Bovine γ-Aminobutyric AcidA Receptor Sequence-Specific Antibodies: Identification of Two Epitopes Which Are Recognised in Both Native and Denatured γ-Aminobutyric AcidA Receptors

Polyclonal antibodies have been raised against synthetic peptides whose sequences correspond to the N-terminal 15 amino acids and the C-terminal 17 amino acids of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit. These antibodies were shown to react with the denatured GABAA receptor alpha subunit, Mr 53,000, in Western blots with both purified receptor and brain membranes as antigens. Also, both antibodies recognised both the purified and detergent-solubilised GABAA receptor as demonstrated by dose-dependent specific immunoprecipitation of the GABA and benzodiazepine binding sites from solution. Evidence is also presented to show brain-regional distribution of the expression of the alpha 1 subunit.     

Autophosphorylation of Mucor rouxii cAMP-dependent protein kinase and its role in holoenzyme activation.

Two phosphoproteins of 53,000 and 63,000 mol. wt detected in partially purified preparations of Mucor rouxii cAMP-dependent protein kinase submitted to phosphorylation conditions with [gamma-32P]ATP are demonstrated to be the result of the autophosphorylation of its regulatory subunit, according to the following criteria: (1) linearity of phosphate incorporation with enzyme sample; (2) independence of phosphate incorporation on temperature; (3) correlation of the phosphoproteins with enzymatic activity in a DEAE-Sepharose chromatography; (4) specific elution of the phosphorylated proteins from cAMP-agarose; (5) phosphorylation of the purified regulatory subunit. Antibodies specific against Mucor regulatory subunit detected an intact subunit of 72,000 mol. wt in crude extracts. Autophosphorylation of the fungal protein kinase A promotes activation of the holoenzyme by cAMP since: (1) under conditions of partial activation, increase of activity is observed when using the phosphoform of the enzyme; (2) release of free catalytic subunit from cAMP-agarose is enhanced when the holoenzyme is previously phosphorylated.     

The regulatory subunit of the type II cAMP-dependent protein kinase in rabbit ovaries is the RII beta isoform.

Based on RII autophosphorylation, photoaffinity labeling with 8-N3[32P]cAMP, and Western blot analysis we have identified the RII isoform found in rabbit corpora lutea as RII beta. The RII beta subunit found in rabbit corpora lutea differs from the RII beta found in rat follicles and corpora lutea in that it migrates at Mr 52,500 on SDS-PAGE and shifts to Mr 53,000 when phosphorylated.     

Phosphorylation and modulation of the enzymic activity of native and protease-cleaved purified hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase by a calcium/calmodulin-dependent protein kinase

A Ca2+/calmodulin-dependent kinase has been purified which catalyzed the phosphorylation and concomitant inactivation of both the microsomal native (100,000 Da) and protease-cleaved purified 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) (53,000 Da) fragments. This low molecular weight brain cytosolic Ca2+/calmodulin-dependent kinase phosphorylates histone H1, synapsin I, and purified HMG-CoA reductase as major substrates. The kinase, purified by sequential chromatography on DEAE-cellulose, calmodulin affinity resin, and high performance liquid chromatography (TSKG 3000 SW) is an electrophoretically homogeneous protein of approximately 110,000 Da. The molecular weight of the holoenzyme, substrate specificity, subunit protein composition, subunit autophosphorylation, subunit isoelectric points, and subunit phosphopeptide analysis suggest that this kinase of Mr 110,000 may be different from other previously reported Ca2+/calmodulin-dependent kinases. Maximal phosphorylation by the low molecular form of Ca2+/calmodulin-dependent kinase of purified HMG-CoA reductase revealed a stoichiometry of approximately 0.5 mol of phosphate/mol of 53,000-Da enzyme. Dephosphorylation of phosphorylated and inactivated native and purified HMG-CoA reductase revealed a time-dependent loss of 32P-bound radioactivity and reactivation of enzyme activity. Based on the results reported here, we propose that HMG-CoA reductase activity may be modulated by yet another kinase system involving covalent phosphorylation. The elucidation of a Ca2+/calmodulin-dependent HMG-CoA reductase kinase-mediated modulation of HMG-CoA reductase activity involving reversible phosphorylation may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.     

Cross-linking of endothelin 1 and endothelin 3 to rat brain membranes: identification of the putative receptor(s)

Affinity-labeling experiments with 125I-endothelin derivatives using bifunctional cross-linking reagents were carried out in an attempt to identify the polypeptide component(s) of the endothelin/sarafotoxin receptors in rat brain tissues. In rat cerebellum, cortex, and caudate putamen, endothelin 1 specifically labeled a major component with a molecular mass of around 53,000. In the same tissues endothelin 3 specifically labeled, in addition to the 53,000 band, a band of molecular mass of 38,000. This result clearly indicates that in the brain the endothelin binding site resides within a polypeptide of apparent Mr = 53,000. The possible presence of receptor subtypes is discussed with reference also to the reported identification of endothelin receptors in chick cardiac membrane and in rat mesangial cells.     

Heritable Variation in a Polypeptide Subunit of the Major Storage Protein of the Bean, Phaseolus vulgaris L

Electrophoretic analysis of the major seed protein, G1 globulin, from four strains of Phaseolus vulgaris L. revealed a three-banded pattern for two strains having a high methionine content (BBL 240 and PI 302,542). The other two strains (PI 207,227 and PI 229,815) known to have a lower seed methionine content, had a two-banded subunit pattern for the G1 globulin. Analytical ultracentrifugation confirmed that globulin from the two-banded strains underwent pH-dependent reversible dissociation similar to that previously found for a three-banded cultivar; additionally, the protomer molecular weight showed that three subunits of about 50,000 molecular weight each were present in the G1 globulin of the two-banded strain. Gel patterns of G1 globulin from the two strains used as parents, BBL 240 and PI 229,815, showed differences in the largest subunit, which appeared as either a 53,000 molecular weight polypeptide known to be present in the three-banded strain, or as a shorter polypeptide having a molecular weight close to 47,000. Analysis of G1 protein from portions of single hybrid seeds showed a banding pattern intermediate between the two- and three-banded types. The subunit pattern from all seeds with intermediate-banded parents segregated in a manner consistent with that expected for control of the polypeptide by a single Mendelian gene. The remaining portions of the seeds were grown to confirm that they represented true crosses. The procedures used are essentially nondestructive, and can be used as a basis for selecting seeds having different protein characters.     

Phosphorylation of the beta subunit of Na+K+-ATPase in Ehrlich ascites tumor by a membrane-bound protein kinase

We have shown previously that proteoliposomes reconstituted with purified Na+K+-ATPase from Ehrlich ascites tumor cells, transport Na+ with low efficiency (Spector, M., O'Neal, S. and Racker, E. (1980) J. Biol. Chem., 255, 5504-5507). We now present evidence that this low efficiency (expressed in the ratio of Na+-transported/ATP-hydrolyzed) is caused by the phosphorylation of the beta subunit of the Na+K+-ATPase by an endogenous protein kinase. On addition of [gamma-32P]ATP, crude tumor plasma membrane preparations phosphorylated the beta subunit of the ATPase, whereas crude mouse brain plasma membranes did not. However, solubilized Na+K+-ATPase from either tumor or brain wre phosphorylated by purified protein kinase from the tumor plasma membrane and dephosphorylated by a phosphatase. In both cases, the phosphorylated enzyme was inefficient; the dephosphorylated enzyme was efficient after reconstitution into liposomes. During isolation of the Na+K+-ATPase from Ehrlich ascites tumor or mouse brain, an endogenous protease partially cleaved from the beta subunit a polypeptide of 29,000 daltons that contained the phosphorylation site. The proteolytic cleavage of the beta subunit was partially inhibited by phenylmethylsulfonyl fluoride and the major site of phosphorylation was then seen in the 53,000-dalton beta subunit of the enzyme. The isolated 29,000-dalton polypeptide from mouse brain ATPase was phosphorylated by tumor protein kinase with a stoichiometry of 1 mol of phosphate/mol of protein. When this 29,000-dalton polypeptide from mouse brain was incorporated into the tumor Na+K+-ATPase after mild proteolytic digestion, a marked increase in efficiency was observed after reconstitution of the Na+ pump.     

COMPARATIVE PEPTIDE MAPPING AND ISOELECTRIC FOCUSING OF ISOLATED SUBUNITS FROM CHICK EMBRYO BRAIN TUBULIN

The α- and β-subunits of chick embryo brain tubulin have been isolated under denaturing conditions and compared with respect to their molecular weight, amino acid composition, tryptic peptide maps, amide content and isoelectric focusing properties. An 8 M-Urea-containing polyacrylamide gel system with varying acrylamide concentrations was used for calculation of the retardation coefficients (K_R) of the tubulin subunits. A molecular weight of 53,000 was estimated for each subunit by comparison to K_R values for standard proteins. Amide contents of approx 41% of the carboxyl groups of α-tubulin and 48% of the carboxyl groups of β-tubulin were calculated using the average PI value, the pK_intrinsic for the ionizable side chains of the amino acids and the amino acid composition of each subunit. Comparative peptide maps of trypsin digested α- and β-tubulin demonstrated 16 peptides unique to each subunit and 23 peptides which comigrate. Both subunits give rise to multiple species on electrofocusing gels. The average isoelectric points for the α- and β-subunits are 5.4 and 5.2, respectively.     

Equal Expression of the Maternal and Paternal Alleles for the Polypeptide Subunits of the Major Storage Protein of the Bean Phaseolus vulgaris L

Discontinuous sodium dodecyl sulfate slab gel electrophoresis of G1 globulin from several strains of Phaseolus vulgaris L. seed permitted clear resolution of the constituent polypeptides. Three strains (Tendergreen, Canadian Wonder, and BBL 240) had subunits of molecular weight 53,000, 47,000 and 43,000 while two strains (Seafarer and PI 229,815) had 50,500, 47,000 and 43,000 molecular weight subunits. F₁ seed from the cross BBL 240 × PI 229,815 showed four polypeptides on dissociation of the G1 protein; however, the amount of each of the 53,000 and 50,500 subunits was half that of the 47,000 subunit. This is interpreted as evidence that both the maternal and paternal loci for these polypeptides are transcribed and translated with similar efficiency. All of the polypeptides were found to have associated sugar residues.     

Localization of the catalytic subunit of a cyclic AMP-dependent protein kinase(S) and acceptor proteins on the external surface of the fat cell membrane.

Cyclic AMP in μM concentrations increases the labeling of a membrane component of approximately 53,000 daltons as well as the labeling of a minor peptide of 18,000 daltons when isolated, intact rat fat cells are incubated with μM concentrations of (γ-³²P)ATP. Controlled tryptic digestion of intact cells followed by incubation with (γ-³²P)ATP results in diminution of labeling of both of these phosphopeptides indicating susceptibility, hence, access of either the catalytic site or the substrates to trypsin action. Addition of the catalytic subunit of the cyclic AMP-dependent protein kinase from beef heart to cells previously treated with trypsin results in the labeling of both phosphopeptides comparable to untreated cells. These findings indicate the catalytic subunit(s) of the cyclic AMP-dependent protein kinase(s) as well as these two phosphopeptides of the intact rat fat cell must be located on the external surface of the plasma membrane. Further, the catalytic subunit(s) of the membrane-located cyclic AMP-dependent protein kinase(s) is susceptible to trypsin action whereas the membrane peptides serving as substrates are not.     

Alpha 2-macroglobulin used to isolate intracellular endopeptidases from mammalian cells in culture.

Extracts of cell cultures labelled with [3H]leucine were incubated with human alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor. The proteinase-alpha 2M complexes were then precipitated with immobilized monoclonal antibodies to alpha 2M and analysed by SDS/polyacrylamide-gel electrophoresis. Parallel experiments were done with methylamine-inactivated alpha 2M to check for unspecific binding of cell proteins to alpha 2M. Several 3H-labelled cell proteins bound to active, but not to inactivated, alpha 2M. Such proteins are likely to be proteinases. Putative endopeptidases of subunit Mr 112000, 78,000, 53,000, and in some experiments 88,000 and 16,000, were trapped by alpha 2M in supernatant fractions from IMR90 human fibroblasts, EBTr bovine fibroblasts and HeLa human carcinoma cells. No additional proteins were trapped in the presence of ATP. The Mr-78,000 endopeptidase was identified as calpain II by immunoblotting. At pH 5.3 putative endopeptidases of subunit Mr 80,000, 53,000 and 28,000-32,000 were trapped from IMR90-fibroblast extracts. Immunoblotting showed that both cathepsin B and cathepsin D were present in the Mr-28,000-32,000 electrophoretic bands. The use of alpha 2M and immobilized antibody to alpha 2M thus allows a rapid enrichment of endopeptidases from cell extracts. Some potentials and limitations of the method are discussed.     

Purification and Properties of Succinyl-CoA:3-Oxo-Acid CoA-Transferase from Rat Brain

Abstract: Rat brain succinyl-CoA:3-oxo-acid CoA-transferase (3-Oxo-acid CoA-transferase, EC 2.8.3.5), the first committed enzyme in the oxidation of ketone bodies in mitochondria, was purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. The enzyme has an apparent molecular weight of 90,000 as determined by (3-150 Sephadex chromatography, and an apparent subunit molecular weight of 53,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was approximately 161 μmol/min/mg of protein. Initial velocity studies of the forward reaction (acetoacetate → acetoacetyl-CoA) are consistent with a "ping pong" mechanism. Substrate inhibition appears above approximately 1 m M acetoacetate. Apparent K_m, values were 70 μM for acetoacetate and 156 μ M for succinyl-CoA (the forward reaction), and 59 μ M for acetoacetyl-CoA and 25 m M for succinate (the reverse reaction). These values are markedly different from those reported for this enzyme from pig heart.     

Chromosome-mediated gene transfer of hydroxyurea resistance and amplification of ribonucleotide reductase activity.

Metaphase chromosomes purified from a hydroxyurea-resistant Chinese hamster cell line were able to transform recipient wild-type cells to hydroxyurea resistance at a frequency of 10(-6). Approximately 60% of the resulting transformant clones gradually lost hydroxyurea resistance when cultivated for prolonged periods in the absence of drug. One transformant was subjected to serial selection in higher concentrations of hydroxyurea. The five cell lines generated exhibited increasing relative plating efficiency in the presence of the drug and a corresponding elevation in their cellular content of ribonucleotide reductase. The most resistant cell line had a 163-fold increase in relative plating efficiency and a 120-fold increase in enzyme activity when compared with the wild-type cell line. The highly hydroxyurea-resistant cell lines had strong electron paramagnetic resonance signals characteristic of an elevated level of the free radical present in the M2 subunit of ribonucleotide reductase. Two-dimensional electrophoresis of cell-free extracts from one of the resistant cell lines indicated that a 53,000-dalton protein was present in greatly elevated quantities when compared with the wild-type cell line. These data suggest that the hydroxyurea-resistant cell lines may contain an amplification of the gene for the M2 subunit of ribonucleotide reductase.     

delta-Aminolevulinate synthase isozymes in the liver and erythroid cells of chicken

Antibodies raised against the purified chicken liver delta-aminolevulinate synthase showed a partial cross-reactivity with the chicken erythroid delta-aminolevulinate synthase. delta-Aminolevulinate synthase synthesized in vitro using polysomes from erythroid cells showed a subunit molecular weight of 55,000, whereas the enzyme synthesized in vitro using liver polysomes had a subunit molecular weight of 73,000. delta-Aminolevulinate synthase isolated from mitochondria of erythroid cells showed a molecular weight of 53,000, while the enzyme in liver mitochondria had a value of 65,000. These observations imply that the erythroid delta-aminolevulinate synthase differs from the hepatic enzyme.     

Effect of plasma biotinyl-peptides on biotinidase activity

Purified biotinidase (enriched 24,000-fold) from fresh human plasma exhibited reduced catalytic activity when incubated with heat-inactivated dialyzed plasma. The polypeptide fractions separated from the heat-inactivated dialyzed plasma using streptavidin-Sepharose resin showed the same effect on purified biotinidase. These inhibitory effects on biotinidase were partial (25-45%) rather than complete. The polypeptide fraction from streptavidin-Sepharose resin was analyzed by SDS-PAGE in the Laemmli system and by various types of HPLC. Analyses by ion-exchange and reversed-phase HPLC revealed the existence of three relatively small mol. wt polypeptides. Each of these peak fractions exhibited similar inhibitory effects on biotinidase activity. SDS-PAGE analysis indicated that the streptavidin affinity resin fraction was composed of four major polypeptides whose mol. wts were 120,000, 76,000, 53,000 and 27,000. The two bands of 120,000 and 76,000 corresponded to the mol. wts of the biotinyl subunit of pyruvate carboxylase, beta-methyl-crotonyl-CoA and/or propionyl-CoA carboxylase respectively. However, the polypeptides of mol. wts 53,000 and 27,000 were found to be two unique biotinyl-peptides present in human plasma. These bands on the gels were transblotted and exhibited a fluorescent activity after incubated with a FITC-avidin. These findings strongly suggest the existence of circulating plasma biotinyl-polypeptides as inhibitory factor(s) on human plasma biotinidase.     

Immunocytochemical Localization of (Na+,K+)-ATPase in the Goldfish Optic Nerve

Antiserum to the catalytic subunit of goldfish brain (Na+, K+)-ATPase has been employed at the electron microscopic level by means of the peroxidase-antiperoxidase immunohistochemical method. In optic nerve, antigenic sites are restricted to the nodes of Ranvier. No reaction product is detected in underlying internodal neurolemma. Outgrowing neurites for cultured retinal explants devoid of glial ensheathment exhibit a continuous distribution of the enzyme subunit. Antibodies against eel electroplax (Na+, K+)-ATPase cross-react with the goldfish brain enzyme and show a similar immunocytochemical distribution pattern.     

Structural studies and two-dimensional gel electrophoresis of gamma-glutamyl transpeptidase

The heterodimeric enzyme gamma-glutamyl transpeptidase (EC 2.3.2.2) was isolated from adult rat kidney and purified to homogeneity for structural studies using papain solubilization and multiple chromatographies. Two-dimensional gel electrophoresis was found to resolve the active papain-purified enzyme into at least 18 components. Seven components with apparent molecular masses of 23,000-26,000 and isoelectric point range of 5.4-7.0 constitute the light subunit, and 11 components with apparent molecular mass of 51,000-53,000 and isoelectric point range of 5.8-7.1 constitute the heavy subunit. Immunoblot analysis of two-dimensional gels showed that all of these components are immunoreactive with a mixture of the two antibodies generated separately against the light and heavy subunits. Preparative subunit separation was achieved using reverse-phase HPLC under acidic but nonreducing conditions. N-Terminal amino acid sequencing of the separated subunits of the papain-purified enzyme yielded sequence information for the first 32 residues of the heavy chain with the N-terminal starting sequence Gly-Lys-Pro-Asp-His-Val-Tyr-Ser-Arg-Ala, and for the first 36 residues of the light subunit with the N-terminal starting sequence Thr-Ala-His-Leu-Ser-Val-Val-Ser-Glu-Asp.     

Structural sequences are conserved in the genes coding for the alpha, alpha' and beta-subunits of the soybean 7S seed storage protein.

Cloned DNAs encoding four different proteins have been isolated from recombinant cDNA libraries constructed with Glycine max seed mRNAs. Two cloned DNAs code for the alpha and alpha'-subunits of the 7S seed storage protein (conglycinin). The other cloned cDNAs code for proteins which are synthesized in vitro as 68,000 d., 60,000 d. or 53,000 d. polypeptides. Hybrid selection experiments indicate that, under low stringency hybridization conditions, all four cDNAs hybridize with mRNAs for the alpha and alpha'-subunits and the 68,000 d., 60,000 d. and 53,000 d. in vitro translation products. Within three of the mRNA, there is a conserved sequence of 155 nucleotides which is responsible for this hybridization. The conserved nucleotides in the alpha and alpha'-subunit cDNAs and the 68,000 d. polypeptide cDNAs span both coding and noncoding sequences. The differences in the coding nucleotides outside the conserved region are extensive. This suggests that selective pressure to maintain the 155 conserved nucleotides has been influenced by the structure of the seed mRNA. RNA blot hybridizations demonstrate that mRNA encoding the other major subunit (beta) of the 7S seed storage protein also shares sequence homology with the conserved 155 nucleotide sequence of the alpha and alpha'-subunit mRNAs, but not with other coding sequences.     

A crystal form of ribulose-1,5-bisphosphate carboxylase/oxygenase from Nicotiana tabacum in the activated state

A new crystal form of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from Nicotiana tabacum has been obtained at alkaline pH with polyethylene glycol 8000 in the presence of a non-ionic detergent, beta-octyl glucoside. The crystals are grown at room temperature by the hanging-drop vapor diffusion technique from a protein solution containing enzyme complexed with CO2, Mg2+, and the transition state analog 2-C-carboxy-D-arabinitol-1,5-bisphosphate. The crystals belong to the the space group P3(1)21 (or P3(2)21) with the cell parameters a = 204.6 A, and c = 117.4 A (1 A = 0.1 nm). The asymmetric unit contains half (L4S4: L, large subunit, 53,000 Mr; S, small subunit, 15,000 Mr) of a hexadecameric molecule (L8S8, 540,000 Mr). The crystals diffract to at least 2.6 A Bragg spacing and are suitable for X-ray structure determination.     

Cross-Reaction of Anti-Rat B-50: Characterization and Isolation of a "B-50 Phosphoprotein" from Bovine Brain

Abstract: Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent M_r 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH_1-24) (10⁻⁵ M and 10⁻⁴ M ) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of M_r 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the M_r of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the M _r of the "B-50 protein" varies from species to species.