Characterization of a phospholipase C beta 2-binding site near the amino-terminal coiled-coil of G protein beta gamma subunits

In previous work (Sankaran, B., Osterhout, J., Wu, D., and Smrcka, A. V. (1998) J. Biol. Chem. 273, 7148-7154), we showed that overlapping peptides, N20K (Asn(564)-Lys(583)) and E20K (Glu(574)-Lys(593)), from the catalytic domain of phospholipase C (PLC) beta2 block Gbetagamma-dependent activation of PLC beta2. The peptides could also be directly cross-linked to betagamma subunits with a heterobifunctional cross-linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. Cross-linking of peptides to Gbeta(1) was inhibited by PLC beta2 but not by alpha(i1)(GDP), indicating that the peptide-binding site on beta(1) represents a binding site for PLC beta2 that does not overlap with the alpha(i1)-binding site. Here we identify the site of peptide cross-linking and thereby define a site for PLC beta2 interaction with beta subunits. Each of the 14 cysteine residues in beta(1) were altered to alanine. The ability of the PLC beta2-derived peptide to cross-link to each betagamma mutant was then analyzed to identify the reactive sulfhydryl moiety on the beta subunit required for the cross-linking reaction. We find that C25A was the only mutation that significantly affected peptide cross-linking. This indicates that the peptide is specifically binding to a region near cysteine 25 of beta(1) which is located in the amino-terminal coiled-coil region of beta(1) and identifies a PLC-binding site distinct from the alpha subunit interaction site.     

Identification of an essential arginine residue in the beta subunit of the chloroplast ATPase

The ATPase activity of soluble chloroplast coupling factor (CF1) was irreversibly inactivated by phenylglyoxal, an arginine reagent. Under the conditions of inactivation, 2.48 mol of [14C]phenylglyoxal were incorporated per 400,000 g of enzyme when the ATPase was inactivated 50% by the reagent. Isolation of the component polypeptide subunits of the [14C]phenylglyoxal-modified enzyme revealed that the distribution of moles of labeled reagent/mol of subunit was the following: alpha, 0.37; beta, 0.40; gamma, 0.08; delta, none; epsilon, 0.03. CNBr treatment of the isolated alpha and beta subunits and fractionation of the peptides by gel electrophoresis revealed that the radioactivity bound to the alpha subunit was nonspecifically associated with several peptides, while a single peptide derived from the beta subunit contained the majority of the radioactivity associated with this subunit. After treating the isolated beta subunit with trypsin and Staphylococcus aureus protease, a major radioactive peptide was isolated with a sequence Arg-Ile-Thr-Ser-Ile-Lys. This sequence, when compared with the primary structure of the CF1 beta subunit as translated from the gene (Zurawski, G., Bottomley, W., and Whitfeld, P. R. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6260-6264) indicated that the arginine marked with the asterisk, the predominant residue modified by phenylglyoxal when the ATPase activity of CF1 is inactivated by the reagent, is Arg 312.     

Covalent structure of collagen: amino acid sequence of three cyanogen bromide-derived peptides from human alpha 1(V) collagen chain

Type V collagen was prepared from human amnionic/chorionic membranes and separated into alpha 1(V) and alpha 2(V) polypeptide chains. The alpha 1(V) chain was digested with cyanogen bromide and nine peptides were obtained and purified. Three of the peptides, alpha 1(V)CB1, CB4, and CB7 having molecular weights of 5000, 8000, and 6000, respectively, were further analyzed by amino acid sequence analysis and thermolytic or tryptic digestions. CB1 contained 54 amino acids and identification of its complete sequence was aided by thermolysin digestion and isolation of two peptides, Th1 and Th2. CB4 contained 81 amino acids and sequence analysis of intact CB4 and five tryptic peptides provided us with its complete amino acid sequence. The peptide CB7 contained 67 amino acids and was cleaved into four tryptic peptides that were used for complete sequence analysis. The above results represent the first available covalent structure information on the alpha 1(V) collagen chain. These data enabled us to establish the location of these peptides within the helical structure of other collagen chains. CB4 was homologous to residues 66-145 in the collagen chain while CB1 represented residues 146-200 and CB7 was homologous with residues 201-269. This alignment was facilitated by identification of a helical collagen crossing site consisting of Hyl-Gly-His-Arg located at positions 87-90 in all collagen chains of this size thus far identified. Seventy-one percent homology (excluding Gly residues) was found between amino acids in this region of the alpha 1(XI) and of alpha 1(V) collagen chains while only 21 and 19% identity was calculated for the same region of alpha 2(V) and alpha 1(I) collagen chains, respectively.     

Binding site for fungal beta-lactone hymeglusin on cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase

We studied the molecular mechanism through which the fungal beta-lactone, hymeglusin, potently and specifically inhibits 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. [(14)C]Hymeglusin covalently bound to purified rat liver and to recombinant hamster cytosolic HMG-CoA synthases. The enzyme activity was completely inhibited at a binding ratio of 1.6-2.0 mol [(14)C]hymeglusin/mol HMG-CoA synthase. Incubating the enzyme with 2 mM iodoacetamide (IAA) or 2 mM N-ethylmaleimide (NEM) but not with 1.0 mM diisopropyl fluorophosphates (DFP) completely inhibited the binding, suggesting that hymeglusin binds to a Cys residue of HMG-CoA synthase. Recombinant hamster HMG-CoA synthase labeled with [(3)H]hymeglusin was digested with V8 protease, and the [(3)H]peptide was purified by high performance liquid chromatography (HPLC). The sequence of the peptide was Ser-Gly-Asn-Thr-Asp-Ile-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-[(3)H]hymeglusyl Cys-Tyr-Gly-Gly-Thr-Ala-Ala-Val-Phe-Asn-Ala-Val-Asn-, which corresponds to the active site sequence (from Ser 115 to Asn 141) of hamster HMG-CoA synthase. These findings showed that hymeglusin inhibits hamster cytosolic HMG-CoA synthase by covalently modifying the active Cys 129 residue of the enzyme.     

Biosynthesis in vitro of a globoside containing a 2-acetamido-2-deoxy-beta-D-galactopyranosyl group (1----3)-linked and Forssman glycolipid by two N-acetylgalactosaminyltransferases from chemically transformed guinea pig cells

Two N-acetylgalactosaminyltransferase activities (GalNAcT-2 and GalNAcT-3) have been characterized in chemically transformed, cultured guinea-pig cell lines (104C1 and 106B). Line 104C1 is a benz[a]pyrene-transformed tumorigenic variant, whereas line 106B is a 7,12-dimethylbenz[a]anthracene-transformed nontumorigenic variant obtained from fetal guinea-pig cells at 43 days of gestation. The GalNAcT-2 (UDP-GalNAc:GbOse3Cer beta-N-acetylgalactosaminyltransferase) isolated from both 104C1 and 106B cells catalyzed the transfer of Gal-NAc from UDP-GalNAc to the 3H-labeled terminal galactose group of Gb3 [( 6-3H]Gal alpha 1----4Gal beta 1----4Glc----Cer). The 3H-labeled globoside was purified and then subjected to exhaustive methylation. After acetolysis, the partially methylated sugars were separated by two-dimensional, thin-layer chromatography. 3H-Label was detected in two major areas, 2,4,6-tri-O-Me-Gal (40%) and 2,3,4,6-tetra-O-Me-Gal (46%). In a separate experiment, 80% of the GalNAc was released when labeled GbOse4Cer [( 3H]GalNAc----Gal alpha 1----4Gal beta 1----4Glc----Cer) was treated with purified clam beta-hexosaminidase. The present results establish the formation of a beta-D-GalpNAc-(1----3) linkage in the terminal region of the biosynthesized globoside. GalNAcT-3 activity (UDP-GalNAc:GbOse4Cer alpha-GalNAc-transferase), which catalyzes the transfer of GalNAc from UDP-[14C]- or -[3H]GalNAc to GbOse4Cer (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc----Cer), was three times higher in 106B cells than in 104C1 cells. The isolated, purified radioactive product formed an immunoprecipitin line against rabbit anti-Forssman antibody.     

Affinity labeling of Escherichia coli phenylalanyl-tRNA synthetase at the binding site for tRNAPhe

Periodate-oxidized tRNA(Phe) (tRNA(oxPhe)) behaves as a specific affinity label of tetrameric Escherichia coli phenylalanyl-tRNA synthetase (PheRS). Reaction of the alpha 2 beta 2 enzyme with tRNA(oxPhe) results in the loss of tRNAPhe aminoacylation activity with covalent attachment of 2 mol of tRNA dialdehyde/mol of enzyme, in agreement with the stoichiometry of tRNA binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the PheRS-[14C]tRNA(oxPhe) covalent complex indicates that the large (alpha, Mr 87K) subunit of the enzyme interacts with the 3'-adenosine of tRNA(oxPhe). The [14C]tRNA-labeled chymotryptic peptides of PheRS were purified by both gel filtration and reverse-phase high-performance liquid chromatography. The radioactivity was almost equally distributed among three peptides: Met-Lys[Ado]-Phe, Ala-Asp-Lys[Ado]-Leu, and Lys-Ile-Lys[Ado]-Ala. These sequences correspond to residues 1-3, 59-62, and 104-107, respectively, in the N-terminal region of the 795 amino acid sequence of the alpha subunit. It is noticeable that the labeled peptide Ala-Asp-Lys-Leu is adjacent to residues 63-66 (Arg-Val-Thr-Lys). The latter sequence was just predicted to resemble the proposed consensus tRNA CCA binding region Lys-Met-Ser-Lys-Ser, as deduced from previous affinity labeling studies on E. coli methionyl- and tyrosyl-tRNA synthetases [Hountondji, C., Dessen, P., & Blanquet, S. (1986) Biochimie 68, 1071-1078].     

Binding of 1alpha,25-dihydroxyvitamin D(3) to annexin II: effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1alpha,25-(OH)(2)D(3) and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [(14)C]-1alpha,25-(OH)(2)D(3) bromoacetate to purified annexin II was inhibited by 1alpha, 25-(OH)(2)D(3) in a concentration-dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1alpha, 25-(OH)(2)D(3) at 24 microM, 18 microM, and 12 microM and blunted by 6 microM and 3 microM. At a concentration of 12 microM, 1beta, 25-(OH)(2)D(3) also diminished the binding of [(14)C]-1alpha, 25-(OH)(2)D(3) bromoacetate to annexin II, but cholecalciferol, 25-(OH)D(3), and 24,25-(OH)(2)D(3) did not. Saturation analyses of the binding of [(3)H]-1alpha,25-(OH)(2)D(3) to purified annexin II showed a K(D) of 5.5 x 10(-9) M, whereas [(3)H]-1beta,25-(OH)(2)D(3) exhibited a K(D) of 6.0 x 10(-9) M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration-dependent effect on [(14)C]-1alpha,25-(OH)(2)D(3) bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1alpha,25-(OH)(2)D(3) binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1beta,25-(OH)(2)D(3) to inhibit the binding of [(14)C]-1alpha, 25(OH)(2)D(3) bromoacetate to annexin II provides a biochemical explanation for the ability of the 1beta-epimer to inhibit the rapid actions of the hormone in vitro.     

Biosynthesis of peptides containing α-aminoadipic acid and cysteine in extracts of a Cephalosporium sp

1. Three intracellular peptides found in small amount in a Cephalosporium sp. were rapidly labelled when dl-[(14)C]valine was added to a shaken suspension of the organism. More (14)C was incorporated into peptide P3, delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine, than into peptide P2 (containing alpha-aminoadipic acid, cysteine, valine and glycine) or peptide P1 (containing beta-hydroxyvaline in place of the valine in peptide P2). 2. Peptides P3 and P2, but not peptide P1 were formed in a broken-cell system from the Cephalosporium sp. in the presence of delta-(l-alpha-aminoadipyl)-l-cysteine and dl-[(14)C]valine. No synthesis was observed in the presence of delta-(d-alpha-aminoadipyl)-l-cysteine or of dl-alpha-amino[(14)C]adipic acid and l-cysteinyl-l-valine or l-cysteinyl-d-valine. 3. The biosynthesis of these peptides was catalysed by the particulate fraction of the broken-cell system, whereas that of glutathione was catalysed by the supernatant fraction. 4. These results are discussed in relation to penicillin N and cephalosporin C biosynthesis.     

Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme.

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-alpha,alpha-1,1-glucose) and maltose (glucosyl-alpha1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of approximately 68 kDa. However, active enzyme exhibited a molecular mass of approximately 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42-45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in >or= 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8-10% free glucose. The K(m) for maltose was approximately 10 mm, whereas for trehalose it was approximately 90 mm. While beta,beta-trehalose, isomaltose (alpha1,6-glucose disaccharide), kojibiose (alpha1,2) or cellobiose (beta1,4) were not substrates for TreS, nigerose (alpha1,3-glucose disaccharide) and alpha,beta-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [(3)H]Trehalose is converted to [(3)H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [(14)C]maltose produces [(14)C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as [(3)H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of [(3)H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry.     

Human placenta type V collagens. Evidence for the existence of an alpha 1(V) alpha 2(V) alpha 3(V) collagen molecule

Human type V collagen was purified from placenta and found to contain alpha 1(V), alpha 2(V), and alpha 3(V) chains in varying ratios. Using any of three independent nondenaturing methods (phosphocellulose chromatography, high-performance ion-exchange chromatography on IEX-540 DEAE, and ammonium sulfate precipitation), this preparation could be resolved into two fractions. Analysis of the two fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that one fraction contained alpha 1(V) and alpha 2(V) in a 2:1 ratio and the other contained alpha 1(V), alpha 2(V), and alpha 3(V) in a 1:1:1 ratio. When the crude placental type V collagen was electrophoresed under nondenaturing conditions, two bands were observed, one co-migrating with purified (alpha 1(V]2 alpha 2(V) and the other co-migrating with the fractions containing alpha 1(V), alpha 2(V), and alpha 3(V) chains in a 1:1:1 ratio. Electrophoresis in a second dimension under denaturing conditions confirmed that the fast-migrating band contained (alpha 1(V]2 alpha 2(V) and that the slow-migrating band contained the three chains in equimolar ratio. CD spectra of the two fractions and resistance to trypsin-chymotrypsin digestion confirmed that the two fractions contain triple helical collagen. Thermal denaturations were monitored by the changes in CD signal at 221 nm. The two fractions purified by ammonium sulfate precipitation melted at 39.1 and 36.4 degrees C for the (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) fractions, respectively. Trypsin cleavage of these two native fractions at temperatures near melting produced completely different fragmentation patterns, indicating different partial unwinding sites of the alpha 1(V) and alpha 2(V) chains in the two preparations and thus different molecular assemblies. Our data demonstrate the existence of two different molecular assemblies of type V collagen in human placenta consisting of (alpha 1(V]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V) heterotrimers.     

Biosynthesis of Glycosyldiglycerides in Mycobacterium smegmatis

A particulate enzyme preparation from Mycobacterium smegmatis catalyzes the transfer of [(14)C]galactose from uridine 5'-diphosphate (UDP)-[(14)C]galactose and of [(14)C]glucose from UDP-[(14)C]glucose into chloroform-soluble products. The radioactive neutral lipids were purified by passage through diethylaminoethyl-cellulose, followed by thin-layer chromatography. When UDP-glucose was used as substrate, two major radioactive lipids were obtained; one had a hexose-glucose-glycerol ratio of 1:1:1. The second product had a hexose-glycerol ratio of 2:1 and, in addition to glucose, contained lesser amounts of mannose and galactose. With UDP-galactose as substrate, two radioactive products were observed that were chromatographically indistinguishable from the [(14)C]glucosyl-labeled mono- and diglycosyldiglyceride. Palmitate and oleate were the predominant fatty acid constituents in these lipids and were present in equimolar amounts in all of the products examined. The products have thus been identified as monoglycosyldiglyceride and a diglycosyldiglyceride containing glucose as the major hexose along with mannose and galactose. Properties of the galactosyl and glucosyl transferases are described.     

Photosensitized cleavage of dynein heavy chains. Cleavage at the V2 site by irradiation at 365 NM in the presence of oligovanadate

Irradiation of the outer-arm dynein ATPase from sea urchin sperm flagella at 365 nm in the presence of 50-200 microM vanadate (Vi) and 1 mM manganese acetate, in the absence of ATP, cleaves the alpha and beta heavy chains at a specific site, termed the V2 site, to form discrete peptides of Mr approximately 260,000 and 170,000 from the alpha chain and of Mr approximately 255,000 and 175,000 from the beta chain, with a yield of 80%. This cleavage at the V2 site is not correlated with any direct effect on the dynein ATPase activity. In the presence of 100 microM Vi, the half-times for cleavage of the alpha and beta chains are about 12 and 50 min, respectively. The rate of heavy chain cleavage shows a sigmoidal dependence upon Vi concentration, with half-maximal rate occurring at 58 +/- 7 microM, consistent with the chromophore responsible for cleavage being tri-vanadate. Addition of 10 microM ATP or ADP, or of 100 microM CTP or UTP, to the irradiation medium inhibits cleavage at the V2 site, and results in a slow cleavage occurring at the V1 site described previously. The peptides produced by sequential cleavage at the V2 and then the V1 sites indicate that the sites are separated by about 100,000 Da along the length of each heavy chain. Photoaffinity labeling with [alpha-32P] 8-azidoadenosine 5'-triphosphate (8-N3ATP) gives specific incorporation of 32P into both the Mr 255,000 and 175,000 peptides of the beta chain but into only the Mr 260,000 peptide of the alpha chain. These results suggest that V2 cleavage occurs on a loop of the heavy chain that forms part of the ATP-binding site, close to the locus of 8-N3ATP attachment.     

Labelling of the cytoplasmic domains of ovine rhodopsin with hydrophilic chemical probes

The disposition of polypeptide chain of ovine rhodopsin in the photoreceptor disc membrane was investigated by using two hydrophilic reagents, 3,5-di-[125I]iodo-4-diazobenzenesulphonate [( 125I]DDISA) and [14C]succinic anhydride. Both reagents were used to modify rhodopsin in intact disc membranes under conditions where no loss of A500 occurred. Reaction of [125I]DDISA with rhodopsin approached completion after 30 min. Binding was saturated at a 75-fold molar excess of reagent, which gave binding ratios of up to 2 mol/mol of rhodopsin. Proteolysis of rhodopsin, using Staphylococcus aureus V8 proteinase, yielded two membrane-bound fragments, both of which contained bound radioactive probe. Subsequent CNBr cleavage of these fragments produced five radiolabelled peptides which corresponded to the C-terminal region and cytoplasmic loops of rhodopsin. Similar studies with [14C]-succinic anhydride also gave binding ratios of up to 2 mol/mol of rhodopsin. Sequencing of the [14C]succinylated peptides identified the location of the reactive sites as lysine residues 66, 67, 141, 245, 248, 311, 325 and 339 in the polypeptide chain. Non-permeability of both probes was demonstrated by the absence of any radioactivity associated with the intradiscal N-terminal glycopeptide. Sonication of membranes in the presence of [125I]DDISA led to the incorporation of label in this peptide.     

Isolation and structural elucidation of a novel type of ganglioside, deaminated neuraminic acid (KDN)-containing glycosphingolipid, from rainbow trout sperm. The first example of the natural occurrence of KDN-ganglioside, (KDN)GM3

Rainbow trout sperm contained almost exclusively monoanionic ganglioside fraction as a major acidic glycosphingolipid. Two monoacidic gangliosides were isolated and purified in this study and designated as sperm ganglioside 1 and 2 (sg-1 and sg-2). The two gangliosides, sg-1 and sg-2, contained the same neutral sugars, galactose and glucose in molar ratio of 1:1 and no GalNAc except for the presence of N-acetyl-neuraminic acid (NeuAc) in sg-1 and deaminated neuraminic acid (KDN; 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) in sg-2. The complete structures of these gangliosides were determined by a combination of methylation analysis, fast atom bombardment mass spectrometry, 400-MHz one- and two-dimensional 1H nuclear magnetic resonance spectroscopy, fatty acid analysis, and endoglycoceramidase digestion NeuAc alpha 2----3Gal beta 1----4Glc beta 1----Cer sg-1 [(NeuAc)GM3] KDN alpha 2----3Gal beta 1----4Glc beta 1----Cer sg-2 [(KDN)GM3] where, for both sg-1 and sg-2, the ceramide moieties (Cer) were found to be made up of 4-sphingenine and mainly C16:0 fatty acid (palmitate; 95%) with a minor amount of C24:1 fatty acyl chain (nervonate, 5%). The structure of sg-2 is novel and represents the first example of a new class of gangliosides, i.e. KDN-gangliosides.     

Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons.

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.     

Identification of an essential glutamic acid residue in the beta subunit of the adenosine triphosphatase from the thermophilic bacterium PS3

The TF1-ATPase from the thermophilic bacterium, PS3, is inactivated by dicyclohexylcarbodiimide (DCCD). This inactivation is stimulated by ADP and other specific nucleotides and is inhibited by Mg2+. When the inactivation is carried out with [14C]DCCD, about 2 g atoms of 14C are bound/mol of TF1 when the enzyme is nearly completely inactivated. The isolated subunits from TF1 inactivated with [14C]DCCD contain the following amounts of 14C/mol: alpha, 0.12 g atom; beta, 0.47 g atom; gamma, approximately 0.04 g atom; delta, none; and epsilon, 0.05 g atom. Fractionation of tryptic digests have shown that the 14C bound to the alpha subunit is nonspecifically associated with several peptides, and that the 14C bound to the beta subunit is associated with a single tryptic peptide with the amino acid sequence Ala-Gly-Val-Gly-Glu-Arg, where Glu represents the N-gamma-glutamyl derivative of dicyclohexyl[14C]urea.     

Human placental estradiol 17 beta-dehydrogenase. Identification of a single histidine residue affinity-labeled by both 3-bromoacetoxyestrone and 12 beta-bromoacetoxy-4-estrene-3,17-dione

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.     

Transforming growth factor beta (TGF-beta) type V receptor has a TGF-beta-stimulated serine/threonine-specific autophosphorylation activity.

The transforming growth factor beta (TGF-beta) type V receptor, a newly identified high molecular weight TGF-beta receptor (M(r) approximately 400,000) has been purified from bovine liver plasma membranes (O'Grady, P., Kuo, M.-D., Baldassare, J. J., Huang, S. S., and Huang, J. S. (1991) J. Biol. Chem. 266, 8583-8589). The purified TGF-beta type V receptor underwent autophosphorylation at serine residues when incubated with [gamma-32P]ATP in the presence of 0.1% beta-mercaptoethanol and 2.5 mM MnCl2. This phosphorylation was stimulated by preincubation with TGF-beta. The preferred exogenous substrate for the Ser/Thr-specific phosphorylation activity of the type V receptor was found to be bovine casein. The TGF-beta type V receptor could be affinity-labeled with 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine. Polylysine appeared to stimulate the autophosphorylation of the TGF-beta type receptor in the presence of [gamma-32P]ATP and the incorporation of 5'-p-[adenine-8-14C]fluorosulfonylbenzoyl adenosine into the TGF-beta type V receptor. The amino acid sequence analysis of the peptide fragments produced by cyanogen bromide cleavage of the purified TGF-beta type V receptor revealed that a peptide, namely CNBr-19, contained an amino acid sequence which shows homology to the putative ATP binding site of the receptors for activin, the Caenorhabditis elegans daf-1 gene product, and TGF-beta type II receptor (Lin, H. Y., Wang, Y.-F., Ng-Eaton, E., Weinberg, R. A., and Lodish, H. F. (1992) Cell 68, 775-785). These results suggest that the TGF-beta type V receptor is a Ser/Thr-specific protein kinase and belongs to the new class of membrane receptors associated with a Ser/Thr-specific protein kinase activity.     

Affinity purification of the C alpha and C beta isoforms of the catalytic subunit of cAMP-dependent protein kinase

A synthetic peptide of 18 amino acids corresponding to the inhibitory domain of the heat-stable protein kinase inhibitor was synthesized and shown to inhibit both the C alpha and C beta isoforms of the catalytic (C) subunit of cAMP-dependent protein kinase. Extracts from cells transfected with expression vectors coding for the C alpha or the C beta isoform of the C subunit required 200 nM protein kinase inhibitor peptide for half-maximal inhibition of kinase activity in extracts from these cells. An affinity column was constructed using this synthetic peptide, and the column was incubated with protein extracts from cells overexpressing C alpha or C beta. Elution of the affinity column with arginine allowed single step isolation of purified C alpha and C beta subunits. The C alpha and C beta proteins were enriched 200-400-fold from cellular extracts by this single step of affinity chromatography. No residual inhibitory peptide activity could be detected in the purified protein. The purified C subunit isoforms were used to demonstrate preferential antibody reactivity with the C alpha isoform by Western blot analysis. Furthermore, preliminary characterization showed both isoforms have similar apparent Km values for ATP (4 microM) and for Kemptide (5.6 microM). These results demonstrate that a combination of affinity chromatography employing peptides derived from the heat-stable protein kinase inhibitor protein and the use of cells overexpressing C subunit related proteins may be an effective means for purification and characterization of the C subunit isoforms. Furthermore, this method of purification may be applicable to other kinases which are known to be specifically inhibited by small peptides.     

Identification of a novel binding site for platelet integrins alpha IIb beta 3 (GPIIbIIIa) and alpha 5 beta 1 in the gamma C-domain of fibrinogen

The interactions of platelets with fibrinogen mediate a variety of responses including adhesion, platelet aggregation, and fibrin clot retraction. Whereas it was assumed that interactions of the platelet integrin alpha IIb beta 3 with the AGDV sequence in the gamma C-domain of fibrinogen and/or RGD sites in the A alpha chains are involved in clot retraction and adhesion, recent data demonstrated that fibrinogen lacking these sites still supported clot retraction. These findings suggested that an unknown site in fibrinogen and/or other integrins participate in clot retraction. Here we have identified a sequence within gamma C that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365-383 sequence in gamma C, designated P3, efficiently inhibited clot retraction in a dose-dependent manner. Furthermore, P3 supported platelet adhesion and was an effective inhibitor of platelet adhesion to fibrinogen fragments. Analysis of overlapping peptides spanning P3 and mutant recombinant gamma C-domains demonstrated that the P3 activity is contained primarily within gamma 370-383. Integrins alpha IIb beta 3 and alpha 5 beta 1 were implicated in recognition of P3, since platelet adhesion to the peptide was blocked by function-blocking monoclonal antibodies against these receptors. Direct evidence that alpha IIb beta 3 and alpha 5 beta 1 bind P3 was obtained by selective capture of these integrins from platelet lysates using a P3 affinity matrix. Thus, these data suggest that the P3 sequence in the gamma C-domain of fibrinogen defines a previously unknown recognition specificity of alpha IIb beta 3 and alpha 5 beta 1 and may function as a binding site for these integrins.