Isolation of mitochondrial succinate: ubiquinone reductase, cytochrome c reductase and cytochrome c oxidase from Neurospora crassa using nonionic detergent.

The electron transfer complexes, succinate: ubiquinone reductase, ubiquinone: cytochrome c reductase, and cytochrome c: O2 oxidase were isolated from the mitochondrial membranes of Neurospora crassa by the following steps. Modification of the contents of the complexes in mitochondria by growing cells on chloramphenicol; solubilisation of the complexes by Triton X-100; affinity chromatography on immobilized cytochrome c and ion exchange and gel chromatography. Ubiquinone reductase was obtained in a monomeric form (Mr approximately 130 000) consisting of a flavin subunit (Mr 72 000) an iron-sulfur subunit (Mr 28 000) and a cytochrome b subunit (Mr probably 14 000). Cytochrome c reductase was obtained in a dimeric form (Mr approximately 550 000), the monomeric unit comprising the cytochromes b (Mr each 30 000), a cytochrome c1 (Mr 31 000), the iron-sulfur subunit (Mr 25 000), and six subunits without known prosthetic groups (Mr 9000, 11 000, 14 000, 45 000, 45 000, and 52 000). Cytochrome c oxidase was also isolated in a dimeric form (Mr approximately 320 000) comprising two copies each of seven subunits (Mr 9000, 12 000, 14 000, 18 000, 21 000, 29 000, and 40 000). The complexes were essentially free of phospholipid. Each bound one micelle of Triton X-100 (Mr approximately 90 000). After isolation, the bound Triton X-100 could be replaced by other nonionic detergents such as: alkylphenyl polyoxyethylene ethers, alkyl polyoxyethylene ethers and acyl polyoxyethylene sorbitan esters.     

Transport of proteins into chloroplasts. The precursor of small subunit of ribulose bisphosphate carboxylase is processed to the mature size in two steps

The precursor of the small subunit of ribulose bisphosphate carboxylase in Pisum sativum (relative molecular mass 20 000) is processed to the mature size (relative molecular mass 14 000) by the purified processing enzyme in two steps. The maturation proceeds via an intermediate of Mr 18 000. Both processing reactions may be carried out by the same enzyme although different residues are involved in the two cleavage sites. The second cleavage is inhibited if the precursor is pre-incubated with iodoacetate. The processing intermediate cannot be detected during the uptake of the precursor by intact isolated chloroplasts but iodoacetate-treated precursor is taken up and converted to a number of polypeptides of Mr 18 000 and below.     

A bifunctional enzyme complex in coenzyme A biosynthesis: purification of pantetheine phosphate adenylyltransferase and dephospho-CoA kinase.

Pantetheine phosphate adenylyltransferase (EC 2.7.7.3) and dephospho-CoA kinase (EC 2.7.1.24) were purified to near homogeneity from pig liver. The purification steps included the use of Sepharose-linked triazine dyes and affinity elution by CoA. Both activities co-purified at every stage of the 18 000-fold purification. An Mr of 115 000 was obtained by gel filtration on Sephadex G-150, and the final preparation yielded one major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, with a subunit Mr of 57 000. It is concluded that pantetheine phosphate adenylyltransferase and dephospho-CoA kinase exist as a bifunctional dimeric protein, which could be designated CoA synthetase.     

Amino acid sequence determination of the ADP,ATP carrier from beef heart mitochondria. The sequence of the C-terminal acidolytic fragment

The primary structure of the C-terminal region (94 residues) of the ADP,ATP carrier of beef heart mitochondria is described. CNBr cleavage results in a large peptide (CB1) with Mr 22 000 and several small peptides (CB2 to CB8). Peptide separation was achieved by gel chromatography with 80% formic acid or with an ethanol/formic acid mixture. The amino acid sequence of the small CNBr peptides was determined by solid-phase techniques. Hydrolysis in formic acid cleaves the carrier protein into an Mr 23 000 fragment (A1) with the blocked N-terminus and an Mr 10 000 fragment (A2) starting with proline. The alignment of two CNBr fragments was possible by degradation of A2 by solid-phase methods for 34 steps. The remaining CNBr fragments were arranged by sequencing the tryptic peptides of citraconylated A2.     

Biogenesis, transit, and functional properties of the insulin proreceptor and modified insulin receptors in 3T3-L1 adipocytes. Use of monensin to probe proreceptor cleavage and generate altered receptor subunits

The biogenesis, intracellular transport, and functional properties of the insulin proreceptor and modified insulin receptors were studied in hormone-responsive 3T3-L1 adipocytes. After control cells were labeled with [35S]Met for 7 min, the principal polypeptide that was precipitated by anti-insulin receptor antibodies had a molecular weight (Mr) of 180 000. This initial precursor was rapidly converted (t1/2 = 35 min) to a 200-kilodalton (kDa) polypeptide, designated the insulin proreceptor, by the apparent posttranslational addition of N-linked, high mannose core oligosaccharide units. Mature alpha (Mr 130 000) and beta (Mr 90 000) subunits were derived from sequences within the proreceptor by proteolytic cleavage and late processing steps, and these subunits appeared on the cell surface 2-3 h after synthesis of the 180-kDa precursor. The cation ionophore monensin was used in combination with metabolic labeling, affinity cross-linking, and external proteolysis to probe aspects of proreceptor function, transit, and the development of insulin sensitivity at the target cell surface. At 5 micrograms/mL, monensin potently inhibited the proteolytic cleavage step, and the 200-kDa polypeptide accumulated. Lower concentrations of the ionophore selectively blocked late processing steps in 3T3-L1 adipocytes so that apparently smaller alpha' (Mr 120 000) and beta' (Mr 85 000) subunits were produced. Proreceptor and alpha' and beta' subunits were translocated to the cell surface, indicating that the signal for intracellular transit occurs in the 200-kDa polypeptide and is independent of the posttranslational proteolysis and late processing steps. The alpha' subunit bound insulin both at the surface of intact cells and after solubilization with Triton X-100; the beta' subunit was phosphorylated in an insulin-stimulated manner. The detergent-solubilized 200-kDa proreceptor also exhibited both functional properties. However, the proreceptor that was transported to and exposed on the cell surface was incapable of binding insulin in intact adipocytes. Thus, late processing is not essential for the expression of functions associated with mature alpha and beta subunits. In contrast, it appears that the proteolytic generation of subunits is required for the correct orientation of the hormone binding site in the plasma membrane bilayer and the development of insulin responsiveness in 3T3-L1 adipocytes.     

Characterization and androgenic regulation of major mRNAs coding for epididymal proteins in a lizard (Lacerta vivipara)

During the reproductive period (spring) under the control of testosterone the epididymis of the viviparous lizard secretes a group of major proteins with an approximate Mr of 19,000 named L protein(s). These proteins are recognized by a specific immunoserum and bind to the heads of spermatozoa. During spring, translation in reticulocyte lysate of RNA from secreting epididymis (stage 6) produced 5 immunoprecipitable bands with Mr values from 21,500 to 25,000. Such synthesis is undetectable during sexual rest in summer (stage 1). The 5 bands disappear when translation is performed in the presence of dog pancreas microsomes although a new band of Mr 19 000 becomes prominent. This suggests that synthesis of L protein involves two steps, i.e. synthesis of precursors (L preproteins) followed by a maturation process. At least 11 translation products (including L-preproteins) are involved in annual variations that follow the differentiation of the epididymal epithelial cells and their androgen dependency was studied by castration and in-vitro stimulation by testosterone. In these conditions, testosterone is able to control accumulation of RNA corresponding to L preproteins and to a translation product of Mr 29 000.     

Monomeric pituitary growth hormone and prolactin variants in man characterized by immunoperoxidase electrophoresis

Immunoperoxidase electrophoresis, combining SDS--ME--PAGE and the 'double bridge' immunoperoxidase staining was applied to crude human pituitary homogenates. With anti-hGH and anti-hPL sera, 4 hGH-related monomers were characterized: a Mr 22 000 peptide corresponding to hGH; a Mr 20 000 peptide corresponding to the known hGH variant and two unknown hGH variants (Mr 65 000 and Mr 75 000). With anti-ovine, rat and human PRL sera, 4 PRL-related monomers were immunostained: one comigrated with purified hPRL (Mr 25 000), and 3 were unknown (Mr 29 000; Mr 45 000; Mr 16 000).     

Relative molecular mass determination of a major, highest relative molecular mass extracellular amelogenin of developing bovine enamel

Proteins of developing bovine enamel were fractionated by molecular sieving and ion-exchange chromatography. The major fraction corresponding to the highest Mr amelogenin of Mr approximately 26 000-30 000 was isolated and its Mr determined by SDS-PAGE, molecular sieving on G-100 resin and high performance liquid chromatography and by sedimentation-equilibrium ultracentrifugation, the latter three procedures in guanidine hydrochloride. SDS-PAGE and HPLC molecular sieving, employing commonly used Mr standards, gave Mr values of approximately 22 000-26 000. SDS-PAGE and HPLC molecular sieving, using proline-rich CNBr peptides of collagen as standards, and sedimentation-equilibrium ultracentrifugation, gave Mr values of approximately 15 000-18 000 and approximately 17 385, respectively. These latter values correspond well with those reported earlier and with the Mr of the major amelogenin computed from recent amino acid sequence data (approximately 19 000). It is concluded that the recently described, highest Mr amelogenin of Mr = 26 000-30 000 is not a new component but is identical to the proline-rich components having relative molecular masses ranging from 15 000 to 18 000 described much earlier by several groups of workers.     

Characterization of a Platelet-Derived Growth Factor Receptor on Swiss 3T3 Cells by Affinity Crosslinking

Abstract

Crosslinking experiments with various bifunctional reagents were used to investigate the nature and fate of the platelet growth factor (PDGF) receptor on Swiss mouse 3T3 cells. With ethylene glycol bis succinimidyl succinate (EGS) two bands with Mr 205′000 and Mr 190′000 were labeled at equal intensity, while with disuccinimidyl suberate (DSS) and the photoactivatable pazidophenylglyoxal (pAPG) almost exclusively the latter band was labeled, when analyzed by SDS polyacrylamide gel electrophoresis under reducing conditions. Evidence is presented that the Mr 190′000 band represents a Mr 175′000 receptor protein crosslinked to a single chain of the PDGF-dimer and the Mr 205′000 species the same Mr 175′000 protein crosslinked to both chains of PDGF. Pretreatment of cells with tunicamycin generated a third labeled band with Mr 150′000, while pretreatment with neuraminidase resulted in a shift of the Mr 205′000 and 190′000 bands by 5′000. This shows that the PDGF receptor is a sialoglycoprotein, consisting of a Mr β 135′000 proteinaceous core and a Mr β 40′000 carbohydrate moiety containing sialic acid. The virtually unchanged labeling intensity seen with tunicamycin and neuraminidase pretreated cells further suggests that the carbohydrate portion of the receptor is not required for PDGF binding. Finally, the crosslinking technique was used to show that at 37°C preformed ¹²⁵I-PDGF receptor complexes disappear from the cell surface with a t_1/2 β 8 min.     

Purification and characterization of a plasminogen activator secreted by a pig kidney cell line

The plasminogen activator secreted by calcitonin-treated pig kidney cells was purified, characterized and compared with human urinary urokinase. The purification procedure was based on the following steps: sulphopropyl-Sephadex chromatography, p-aminobenzamidine-Sepharose chromatography, preparative sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectrofocusing. The purified enzyme was obtained from the conditioned medium with a yield of 13% and a purification factor of 390-fold. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under non-reducing conditions showed one closely spaced doublet with an Mr of 50 000; in the presence of reducing agents, two additional bands of Mr 30 000 and 20 000 appeared. The purified enzyme resembles the 53 000-Mr components of human urinary urokinase in amino acid composition and two-dimensional tryptic peptide maps and in its catalytic properties, and the two enzymes cross-react immunologically with rabbit antibodies raised against either. The enzyme appears to be different from tissue plasminogen activator secreted by HeLa cells.     

Characterization of C1q, C1s and C-1 Inh synthesized by stimulated human monocytes in vitro

C1q, C1s and C1 Inh synthesized and secreted by human monocytes were characterized by SDS-PAGE. C1q is formed of three chains A (Mr approximately 35 000), B (Mr approximately 33 000) and C (Mr approximately 25 000) which are associated in two subunits A-B and C-C. It appears identical to C1q purified from plasma. C1s is secreted as a non-activated, monocatenar protein of Mr approximately 87 000 identical to proenzymic C1s from plasma. Secreted C1 Inh (Mr approximately 100 000) has a slightly higher Mr than purified plasmatic C1 Inh. Monensin treatment of the cells favours the intracytoplasmic accumulation of products at various glycosylation stages.     

Limited proteolysis of elongation factor Tu from Escherichia coli, Multiple intermediates.

Limited proteolysis of native elongation factor Tu (Mr 44 000) by trypsin occurs in at least three distinct steps. The first intermediate arises through cleavage at a site about 65 residues from the amino-terminal end of the protein. It is functionally active [Jacobson, G. R. & Rosenbusch, J. P. (1976) Biochemistry, 15, 5105-5110] and is partially protected from further degradation by the antibiotic kirromycin. The second step converts this intermediate to one of similar size (Mr 37 000) which now is partially inactivated. It is likely to be identical with the intermediate described by Arai et al. [(1976) J. Biochem. Tokyo, 79, 69-83]. In the third step, the partially inactive intermediate is cleaved without any apparent change in the functional properties tested. The resulting two trypsin-resistant fragments have molecular weights of 24 000 and 14 000, and remain associated under nondenaturing conditions. When either of these polypeptides, after isolation in 8 M urea, is allowed to renature, no significant reactivation of GDP binding is observed unless the isolated fragments are mixed before renaturation. These results show that the two fragments are structurally and functionally interdependent.     

Evidence for biosynthesis of lactase-phlorizin hydrolase as a single-chain high-molecular weight precursor

Precursor forms of lactase-phlorizin hydrolase, sucrase-isomaltase and aminopeptidase N were studied by pulse-labelling of organ-cultured human intestinal biopsies. After labelling the biopsies were fractionated by the Ca2+-precipitation method and the enzymes isolated by immunoprecipitation. The results indicate that the lactase-phlorizin hydrolase is synthesized as a Mr 245 000 polypeptide, which is intracellularly cleaved into its mature Mr 160 000 form. Sucrase-isomaltase is shown to be synthesized as a single chain precursor (Mr 245 000 and 265 000) while the precursor of aminopeptidase N is shown to be of apparently the same size as the mature enzyme (Mr 140 000 and 160 000).     

Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity

Human factor VIII was isolated from commercial factor VIII concentrates and found to consist of multiple polypeptides with molecular weights ranging from 80 000 to 210 000. Immunological and amino acid sequence data identified these polypeptides as subunits of factor VIII. N-Terminal amino acid sequence analysis determined that the Mr 210 000 and 80 000 proteins are derived from the N- and C-terminal portions of factor VIII, respectively; Mr 90 000-180 000 polypeptides are derived from the Mr 210 000 polypeptide by C-terminal cleavages. Treatment of purified factor VIII with thrombin resulted in proteolysis of Mr 80 000-210 000 proteins and the generation of polypeptides of Mr 73 000, 50 000, and 43 000. Maximum coagulant activity of thrombin-activated factor VIII was correlated with the generation of these polypeptides. The proteolysis as well as activation of factor VIII by thrombin was found to be markedly dependent on CaCl2 concentration. Proteolysis of factor VIII with activated protein C (APC) resulted in degradation of the Mr 90 000-210 000 proteins with the generation of an Mr 45 000 fragment. This cleavage correlated with inactivation of factor VIII by APC. The Mr 80 000 protein was not degraded by APC. Factor Xa cleaved the Mr 80 000-210 000 factor VIII proteins, resulting in the generation of fragments of Mr 73 000, 67 000, 50 000, 45 000, and 43 000. Factor Xa was found to initially activate and subsequently inactivate factor VIII.(ABSTRACT TRUNCATED AT 250 WORDS)     

The size of human factor VIII heterodimers and the effects produced by thrombin

The heterodimeric structure of factor VIII was demonstrated by two approaches. First, the native molecular weights of several partially purified fractions of factor VIII were determined by measurement of Stokes radii and sedimentation coefficients to be approx. 237 500, 201 000 and 141 000. These measured molecular weights correlated with those derived from polypeptide chain composition, in which each molecule would consist of a doublet polypeptide of Mr 83 000/81 000 plus one predominant high-Mr polypeptide of either 146 000, 120 000 or 93 000. In addition, immunoadsorption using a monoclonal antibody specific for the light-chain doublet removed all of the heavy chains. Separation of the heavy chains from the light chain by EDTA further illustrated the non-covalent nature of the heterodimers. All forms had coagulant activity which was potentiated 13-15-fold by an equimolar amount of human alpha-thrombin. Thrombin converted the Mr 83 000/81 000 doublet to one of Mr 73 000/71 000, and cleaved the largest polypeptides to a transient intermediate form of Mr 93 000 which was further cleaved to polypeptides of Mr 51 000 and 43 000. Potentiation of coagulant activity was correlated with proteolytic cleavage of either or both the doublet and the Mr 93 000 polypeptides. These data indicate that human factor VIII purified from plasma consists of a group of heterodimers, composed of a light chain of Mr 83 000 (81 000) and a heavy chain which varies in size between Mr 170 000 and 93 000, each form of which is similarly potentiated and cleaved by thrombin.     

Human C1 inhibitor: improved isolation and preliminary structural characterization

An improved procedure for the isolation of the C1 inhibitor (C1-INH) component of human complement is reported. Following preliminary steps to remove plasminogen, fibrinogen, and aggregated material, three conventional chromatographic steps are used to isolate C1-INH in high (70%) overall yield. An extinction coefficient (E 1%, 1 cm 280nm) of 3.60 has been determined. The isolated protein exhibits a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a mobility corresponding to an apparent molecular weight (Mr) of 105 000. After removal of carbohydrate, the protein shows an increased mobility, corresponding to an apparent Mr of 78 000. A total carbohydrate content of 33% has been calculated, and from this and the size of the deglycosylated polypeptide, a true molecular weight of 116 000 was estimated. Further analysis of the carbohydrate has indicated a galactose:mannose ratio of 2:1 and approximately equimolar amounts of N-acetylglucosamine and N-acetylgalactosamine. This composition is unusual for a plasma protein and suggests that much of the carbohydrate is contained in linkages other than the typical N-glycosidic structures. Values found for the amino acid composition are compared with those reported previously. The amino-terminal sequence (40 residues) of C1-INH is also reported. Asparagine lies at the amino terminus. Neither high-performance liquid chromatography of the released phenylthiohydantoin derivative nor back-hydrolysis of the thiazolinone permitted identification of the residue contained at position 3. The sequence around this position is compatible, however, with an N-glycosidic linkage to residue 3.(ABSTRACT TRUNCATED AT 250 WORDS)     

The 35 kDa DCCD-binding protein from pig heart mitochondria is the mitochondrial porin

The protein which can be labelled by low concentrations of dicyclohexylcarbodiimide in the Mr region of 30 000-35 000 has been purified from pig heart mitochondria with a high yield and as a single band of apparent Mr 35 000 in dodecyl sulphate-containing gels. The protein is not identical with the phosphate carrier as suggested before, since the two proteins behave differently during isolation. Incorporation of the isolated 35 kDa dicyclohexylcarbodiimide-binding protein into lipid bilayer membranes causes an increase of the membrane conductance in definite steps, due to the formation of pores. The specific pore-forming activity increases during the purification procedure. The single pore conductance is about 4.0 nS, suggesting a diameter of 1.7 nm of the open pore. The pore conductance is dependent on the voltage across the membrane. Anion permeability of the pore is higher than cation permeability. These properties are similar to those described for isolated mitochondrial and bacterial porins. It is concluded that the 35 kDa dicyclohexylcarbodiimide-binding protein from pig heart mitochondria is identical with porin from outer mitochondrial membrane.     

Identification of a human neutrophil protein of Mr 24 000 that binds N-formyl peptides: co-sedimentation with specific granules

In assaying subcellular fractions of human neutrophils for N-formyl peptide binding sites using the photoaffinity ligand FMLPL-SASD-125I (125I-labelled N-formylmethionylleucylphenylalanyl-N epsilon- (2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionyl)-lysine) several molecular species were observed. We confirmed localization of the N-formyl peptide receptor of Mr 50 000-70 000 in the plasma membrane and specific granule fractions. A species of Mr 33 000-35 000 was detected in the light Golgi/endosomal fraction, whose size is consistent with the deglycosylated form of the receptor. In addition, a major binding species of Mr 24 000 was identified that co-localized on sucrose gradients with specific granule markers. This Mr 24 000 species, which was investigated further, was found to be released upon cell stimulation with phorbol myristate acetate or FMLP in the presence of dihydrocytochalasin B. It had an affinity for FMLPL-SASD of 145 nM (cf. 0.3 nM for the cell surface receptor). The specificity for the formyl group was lost as the nonformylated Met-Leu-Phe was as effective FMLPL in competing with FMLPL-SASD-125I for binding to th Mr 24 000 species. A structurally unrelated peptide, however, did not compete for the binding. The labelling of the Mr 24 000 species was dependent on the presence of Ca2+, as was its apparent Mr, which shifted from 24 000 to 50 000-70 000 in the presence of Ca2+. By incubating photoaffinity-labelled plasma membrane fractions with specific granule fractions, we could generate a receptor fragment of Mr 24 000, although the relationship to this fragment of the specific granule species is unknown at present. The N-terminal sequence of the Mr 24 000 species was determined and it appears to be a novel protein. Further work will allow its relationship to the receptor, if any, to be elucidated and allow assignment of a function to this potentially important molecule.     

Synthesis and degradation in vivo of a phosphoprotein from rat dental enamel. Identification of a phosphorylated precursor protein in the extracellular organic matrix.

The cellular enamel organ and the cell-free organic matrix of developing enamel of female rats injected intravascularly with [3H]serine and [3H]proline were extracted in a number of solvents and examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and h.p.l.c. in 6M-guanidinium chloride at intervals varying from 5 min to 1 week after injection. Three major species soluble in NH4HCO3 with Mr values of approx. 100 000, 25 000 and 11 000 were identified in the cellular enamel organ. The Mr 100 000 and 11 000 components were not secreted but remained intracellular for periods of up to 1 week after injection of the radioactively labelled amino acids. In contrast, the Mr 25 000 species was secreted from the cells and was first detected in the extracellular organic matrix approx. 15-30 min after injection. With time, labelled components, first of Mr approx. 11 000 and subsequently approx. 6500, were detected in the organic matrix concomitant with a relative decrease in the Mr 25 000 component, demonstrating that the lower Mr species were derived from degradation of the putative extracellular precursor protein (Mr 25 000). All of the extracellular components were found to contain O-phosphoserine. No radioactively labelled component with an Mr greater than approx. 25 000, either an amelogenin or an enamelin, was observed in the extracellular organic matrix or in an intracellular component which subsequently was lost from the intracellular pool. The Mr of the highest Mr protein or class of proteins is calculated to be approx. 22 000-26 000 when standard proteins are used as markers, but only 15 000-18 000 when using the CNBr peptides of alpha 1 chains of rat tail tendon collagen as markers.     

Processing of the gp55-116 envelope glycoprotein complex (gB) of human cytomegalovirus.

The processing pathway of the major envelope glycoprotein complex, gp55-116 (gB), of human cytomegalovirus was studied using inhibitors of glycosylation and endoglycosidases. The results of these studies indicated that the mature gp55-116 is synthesized by the addition of both simple and complex N-linked sugars to a nonglycosylated precursor of estimated Mr 105,000. In a rapid processing step, the Mr 105,000 precursor is glycosylated to a protein of Mr 150,000 (gp150) which contains only endoglycosidase H-sensitive sugar linkages. The gp150 is then processed relatively slowly to a Mr 165,000 to 170,000 species (gp165-170), which is then cleaved to yield the mature gp55-116. Monensin prevented the final processing steps of the gp150, including cleavage, suggesting that transport through the Golgi apparatus is required for complete processing. Digestion of the intracellular forms of this complex as well as the virion forms confirmed the above findings and indicated that the mature virion form of gp55 contains 8,000 daltons of N-linked sugars. The virion gp116 contains some 52,000 to 57,000 daltons of N-linked carbohydrates and approximately 5,000 daltons of O-linked sugars.