Protein Of Apparent Mr Research Articles

Mammalian red cell membrane Rh polypeptides are selectively palmitoylated subunits of a macromolecular complex.

Incubation of [3H]palmitic acid, ATP, and CoA with inside-out membrane vesicles prepared from human or other mammalian red cells resulted in nearly exclusive 3H-palmitoylation of the Mr = 32,000 Rh polypeptides. [3H]Palmitic, [3H]myristic, and [3H]oleic acids were comparably esterified onto Rh polypeptides in inside-out membrane vesicles in the presence of ATP and CoA, although [3H]palmitic acid was preferentially incorporated by intact human red cells. Experiments using sulfhydryl reagents or tryptic digestions suggested that multiple sulfhydryl groups on the Rh polypeptides located near the cytoplasmic leaflet of the lipid bilayer were 3H-palmitoylated; the exofacial sulfhydryl group essential for Rh antigenic reactivity was not 3H-palmitoylated. Transfer of fatty acid from [14C]palmitoyl-CoA to sites on the Rh polypeptides occurred even after previous incubation of inside-out membrane vesicles at 95 degrees C or after solubilization of inside-out membrane vesicles in Triton X-100. Hydrodynamic analyses of Triton X-100-solubilized membranes surprisingly demonstrated that 3H-palmitoylated Rh polypeptides behaved as a protein of apparent Mr = 170,000. These in vitro studies suggest that palmitoylation of Rh polypeptides occurs within a macromolecular complex by a highly selective but possibly nonenzymatic mechanism.

Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium

A protein of apparent Mr = 15,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the major plasma membrane substrate for cAMP-dependent protein kinase (PK-A) and protein kinase C (PK-C) in several different tissues. In the work described here, we purified, cloned, and sequenced the canine cardiac sarcolemmal "15-kDa protein." The amino terminus of the purified protein was not blocked, allowing determination of 50 consecutive residues by standard Edman degradation. Overlapping proteolytic phosphopeptides yielded 22 additional residues at the carboxyl terminus. Dideoxy sequencing of the full-length cDNA confirmed that the 15-kDa protein contains 72 amino acids, plus a 20-residue signal sequence. The mature protein has a calculated Mr = 8409. There is one hydrophobic membrane-spanning segment composed of residues 18-37. The acidic amino-terminal end (residues 1-17) of the protein is oriented extracellularly, whereas the basic carboxyl-terminal end (residues 38-72) projects into the cytoplasm. The positively charged carboxyl terminus contains the phosphorylation sites for PK-A and PK-C. In the transmembrane region, the 15-kDa protein exhibits 52% amino acid identity with the "gamma" subunit of Na,K-ATPase. High stringency Northern blot analysis revealed that 15-kDa mRNA is present in heart, skeletal muscle, smooth muscle, and liver but absent from brain and kidney. We propose the name "phospholemman" for the 15-kDa protein, which denotes the protein's location within the plasma membrane and its characteristic multisite phosphorylation.

Characterization of the phosphorylation sites in the chicken and bovine myristoylated alanine-rich C kinase substrate protein, a prominent cellular substrate for protein kinase C

Little is known about the important cellular substrates for protein kinase C and their potential roles in mediating protein kinase C-dependent processes. We evaluated the protein kinase C phosphorylation sites in a major cellular substrate for the kinase, a protein of apparent Mr 80,000 in bovine and 60,000 in chicken tissues; we have recently determined the primary sequences of these proteins and tentatively named them the myristoylated alanine-rich C kinase substrates. The proteins were purified to apparent homogeneity from bovine and chicken brains, phosphorylated with protein kinase C, digested with trypsin, and the phosphopeptides purified and sequenced. Four distinct phosphopeptides were identified from both the bovine and chicken proteins. Two of the phosphorylated serines were contained in the repeated motif FSFKK, one in the sequence LSGF, and one in the sequence SFK. All four sites were contained within a basic domain of 25 amino acids which was identical in the chicken and bovine proteins. All of the sites phosphorylated in the cell-free system appeared to be phosphorylated in intact cells; an additional site may have been present in the proteins from intact cells. The identity of the phosphorylation site domains from two proteins of overall 65% amino acid sequence identity suggests a potential role for this domain in the physiological function of the myristoylated alanine-rich C kinase substrate proteins.

Steroidogenesis Activator Polypeptide May be a Product of Glucose Regulated Protein 78 (GRP78)

Cholesterol side-chain cleavage is sensitive to antibiotic inhibitors of protein synthesis, suggesting that a labile protein may play a regulatory role in this process. We have previously characterized such a factor--steroidogenesis activator polypeptide (SAP). Given the low molecular weight of SAP (Mr 3215), a SAP precursor has been sought. Using immunoblotting techniques with two polyclonal antisera directed against portions of the SAP sequence, a single protein of apparent Mr 82,000 (p82) can be detected in rat adrenocortical tissue. Our data suggest that adrenal p82 is most likely the widely-distributed minor heat shock protein, glucose regulated protein 78 (GRP78). The two proteins share biochemical attributes, including pI (5.2) and ATP affinity, and the reported amino acid sequences for SAP and for the carboxyl-terminal end of GRP78 are nearly identical. We propose that SAP is cleaved from GRP78--or a cognate protein--and that this proteolysis is regulated in a manner characteristic of steroidogenic tissues.

The brain corticotropin-releasing factor (CRF) receptor is of lower apparent molecular weight than the CRF receptor in anterior pituitary. Evidence from chemical cross-linking studies.

The ligand binding subunits of the corticotropin-releasing factor (CRF) receptors in brain and anterior pituitary of a number of species have been identified by chemical affinity cross-linking using the homobifunctional cross-linking agent disuccinimidyl suberate and 125I-Tyr0-oCRF (ovine CRF). In homogenates of rat, monkey, and human cerebral cortex, 125I-Tyr0-oCRF was covalently incorporated into a protein of Mr = 58,000. Under identical conditions in the anterior pituitary of rat, monkey, cow, and pig, 125I-Tyr0-oCRF was incorporated into a protein of apparent Mr = 75,000. The specificity of the labeling was typical of the CRF binding site since both the cerebral cortex- and pituitary-labeled proteins exhibited the appropriate pharmacological rank order profile characteristic of the CRF receptor (Nle21,Tyr32-oCRF approximately equal to rat/human CRF approximately equal to ovine CRF approximately equal to alpha-helical CRF(6-41) greater than alpha-helical oCRF(9-41) greater than or equal to oCRF(7-41) greater than rat/human CRF(1-20) approximately equal to vasoactive intestinal peptide). In addition to the major labeled proteins, 125I-Tyr0-oCRF was incorporated into higher molecular weight peptides which may represent precursors and into lower molecular weight components which may represent fragments of the major labeled proteins or altered forms of the CRF binding subunit. In summary, these data indicate a heterogeneity between brain and pituitary CRF receptors with the ligand binding subunit of the brain CRF receptor residing on a Mr = 58,000 protein, while in the anterior pituitary, the identical binding subunit resides on a protein of apparent Mr = 75,000.

The cloning, DNA sequence, and overexpression of the gene araE coding for arabinose-proton symport in Escherichia coli K12.

A lambda placMu1 insertion was made into araE, the gene for arabinose-proton symport in Escherichia coli. A phage containing an araE'-'lacZ fusion was recovered from the lysogen and its restriction map compared with that of the 61-min region of the E. coli genome to establish the gene order thyA araE orf lysR lysA galR; araE was transcribed toward orf. A 4.8-kilobase SalI-EcoRI DNA fragment containing araE was subcloned from the phage lambda d(lysA+ galR+ araE+) into the plasmid vector pBR322. From this plasmid a 2.8-kilobase HincII-PvuII DNA fragment including araE was sequenced and also subcloned into the expression vector pAD284. The araE gene was 1416-base pairs long, encoding a hydrophobic protein of 472 amino acids with a calculated Mr of 51,683. The amino acid sequence was homologous with the xylose-proton symporter of E. coli and the glucose transporters from a human hepatoma HepG2 cell line, human erythrocytes, and rat brain. The overexpressed araE gene product was identified in Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell membranes as a protein of apparent Mr 35,000 +/- 1,150. Arabinose protected this protein against reaction with N-ethylmaleimide.

Characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Evidence that the initiation factor is a monomeric protein of Mr of about 58,000-62,000.

Eukaryotic initiation factor 5 (eIF-5) has been purified from the ribosomal salt-wash proteins of rabbit reticulocyte lysates. The purified factor migrates as a single polypeptide upon sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of about 58,000-62,000. In contrast, less pure preparations of reticulocyte eIF-5 behave in gel filtration columns and in glycerol gradient centrifugation in buffers containing 75-100 mM KCl as a protein of apparent Mr = 140,000-160,000. Presumably, this is due to association of the factor with other proteins, since eIF-5 activity present in such preparations can also be shown by (a) glycerol gradient centrifugation in buffers containing 500 mM KCl or (b) gel electrophoresis under denaturing conditions, to be associated with a 58,000-62,000-dalton protein. Furthermore, eIF-5 purified from rabbit reticulocyte lysates in the absence or presence of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity. It can be calculated that 1 pmol of the purified eIF-5 catalyzes the formation of nearly 50 pmol of 80 S initiation complex under in vitro initiation reaction conditions. Because of the highly catalytic activity of eIF-5 in initiation reactions, the presence of even low levels of eIF-5 in eIF-2 preparations causes hydrolysis of GTP bound to the 40 S initiation complex. This results in destabilization of Met-tRNA(f) bound to the 40 S complex in sucrose gradient centrifugation.

Coordinate expression of a small polypeptide with the lactose carrier of Escherichia coli.

Induction of the lac operon in wild type Escherichia coli strains results in synthesis of a 16-kDa inner membrane protein in addition to the known products of the lacZ, lacY, and lacA genes. Cells carrying the lacY gene on a multicopy plasmid overproduce this 16-kDa polypeptide as well as the Lac carrier, the membrane protein product of the lacY gene. However, [35S]methionine labeling of minicells carrying the lacY plasmid shows that the 16-kDa protein is not synthesized from the plasmid DNA. We have purified and partially characterized the 16-kDa protein. It is an acidic membrane protein of apparent Mr = 15,800 whose amino-terminal sequence (NH2-Met-Arg-Asn-Phe-Asp-Leu-) does not match any known lac operon DNA sequence. Using antibody prepared to the purified 16-kDa protein, we have quantitatively analyzed conditions under which this protein is made and have shown that the amount of 16-kDa protein which appears in the membrane is proportional to lac operon expression. Hybridization of a synthetic oligodeoxyribonucleotide probe complementary to the 5' end of 16-kDa protein mRNA shows that its synthesis is regulated at the level of transcription.

Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli.

A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4.

Identification of the GalP galactose transport protein of Escherichia coli.

Escherichia coli strains have been isolated with a transposon 10 insertion or an amber mutation inactivating the galP gene, which specifies the galactose-H+ (GalP) transport system. Comparison of the membrane proteins between these strains and their GalP+ parents by dual isotope analysis showed that a component of Mr = 34-39,000 was consistently absent from the GalP- mutants. Galactose, methyl-beta-D-galactoside, and talose protected the GalP transport system from inactivation by N-ethylmaleimide. A membrane protein of Mr = 34-38,000 was modified by N-([2-3H]ethyl)maleimide at the binding site of these sugars. Two-dimensional gel electrophoresis of the membrane proteins has resolved a component of Mr = 35-38,000 (average apparent pI = 5.7) present in parent strains (GalP+) but not in the GalP- mutants. These observations identified a protein of apparent Mr = 37,000 as the product of the galP gene of E. coli.

In vitro biosynthesis and processing of immunologically identified methionine-enkephalin precursor protein.

The biosynthesis and initial processing of the methionine-enkephalin precursor preproenkephalin A were examined by cell-free translation of mRNA from brain and adrenal medulla. A novel antiserum raised against Met-enkephalin-Arg6-Phe7 was shown to react with bovine adrenal medulla fractions (apparent Mr 34,000) containing proenkephalin A. Affinity-purified antibodies from this antiserum were used to immunoprecipitate cell-free translated [35S]Met-enkephalin-containing protein. A protein of apparent Mr 30,000 +/- 500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was the only Met-enkephalin precursor consistently synthesized by translation of mRNA from bovine or guinea pig striatum, rat brain, or bovine adrenal medulla. The presence of [35S]Met-enkephalin sequences in this protein was confirmed by high pressure liquid chromatography of trypsin/carboxypeptidase B digests. Dog pancreas endoplasmic reticulum membranes converted the Mr 30,000 protein to an immunoreactive protein of apparent Mr 28,500 that lacked significant core glycosylation. These results suggest that 1) a protein similar or identical to bovine adrenal medullary preproenkephalin A is the major Met-enkephalin precursor synthesized in brain as well as adrenal medulla, and 2) preproenkephalin A is converted to a protein resembling proenkephalin A, presumably by removal of a signal peptide.

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.

Enzymatic synthesis of a glucose-containing oligosaccharide-lipid involved in glycosylation of proteins

Earlier studies on the biosynthesis of an oligosaccharide-lipid with the structure (alpha-Man)4-6-beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)-GlcNAc-P-P-dolichol have been extended to show that enzymes in membrane preparations of hen oviduct catalyze synthesis of a Glc-containing oligosaccharide-lipid that is similar in properties to the Glc-free oligosaccharide-lipid. Conditions that enable enzymatic preparation of Man-, GlcNAc-, or Glc-labeled oligosaccharide-lipid have been established. Experiments with isolated [Glc-14C]oligosaccharide-lipid as substrate demonstrated that the oligosaccharide chain was transferred en bloc from the lipid to an endogenous membrane protein of apparent Mr = 25,000. In addition, an exogenous soluble protein, S-carboxymethylated alpha-lactalbumin, was shown to serve as acceptor of the oligosaccharide chain from the Glc-containing oligosaccharide-lipid. Studies on the metabolic fate of the Glc residues in these proteins are reported in the accompanying paper (Chen, W. W., and Lennarz, W. J. (1978) J. Biol. Chem. 253, 5780-5785).