Purification and Characterization of the Major Lipoprotein (P28) of Spiroplasma apis

The plasma membrane of Spiroplasma apis contains a 28-kDa major protein (P28), like other spiroplasmas which also possess a main 26- to 28-kDa membrane polypeptide, called spiralin. In the work described here, we have developed a simple and efficient method for the purification of P28 of this mollicute, a wall-less eubacteria. Proteins were first selectively extracted from the isolated membrane with the mild detergents (i) sodium N-lauroylsarcosinate (Sarkosyl) and (ii) 3-[(3-cholamidopropyl)dimethylamonio]-1-propyl sulfonate (Chaps) and subjected to size-exclusion HPLC in the presence of Chaps. The P28-enriched fraction was thereafter subjected to the second chromatographic step involving cation exchange HPLC in the presence of the same detergent. P28 was purified at the milligram level (yield, 40%). Metabolite labeling with [¹⁴C]palmitic acid and chemical analysis of P28 indicated that it is covalently modified by two O-ester-bound fatty acids and one amide-linked chain and contains a S-glycerylcysteine at the N-terminus. By charge-shift electrophoresis, Triton X-114 phase separation, and growth inhibition tests it was shown that P28 is a typical amphiphilic protein exposed, at least partly, at the cell surface. Together, our data provided evidence that P28 is a “classical” lipoprotein (i.e., triacylated) like the members of the spiralin family.     

Chemical analysis of processing of spiralin, the major lipoprotein of Spiroplasma melliferum

The plasma membrane of Spiroplasma melliferum contains a major membrane-associated lipoprotein called spiralin. In this study, the processing pathway of spiralin was investigated by chemical analysis of the purified protein and by using [35S]cysteine, [35S]methionine, [14C]myristic acid (14C-14:0), [14C]palmitic acid (14C-16:0), and globomycin. SDS-PAGE analysis of membrane proteins showed the leader peptide cleavage of prospiralin and provided evidence for an apparent selectivity in the acylation: the unprocessed protein was labelled with 14C-16:0 only (O-ester-linked acyl chains), and the mature form with both 14C-labelled fatty acids (O-ester-linked + amide-linked chains). Chemical analysis of the purified protein revealed that spiralin contains S-glycerylcysteine and is covalently modified with two O-ester-linked acyl chains and one amide-linked fatty acid chain. However, a specific selectivity in the O- and the N-acylations was not confirmed; palmitate and stearate were the major components. The amounts of O-ester- and amide-linked acyl chains, the resistance to Edman degradation and the presence of S-glycerylcysteine together indicate that spiralin is a "classical" lipoprotein (i.e. is triacylated) and is probably processed by a mechanism similar to that described for gram-negative eubacteria. On the basis of these findings, a biogenesis pathway for spiralin is proposed.     

Topology and acylation of spiralin.

Of the 51 polypeptides detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the plasma membrane of the helical mollicute Spiroplasma melliferum, 21 are acylated, predominantly with myristic (14:0) and palmitic (16:0) chains. This is notably the case for spiralin, the major membrane protein of this bacterium, which contains an average of 0.7 acyl chains per polypeptide, attached very probably by ester bonds to alcohol amino acids. The amphiphilicity of spiralin was demonstrated by the behavior of the protein in charge-shift electrophoresis, its incorporation into liposomes, and its ability to form in the absence of lipids and detergents, globular protein micelles (diameter, approximately 15 nm). The presence of epitopes on the two faces of the cell membrane, as probed by antibody adsorption and crossed immunoelectrophoresis, and the strong interaction between spiralin and the intracytoplasmic fibrils show that spiralin is a transmembrane protein. The mean hydropathy of the amino acid composition of spiralin (-0.30) is on the hydrophilic side of the scale. Surprisingly, the water-insoluble core of spiralin micelles, which is the putative membrane anchor, has a still more hydrophilic amino acid composition (mean hydropathy, -0.70) and is enriched in glycine and serine residues. Taking into account all these properties, we propose a topological model for spiralin featuring a transbilayer localization with hydrophilic domains protruding on the two faces of the membrane and connected by a small domain embedded within the apolar region of the lipid bilayer. In this model, the membrane anchoring of the protein is strengthened by a covalently bound acyl chain.     

Purification and characterization of 55-kDa protein with 3,5,3'-triiodo-L-thyronine-binding activity and protein disulfide-isomerase activity from beef liver membrane

Beef liver membranes were shown to have different kinds of 3,5,3'-triiodo-L-thyronine binding proteins including the 55-kDa protein which had been reported to have this activity in many cells by affinity labelling with N-bromoacetyl-3,5,3'-[125I]triiodo-L-thyronine. In order to characterize the molecular features of these binding proteins, the 55-kDa protein was purified from a beef liver membrane fraction abundant in the plasma membrane. The protein was solubilized with 0.5% Chaps and purified by chromatography on gel filtration, hydroxyapatite, and Mono Q anion-exchange columns. The purity was confirmed with reversed-phase HPLC and SDS/PAGE. Consequently, 0.4% of the total proteins in the membrane fraction was recovered as the 55-kDa protein. One fourth of the amino acid composition of this protein was Glx (14.6%) plus Asx (11.7%) and the pI of this protein was 4.5. The purified protein has triiodothyronine-binding activity with a Kd of 57 nM which is similar to the high-affinity binding site of the membranes. The anti-(55-kDa protein) sera specifically recognized the 55-kDa protein of beef, rat and human cells. The immunoglobulin G fraction of the anti-(55-kDa protein) sera inhibited triiodothyronine binding to the beef liver membrane fraction. The purified protein also showed the activity of protein disulfide-isomerase (EC 5.3.4.1) as determined by reactivating scrambled ribonuclease. These data strongly suggested that the multi-functional 55-kDa protein which has triiodothyronine-binding activity and the activity of protein disulfide-isomerase, which is also reported to be the beta subunit of prolyl-4-hydroxylase, glycosylation-site-binding protein of oligosaccharyl transferase and iodothyronine 5'-monodeionidase, could be significant in the action of triiodothyronine towards the target cells.     

Solubilization of somatostatin receptors in hamster pancreatic beta cells. Characterization as a glycoprotein interacting with a GTP-binding protein

Somatostatin receptors of plasma membranes from beta cells of hamster insulinoma were covalently labelled with 125I-[Leu8,D-Trp22,Tyr25]somatostatin-28 (125I-somatostatin-28) and solubilized with the non-denaturing detergent Triton X-100. Analysis by SDS/PAGE and autoradiography revealed three specific 125I-somatostatin-28 receptor complexes with similar molecular masses (228 kDa, 128 kDa and 45 kDa) to those previously identified [Cotroneo, P., Marie, J.-C. & Rosselin, G. (1988) Eur. J. Biochem. 174, 219-224]. The major labelled complex (128 kDa) was adsorbed to a wheat-germ-agglutinin agarose column and eluted by N-acetylglucosamine. Also, the binding of 125I-somatostatin-28 to plasma membranes was specifically inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate) (GTP[S]) in a dose-dependent manner. Furthermore, when somatostatin-28 receptors were solubilized by Triton X-100 as a reversible complex with 125I-somatostatin-28, GTP[S] specifically dissociated the bound ligand to a larger extent from the soluble receptors than from the plasma-membrane-embedded receptors, the radioactivity remaining bound after 15 min at 37 degrees C being 30% and 83% respectively. After pertussis-toxin-induced [32P]ADP-ribosylation of pancreatic membranes, a 41-kDa [32P]ADP-ribose-labelled inhibitory guanine nucleotide binding protein coeluted with the 128-kDa and 45-kDa receptor complexes. The labelling of both receptor proteins was sensitive to GTP[S]. The labelling of the 228-kDa band was inconsistent. These results support the conclusion that beta cell somatostatin receptors can be solubilized as proteins of 128 kDa and 45 kDa. The major labeled species corresponds to the 128-kDa band and is a glycoprotein. The pancreatic membrane contains a 41-kDa GTP-binding protein that can complex with somatostatin receptors.     

Convulsant/barbiturate activity on the soluble gamma-aminobutyric acid-benzodiazepine receptor complex

Cage convulsant t-butyl bicyclophosphoro[35S]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (Chaps) and shown to co-purify with the benzodiazepine--gamma-aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X-100, or by addition of Triton X-100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA-benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X-100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3-5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.     

Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation.

Spiralin is defined as the major membrane protein of the helical mollicute Spiroplasma citri. According to the S. citri strain used, spiralin shows polymorphism in its electrophoretic mobility. The spiralin gene sequences of eight S. citri strains were determined by direct sequencing of the PCR-amplified genes. All spiralins were found to be 241 amino acids long, except for the spiralin of strain Palmyre, which is 242 amino acids long. The molecular masses calculated from these sequences did not explain the differences observed in the electrophoretic mobilities. In all of the spiralins examined, the first 24 N-terminal amino acids were conserved, including a cysteine at position 24, and had the features of typical signal peptides of procaryotic lipoproteins. When S. citri strains were grown in the presence of [3H]palmitic acid, at least 10 proteins, including spiralin, became labeled. In the presence of globomycin, a lipoprotein signal peptidase inhibitor in eubacteria, apparently unprocessed spiralin could be detected. Formic acid hydrolysis of the [3H]palmitic acid-labeled spiralins of four representative S. citri strains yielded two peptide fragments for each spiralin, as expected from the gene sequence. On fragment was [3H]palmitic acid labeled, and it had almost the same electrophoretic mobility irrespective of the spiralins used. Samples of the unlabeled peptide fragments from the four representative strains had slightly different electrophoretic mobilities (delta Da approximately equal to 800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (delta Da approximately equal to 8,000 Da). These results suggest that spiralin polymorphism in S. citri is not due to differences in posttranslational modification by palmitic acid and is certainly a structural property of the whole protein or could result from an unidentified posttranslational modification of spiralin.     

Isolation of the adherence protein of Mycoplasma pneumoniae by fractionated solubilization and size exclusion chromatography

The 168-kDa adherence protein of M. pneumoniae was solubilized and purified to homogeneity. Optimal yield was obtained by pretreatment of whole M. pneumoniae cells with buffer containing 1% Chaps and subsequent extraction with octylglucosid at a detergent to protein ratio of 5 and at octylglycoside concentrations between 1.5 and 2%. Contaminating membrane proteins with high molecular masses were removed by pretreatment with 1% Chaps and proteins of low molecular masses by size exclusion chromatography.     

A protein partially expressed on the surface of HepG2 cells that binds lipoproteins specifically is nucleolin

Nucleolin, a major nucleolar protein of rapidly growing eukaryotic cells, has been thought to be predominantly if not exclusively located in the nucleolus. Recent data however [Borer, R.A., Lehner, C.F., Eppenberger, H.M., & Nigg, N.A. (1989) Cell 56, 379-390] suggest that the protein shuttles constantly between the nucleus and cytoplasm. Ligand blotting studies of whole cell extracts of HepG2 cells identified, in addition to the LDL receptor, another LDL binding protein of Mr 109,000. The 109-kDa protein was partially purified by HPLC and, like the LDL receptor, bound apoB- and apoE-containing lipoproteins but not HDL. However, unlike the LDL receptor, the 109-kDa protein bound lipoproteins in the presence of EDTA and reducing agents, had a lower affinity for lipoproteins than the LDL receptor, and did not react with two antibodies raised against the LDL receptor. The protein sequences of three separate peptides derived from the partially purified 109-kDa species were determined and were identical except for one residue to three separate regions of the published sequence of nucleolin. On immunoblot analysis the 109-kDa protein reacted with a nucleolin-specific antibody, and purified nucleolin reacted both with anti-109-kDa antibody and with LDL. When intact HepG2 cells were treated with Pronase before harvest, there was a 46% decrease in 109-kDa protein while recovery of actin, an intracellular protein, was unaffected. When intact HepG2 cells were surface iodinated and the proteins subjected to HPLC fractionation, the 109-kDa protein was found to be iodinated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation of Serine 41 in the Neuron-Specific Protein B-50 (GAP-43) Prohibits Phosphorylation by Protein Kinase C

The neuron-specific, calmodulin-binding protein B-50 (also known as GAP-43, F1, or neuromodulin) is an endogenous substrate of protein kinase C (PKC). PKC exclusively phosphorylates Ser residues in B-50. As potential phosphorylation sites for PKC, Ser41, Ser110, and Ser122 were indicated, of which Ser41 is contained in the sequence ASF, which matches with the sequence of a synthetic PKC substrate. N-terminally 35S-labeled B-50, produced from cDNA, was subjected to digestion with Staphylococcus aureus V8 protease (SAP). Consecutively, 35S-labeled 28- and 15-kDa fragments were formed, similar to those after digestion of 32P-labeled B-50. In a previous study, we showed that the 32P-labeled 15-kDa SAP fragment contains all 32P radioactivity. The present data indicate that it contains the N-terminus of B-50 as well. The 15-kDa fragment, with a calculated length ranging from amino acid residue 1 to 65, contains only one potential PKC phosphorylation site, at Ser41. Mutagenesis of Ser41 into Thr or Ala resulted in recombinant B-50 products with mobilities on two-dimensional electrophoresis similar to those of the nonmutated recombinant B-50 and the rat brain B-50. Only [Ser41]B-50 was phosphorylated by PKC, whereas [Thr41]- or [Ala41]B-50 did not show any phosphorylation at the positions indicated on the immunoblots. This leads us to the conclusion that Ser41 is the sole phosphorylation site for PKC in vitro.     

Evidence for opioid receptor-mediated activation of the G-proteins, Go and Gi2, in membranes of neuroblastoma x glioma (NG108-15) hybrid cells.

In membranes of neuroblastoma x glioma (NG108-15) hybrid cells, the photoreactive GTP analog, [alpha-32P] GTP azidoanilide, was incorporated into 39-41-kDa proteins comigrating in urea-containing sodium dodecyl sulfate-polyacrylamide gels with immunologically identified G-protein alpha-subunits, i.e. a 39-kDa Go alpha-subunit, a 40-kDa Gi2 alpha-subunit, and a 41-kDa Gi alpha-subunit of an unknown subtype. The synthetic opioid, D-Ala2,D-Leu5-enkephalin (DADLE), stimulated photolabeling of the 39-41-kDa proteins. In the presence of GDP, which increased the ratio of agonist-stimulated to basal photolabeling, DADLE at a maximally effective concentration stimulated photolabeling of the 39- and the 40-kDa protein 2-3-fold. Somatostatin, adrenaline, and bradykinin were less potent than DADLE and, to varying degrees, stimulated photolabeling of the 40-kDa protein more than that of the 39-kDa protein. Prostaglandin E1 was inactive. The present data represent direct evidence for an activation of endogenous Go and Gi2 via opioid receptors and other receptors in the native membrane milieu.     

Characterization of structural domains of the human epidermal growth factor receptor obtained by partial proteolysis

Partial cleavage with trypsin has been used to study the structure of the epidermal growth factor (EGF) receptor purified from human carcinoma cells. Following affinity labeling of the receptor with 125I-EGF or the ATP analogue 5'-p-fluorosulfonyl benzoyl[14C]adenosine, metabolic labeling with [35S]methionine, [3H]glucosamine, or [32P]orthophosphate, or in vitro autophosphorylation with [gamma-32P]ATP, tryptic cleavage defines the following three regions of the 180-kDa receptor protein: 1) a 125-kDa trypsin-resistant domain which contains sites of glycosylation, EGF binding, and an EGF-specific threonine phosphorylation site; 2) an adjacent 40-kDa fragment which contains serine and threonine phosphorylation sites and is further cleaved to a 30-kDa trypsin-resistant domain; and 3) a terminal 15-kDa portion of the receptor that contains the sites of tyrosine phosphorylation and is degraded to small fragments in the presence of trypsin. Both the 125- and 40-kDa regions of the EGF receptor appear to be required for receptor-associated protein kinase activity since separation of these regions by tryptic cleavage abolishes this activity, and both regions are specifically labeled with an ATP affinity analogue, suggesting that both are involved in ATP binding. Additional 63- and 48-kDa phosphorylated fragments are generated upon trypsin treatment of EGF receptor from EGF-treated cells. The potential usefulness of partial tryptic cleavage in studying the EGF receptor and the possible biological function of the 30-kDa trypsin-resistant fragment of the receptor are discussed.     

Solubilization and characterization of guinea-pig pancreatic somatostatin receptors

The solubilization of somatostatin receptors from guinea-pig pancreas by different non-denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I-[Tyr11]somatostatin or its analogue 125I-[Leu8,DTrp22,Tyr25]somatostatin 28, to pancreatic plasma membranes. The somatostatin-receptor complexes were solubilized in a high yield by Zwittergent 3-14 (3-[tetradecyldimethylammonio]-1-propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X-100, Chaps (3-[cholamidopropyldimethylammonio]-1-propanesulfonate) and octyl beta-D-glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol. In order to characterize solubilized somatostatin-receptor complexes, membranes receptors were covalently labelled using N-5-azido-2-nitrobenzoyloxysuccinimide as cross-linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr = 93,000 which interacted with the two radioligands. In addition, a similar component of Mr = 88,000 was characterized after analysis by SDS-PAGE of membrane receptors covalently cross-linked with 125I-[Leu8,DTrp22,Tyr25]somatostatin 28 by different heterobifunctional reagents: N-5-azido-2-nitrobenzoyloxysuccinimide, N-hydroxysuccinimidyl 4-azidobenzoate, N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate. Optimal cross-linking results were obtained with N-5-azido-2-nitrobenzoyloxysuccinimide. The solubilized somatostatin-receptor complex was adsorbed to wheat-germ agglutinin-agarose column and eluted by specific sugars. We concluded that the guinea-pig pancreatic somatostatin receptor in the membrane and in the non-denaturing detergent solution behaves as a protein monomer of apparent Mr approximately 85,000-90,000. The somatostatin receptor is a glycoprotein which contains complex-type carbohydrate chains.     

Localization of ras antigenicity in rat hepatocyte plasma membrane and rough endoplasmic reticulum fractions

We have examined the antigenicity of plasma membrane (PM) and rough microsomal (RM) fractions from rat liver using anti-ras monoclonal antibodies 142-24EO5 and Y13-259 and immunochemistry as well as electron microscope immunocytochemistry. Proteins immunoprecipitated with monoclonal antibody 142-24E05 were separated using single-dimensional gradient-gel electrophoresis. The separated proteins were then blotted onto nitrocellulose sheets and incubated with [alpha-32P]GTP. Radioautograms of blots indicated the presence of specific 21.5- and 22-kDa labeled proteins in the PM fraction. A 23.5-kDa [alpha-32P] GTP-binding protein was detected in immunoprecipitates of both PM and RM fractions. Monoclonal antibody Y13-259 reacted only with the 21.5-kDa [alpha-32P] GTP-binding protein in the plasma membrane fraction. When anti-ras monoclonal antibody 142-24E05 and the immunogold technique were applied to membrane fractions using a preembedding immunocytochemical method, specific labeling was observed in association with both vesicular structures and membrane sheets in the PM fraction but only with electron-dense vesicular structures in the RM fraction. Thus ras antigenicity is associated with hepatocyte plasma membranes and ras-like antigenicity is probably associated with vesicular (secretory/endocytic) elements in both plasma membrane and rough microsomal preparations.     

Evidence for an Association of the 15-kDa Proteolipid of Mediatophore with a 14-kDa Polypeptide

The present report shows that mediatophore, a nerve terminal membrane protein that translocates acetylcholine on calcium action, forms a complex with a 14-kDa polypeptide. The complex was identified based on the following results. (a) A polyclonal antimediatophore antiserum that immunoprecipitates activity precipitates both the 15- and 14-kDa polypeptides. (b) After HPLC purification of mediatophore, both antigens were found in the same peak. (c) After 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of presynaptic membranes or of the purified mediatophore, an immunoaffinity column made with the anti-14-kDa antigen monoclonal antibody retained both the 14-kDa and the 15-kDa polypeptide. Similarly, immunoprecipitation experiments using protein A-coated beads sedimented an immunocomplex in which both antigens were found. (d) The 14-kDa antigen could be localized in the synaptosomal membrane where mediatophore and its 15-kDa component are found.     

Purification and molecular characterization of ortho-chlorophenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans.

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positive Desulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a Vmax of 28 units/mg protein and a Km of 20 microM. The pH and temperature optimum were 8.2 and 52 degrees C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (Em) = -440 mV), one [3Fe-4S] cluster (Em = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an Em of -370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.     

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.     

Cyclic beta-(1,2)-glucan synthesis in Rhizobiaceae: roles of the 319-kilodalton protein intermediate.

Cyclic beta-(1,2)-glucans are synthesized by members of the Rhizobiaceae family through protein-linked oligosaccharides as intermediates. The protein moiety is a large inner membrane molecule of about 319 kDa. In Agrobacterium tumefaciens and in Rhizobium meliloti the protein is termed ChvB and NdvB, respectively. Inner membranes of R. meliloti 102F34 and A. tumefaciens A348 were first incubated with UDP-[14C]Glc and then solubilized with Triton X-100 and analyzed by polyacrylamide gel electrophoresis under native conditions. A radioactive band corresponding to the 319-kDa protein was detected in both bacteria. Triton-solubilized inner membranes of A. tumefaciens were submitted to native electrophoresis and then assayed for oligosaccharide-protein intermediate formation in situ by incubating the gel with UDP-[14C]Glc. A [14C]glucose-labeled protein with an electrophoretic mobility identical to that corresponding to the 319-kDa [14C]glucan protein intermediate was detected. In addition, protein-linked radioactivity was partially chased when the gel was incubated with unlabeled UDP-Glc. A heterogeneous family of cyclic beta-(1,2)-glucans was formed upon incubation of the gel portion containing the 319-kDa protein intermediate with UDP-[14C]Glc. A protein with an electrophoretic behavior similar to the 319-kDa protein intermediate was "in gel" labeled by using Triton-solubilized inner membranes of an A. tumefaciens exoC mutant, which contains a protein intermediate without nascent glucan. These results indicate that initiation (protein glucosylation), elongation, and cyclization were catalyzed in situ. Therefore, the three enzymatic activities detected in situ reside in a unique protein component (i.e., cyclic beta-(1,2)-glucan synthase). It is suggested that the protein component is the 319-kDa protein intermediate, which might catalyze the overall cyclic beta-(1,2)-glucan synthesis.     

Quantitative solubilization of nonhistone chromosomal proteins without denaturation using zwitterionic detergents

Of three kinds of commercial zwitterionic detergents [SB 12, SB 14, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)], SB 12 and Chaps were more useful than SB 14 because of high solubility and less interference with protein assay. Efficiency for protein solubilization at pH 6-9 was higher for SB 12 than for Chaps with either calf thymus chromatin or rat liver nuclei. At pH 9 and ionic strength (I) = 0.35, 1% SB 12 and 1% Chaps were capable of solubilizing about 70% and about 47% of total proteins in rat liver nuclei, respectively. Core histones in rat liver nuclei were extracted to a lesser extent with Chaps than with SB 12. DNA-dependent RNA polymerase and isopeptidase activities were barely inactivated by 1% Chaps at pH 8-9, but isopeptidase activity was inhibited by 0.3% SB 12. These facts indicate that whereas SB 12 is effective for solubilization of whole nuclear proteins, Chaps is suitable for the selective extraction of nonhistone chromosomal proteins without denaturation.     

Effects of guanine nucleotides on cholera toxin catalyzed ADP-ribosylation in rat adipocyte plasma membranes

ADP-ribosylation of rat adipocyte plasma membrane proteins was investigated following incubation of membranes with [alpha-32P]NAD and cholera toxin in the presence and absence of various guanine nucleotides. In membranes incubated without guanine nucleotides, cholera toxin induced incorporation of 32P into three discrete proteins of 48, 45, and 41 kDa. In membranes containing 100 microM GTP or GDP, toxin-catalyzed incorporation of 32P into the 41-kDa protein was inhibited. GMP and Gpp(NH)p (100 microM) allowed moderate incorporation of 32P into the 41-kDa protein. Toxin-catalyzed labeling of all proteins was rapid, reaching maximal levels between 5 and 10 min. Toxin-catalyzed ADP-ribosylation of the 48- and 45-kDa proteins was stimulated by GTP, reaching maximal levels at 10(-5) M GTP. Inhibition of toxin-dependent labeling of the 41-kDa protein required GTP concentrations above 10(-7) M with complete inhibition occurring between 10(-5) and 10(-4) M GTP. Cholera toxin catalyzed ADP-ribosylation was increased up to 2-fold in membranes supplemented with adipocyte cytosol. These results indicate that cholera toxin catalyzes ADP-ribosylation of three distinct adipocyte plasma membrane proteins, each of which is regulated by the amount and type of added guanine nucleotides.