthesized as a single species of 73,000 at approximately the same rate as the lower molecular weight species of the membranous enzyme. A constitutively secreted third form of the enzyme with an intermediate apparent molecular weight also incorporated [35S]sulfate, whereas no significant amount of [35S]sulfate was observed in the cellular forms of the enzyme. The [35S]sulfate was incorporated on N-linked oligosaccharides. Approximately 12% of the enzyme is released constitutively within 1 h. These results demonstrate that neuronal cells have the ability to constitutively secrete a specific form of dopamine beta-hydroxylase which may contribute to the levels of this enzyme found in plasma.     

Calmodulin-Binding Proteins Within the Slow Phase of Axonal Transport in the Rabbit Vagus Nerve Per Ekstrom and Martin Kanje

Calmodulin-binding proteins (CBPs) in the rabbit vagus nerve were studied by means of calmodulin-Sepharose affinity chromatography and polyacrylamide gel electrophoresis. The soluble fraction (10(5) g supernatant) of a nerve homogenate contained four CBPs with molecular weights of 44, 55, 91, and 93 kD, respectively. Slowly transported proteins were recovered in the vagus 3 days after injection of [35S]methionine into the nodose ganglion. Four labelled CBPs with molecular weights of 44, 55, 69, and 83 kD, respectively were found. The nodose ganglion contained two labelled CBPs, 44 and 55 kD. The 55-kD CBP was identified as tubulin after immunoblotting. In separate experiments it was also shown that bovine brain tubulin bound to the calmodulin column.