Structure of thrombospondin

The NH2-terminal amino acid sequences of thrombospondin and of a 30,000-Da heparin-binding peptide derived from thrombospondin by treatment with plasmin are identical. The heparin-binding peptide is homogeneous in size but slightly heterogeneous in charge with the predominant isoelectric points being 6.1 and 5.7. Electron microscopy of tungsten replicas of thrombospondin reveals a tripartite structure resembling a "bola" which is about 60 nm across when fully extended. Each part of the molecule terminates in a globular node or head which disappears upon limited plasmin digestion, suggesting that the heparin-binding peptide is located in the head region. In addition to the heparin-binding peptide, a 20,000-Da peptide also apparently associated with the head region is liberated during proteolysis. The electron micrographs indicate that the legs of the bola-like structure must be folded into an extended, flexible, tertiary structure. These legs, each of about 65,000 Da, appear to be attached near the ends opposite the heads, probably by disulfide bonds. Each leg possesses a tab or protein (approximately 20,000 Da) which juts out from this attachment point.     

Purification and characterization of the trifunctional beta-subunit of anthranilate synthase from Neurospora crassa

The trifunctional beta-subunit of anthranilate synthase complex of Neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. The isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on Sephacryl S-300 compared with a monomer molecular weight of approximately 84,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The purified subunit was cleaved by elastase, trypsin, or chymotrypsin into fragments which retained the three enzyme activities. After elastase digestion, two active fragments were separated by gel filtration and ion exchange chromatography. A 30,000-Da fragment, which behaved as a monomer on gel filtration, interacted with free alpha-subunit to produce glutamine-dependent anthranilate synthase activity. A second 56,000-Da fragment, which behaved as an asymmetric dimer (apparent molecular weight 140,000) on gel filtration, retained both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthase activity. The failure to detect an NH2-terminal amino acid residue on either the intact beta-subunit or the 30,000-Da complementing fragment, while the 56,000-Da fragment possessed an NH2-terminal histidine residue, indicated that the complementing fragment was derived from the NH2-terminal sequence of the beta-subunit.     

Biochemical and ultrastructural characterization of the 1,4-dihydropyridine receptor from rabbit skeletal muscle. Evidence for a 52,000 Da subunit

The 1,4-dihydropyridine receptor purified from rabbit skeletal muscle contains four polypeptide components of 175,000 Da (nonreduced)/150,000 Da (reduced), 170,000, 52,000, and 32,000 Da (Leung, A. T., Imagawa, T., and Campbell, K. P. (1987) J. Biol. Chem. 262, 7943-7946). A monoclonal antibody specific to the 52,000-Da polypeptide component of the dihydropyridine receptor has been produced and used in immunoprecipitation and immunoblotting experiments to demonstrate that the 52,000-Da polypeptide is an integral subunit of the purified dihydropyridine receptor. Peptide mapping experiments with 32P-labeled dihydropyridine receptor have also demonstrated that the 52,000-Da polypeptide is distinct from and not a proteolytic fragment of the 170,000-Da subunit. Densitometric scanning of Coomassie Blue-stained sodium dodecyl sulfate-polyacrylamide gels of the purified dihydropyridine receptor has demonstrated that the 52,000-Da polypeptide exists in a 1:1 stoichiometric ratio with the 170,000-, 175,000/150,000-, and 32,000-Da subunits of the dihydropyridine receptor. Electron microscopy of the freeze-dried, rotary-shadowed dihydropyridine receptor has shown that the preparation contains a homogeneous population of 16 x 22-nm ovoidal particles large enough to contain all four polypeptides of the dihydropyridine receptor. The particles have two distinct components of similar size which may represent the location in the molecule of the two larger subunits.     

Structural characterization of the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel from rabbit skeletal muscle. Evidence for two distinct high molecular weight subunits

The 1,4-dihydropyridine receptor purified from rabbit skeletal muscle triads was shown to contain four protein components of 175,000, 170,000, 52,000, and 32,000 Da when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Monoclonal antibodies capable of specifically immunoprecipitating the [3H]PN200-110-labeled dihydropyridine receptor from digitonin-solubilized triads recognized the 170,000-Da protein on nitrocellulose transfers of skeletal muscle triads, transverse tubular membranes, and purified dihydropyridine receptor. Wheat germ agglutinin peroxidase stained the 175,000-Da protein on similar nitrocellulose transfers, demonstrating that the 175,000-Da protein is the glycoprotein subunit of the purified dihydropyridine receptor. The apparent molecular weight of the Mr 170,000 protein remained unchanged with reduction, whereas the apparent molecular weight of the glycoprotein subunit shifted from 175,000 to 150,000 upon reduction. These results demonstrate that the 1,4-dihydropyridine receptor of the voltage-dependent Ca2+ channel from rabbit skeletal muscle contains two distinct high molecular weight subunits of 175,000 and 170,000.     

Molecular organization of 19 S calf thyroglobulin

Controlled freezing and thawing of 19 S calf thyroglobulin resulted in a specific and reproducible breakdown of the protein. Beside the elementary chain (300,000 Da), new, discrete bands are revealed by gel electrophoresis in sodium dodecyl sulfate under reducing conditions. These new species consist of a major peptide of 100,000 Da and several faster-migrating bands. Most of these polypeptides were purified by preparative gel electrophoresis and individually digested in formic acid with CNBr. A comparative gel electrophoresis under denaturating and reducing conditions of (i) the fragments obtained from the native protein, (ii) the electrophoretically purified elementary chain, (iii) the 100,000-Da peptide, and (iv) a smaller fragment (of about 50,000) was performed. It revealed a very close homology among the peptide maps of the intact 19 S, the elementary chain, and the 100,000-Da peptide. Furthermore, it was shown that the digestion products of the smaller fragment, present in the peptide map of the native protein, were absent in both the elementary chain and the 100,000-Da species. These results support the idea that calf thyroglobulin, even though it has an apparently complex molecular organization, contains structural motifs which are repeated in the elementary chain.     

Analysis of inter-alpha-trypsin inhibitor and a novel trypsin inhibitor, pre-alpha-trypsin inhibitor, from human plasma. Polypeptide chain stoichiometry and assembly by glycan

The polypeptide chain composition of protein material referred to in the literature as "inter-alpha-trypsin inhibitor" was investigated. The material was found to consist of distinct proteins of 125,000 and 225,000 Da, each of which contained more than one polypeptide chain. The links that assemble each protein were found to be stable to various strong denaturants, but susceptible to treatment with trifluoromethanesulfonic acid or hyaluronidase, indicating a glycan nature. The 225,000-Da protein migrated with inter-alpha mobility on agarose gel electrophoresis and is designated inter-alpha-trypsin inhibitor, whereas the 125,000-Da protein migrated with pre-alpha mobility, and we designate it pre-alpha-trypsin inhibitor. Analysis of the proteins, the separated chains, and proteolytic derivatives thereof revealed that each protein contained a single, identical, trypsin-inhibitory chain of 30,000 Da. Inter-alpha-trypsin inhibitor contains noninhibitory heavy chains of 65,000 and 70,000 Da, whereas pre-alpha-trypsin inhibitor contains a heavy chain of 90,000 Da. Our data allow identification of several recently reported cDNA clones and clarify the confusion surrounding the composition of plasma proteins referred to as inter-alpha-trypsin inhibitor.     

Surface expression and partial characterization of an arsonate hapten-specific idiotype-bearing T-cell receptor

Anti-idiotype antibodies raised against the arsonate hapten idiotype have been used to detect arsonate-binding receptors on the surface of peripheral T cells of A/J mice and to isolate this material after biosynthetic labeling for partial chemical characterization. It was found that 2-3% of splenic T cells from arsonate-immune mice specifically bound the hapten using immunofluorescent keyhole limpet hemocyanin as a carrier. In double-immunofluorescence labeling experiments, a high proportion (approximately equal to 70%) of these cells also bound the (Fab')2 fragment of rabbit anti-idiotype antibody in exactly the same patches on the cell as the arsonate hemocyanin antigen. In addition, the anti-idiotype antibody inhibited the binding of the hapten-carrier complex to T cells by approximately equal to 70%. In parallel experiments, fowl antibodies against mouse (Fab')2 fragments bound to 100% of arsonate-binding T cells in the same cell-surface patches as the hapten, and were capable of inhibiting 100% of the hapten-binding cells. Capping, shedding, and resynthesis experiments indicated that the T cells synthesized their antigen-binding idiotype-bearing receptors. Immunoblots of unreduced detergent extracts of purified splenic T cells developed with anti-idiotype antibodies showed bands at 150,000 and 94,000 Da. Equal amounts of protein extracted from liver and analyzed in the same gels as the T-cell material failed to show any reactivity with anti-idiotype antibodies. To confirm the biosynthetic origin of the idiotype-positive materials, detergent extracts from 75Se-methionine- or [3H]leucine-labeled Con A-treated splenic T cells were reacted with anti-idiotype antibodies and the bound material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of 2-mercaptoethanol the major band was at 68,000 Da, with variable minor levels of material at 45,000 Da, while when hapten was used to isolate the receptor a dominant 25,000- to 30,000-Da band was seen. We believe that the higher-molecular-weight materials are multimers of the 25,000-30,000 subunit.     

Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin

Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an Mr 25,000 fragment from the thermolysin digest and an Mr 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the Mr 180,000 TSP peptide chain.     

Rapid purification and partial characterization of human platelet glycoprotein IIIb. Interaction with thrombospondin and its role in platelet aggregation

Glycoprotein IIIb (also known as glycoprotein IV) is a major glycoprotein present on the surface of human platelets. Recent studies suggest that glycoprotein IIIb may be a receptor site for thrombospondin. Thrombospondin, a multifunctional adhesive glycoprotein released from stimulated platelets, plays an important role in the stabilization of platelet aggregates. In this study, a new method for the purification of glycoprotein IIIb is described. Glycoprotein IIIb was isolated from Triton X-114 platelet membrane extracts, under nondenaturing conditions, by tandem anion-exchange and size exclusion fast protein liquid chromatography. The purified glycoprotein had the same apparent molecular mass (88 kDa) under nonreducing or reducing conditions. The tryptic peptide map of the purified protein was identical to that of bona fide glycoprotein IIIb as isolated from two-dimensional polyacrylamide gels of platelet membrane proteins. In addition, the purified glycoprotein was recognized by an anti-GPIIIb monoclonal antibody (OKM5). The purified glycoprotein specifically bound to thrombospondin in the presence of calcium. Monospecific anti-GPIIIb antibodies interfered with the expression of endogenous thrombospondin on thrombin-activated platelets and partially inhibited collagen- and thrombin-induced platelet aggregation without a significant effect on platelet secretion. Glycoprotein IIIb, by interacting with thrombospondin on the activated platelet surface, may play an important role in the platelet aggregation process.     

Purification and characterization of hepatic and intestinal phenol sulfotransferase with high affinity for benzo[a]pyrene phenols from channel catfish, Ictalurus punctatus

Cytosol from channel catfish liver and intestinal mucosa has high sulfotransferase activity with low concentrations of 3-, 7-, or 9-hydroxybenzo[a]pyrene. To further investigate this conjugation pathway, sulfotransferase activity toward 9-hydroxybenzo[a]pyrene was isolated from catfish intestinal and hepatic cytosol by chromatography on anion exchange and PAP-agarose affinity columns. SDS-PAGE of the active fractions showed that one major band with molecular size of about 41,000 Da was isolated from intestine, while two bands of about 41,000 and 31,000 Da were obtained from liver. Antibodies against human phenol-sulfating sulfotransferase cross-reacted strongly with the 41,000-Da bands from liver and intestine, but weakly with the hepatic 31,000-Da protein. N-Terminal sequence information could not be obtained from the pure proteins. Following digestion, an internal sequence of 20 amino acid residues was obtained from the hepatic 41,000-Da protein, which matched a sequence found in several mammalian sulfotransferases. No fish sulfotransferase sequences were available for comparison. The identity of the hepatic 31,000-Da protein was not established. The purified 41,000-Da proteins had very high activities with 3-, 7-, or 9-hydroxybenzo[a]pyrene, with K(m) values in the 40-100 nM range and V(max) 125-300 nmol/min/mg of protein. Substrate inhibition was observed when the concentrations of hydroxylated benzo[a]pyrenes were above 0.5 microM. As well as benzo[a]pyrene phenols, the purified 41,000-Da sulfotransferases catalyzed sulfation of 2-naphthol, 4-nitrophenol, 4-methylumbelliferone, 7-(hydroxymethyl)-12-methylbenz[a]anthracene, dehydroepiandrosterone, estrone, and 17beta-estradiol. Phenolic compounds were the preferred substrates for the purified enzymes.     

Characterization of the 1,4-dihydropyridine receptor using subunit-specific polyclonal antibodies. Evidence for a 32,000-Da subunit

The purified receptor for the 1,4-dihydropyridine Ca2+ channel blockers from rabbit skeletal muscle contains protein components of 170,000 Da (alpha 1), 175,000 Da (alpha 2), 52,000 Da (beta), and 32,000 Da (gamma) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Subunit-specific polyclonal antibodies have now been prepared and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. Immunoblot analysis of fractions collected during purification of the dihydropyridine receptor shows that the 32,000-Da polypeptide copurified with alpha 1 and alpha 2 subunits at each step of the purification. In addition, monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex which includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. In addition, polyclonal antibodies against the rabbit skeletal muscle 32,000-Da polypeptide specifically react with similar proteins in skeletal muscle of other species including avian and amphibian species. Thus, our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.     

Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase.

The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.     

The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14). We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl. The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each. The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product. DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6621-6625). Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da. The data suggest that the enzyme is post-translationally processed during protein purification.     

Expression and initial characterization of a recombinant human thrombospondin heparin binding domain

Thrombospondin (TSP) is a trimeric glycoprotein of Mr 420,000. It was originally described as a major component of human platelet alpha granules and is essential for the secondary phase of platelet aggregation. TSP is also synthesized and secreted by a variety of nucleated cells where it functions in processes involving growth and adhesion of cells to the extracellular matrix. Many of these processes are heparin-inhibitable and are mediated by a proteolytic fragment of TSP called the heparin binding domain (HBD). In order to facilitate the analysis of the structure and function(s) of this domain, we have expressed this molecule in Escherichia coli. A fragment of a TSP cDNA that encodes the heparin binding domain was inserted into the prokaryotic expression vector pJBL6. In bacterial cells grown at 42 degrees C, this vector directs the synthesis of a 24,000-Da polypeptide. Milligram quantities of this protein were purified to homogeneity from E. coli lysates. The structure of the recombinant HBD was confirmed by protein sequencing. The protein was further characterized by analysis of its conformation and function. The recombinant HBD binds [3H]heparin with a Kd of 71 nM, almost identical to that of TSP-derived HBD (80 nM). Additionally, the recombinant HBD is able to compete for TSP binding to 11B carcinoma cells. These studies indicate that the recombinant HBD is synthesized and purified in a native configuration and is functionally equivalent to thrombospondin-derived HBD. They further indicate that glycosylation of the thrombospondin HBD is not necessary for its interaction with heparin and that sequences essential to this interaction reside within the first 229 amino acids of secreted thrombospondin.     

A chloride- and calcium-dependent glutamate-binding protein from rat brain. Identification as a ubiquitous constituent of the inner mitochondrial membrane

We have recently solubilized and enriched a chloride- and calcium-dependent glutamate-binding protein from rat brain (Brose, N., Halpain, S., Suchanek, C., and Jahn, R. (1989) J. Biol. Chem. 264, 9619-9625). The partially purified protein fraction, containing two major protein components of 51,000 Da and 105,000 Da, was used to generate a rabbit antiserum. This serum quantitatively precipitated the binding activity from membrane extracts. Small amounts of the antiserum inhibited glutamate binding when chloride was absent from the incubation medium. Three protein bands were labeled by the serum on immunoblots. From the affinity purified antibody fractions contained in the serum, only the antibodies directed against a 51,000-Da protein were able to immunoprecipitate the binding activity, indicating that this protein is an essential component of the binding site. A survey of a variety of rat tissues by immunoblot analysis revealed a ubiquitous distribution of the protein. After subcellular fractionation of liver and brain, the 51,000-Da protein copurified with mitochondrial markers. Furthermore, exclusive labeling of mitochondria was observed by light and electron microscopy immunocytochemistry. Subfractionation of purified liver mitochondria resulted in a selective association of the protein with inner mitochondrial membranes. Pharmacological characterization of glutamate binding to liver mitochondrial membranes revealed a pattern almost identical to that of the chloride- and calcium-dependent glutamate-binding site in rat brain.     

Biosynthesis and processing of rat alpha 1-antitrypsin

Various biosynthetic forms of rat alpha 1-antitrypsin (alpha 1AT) have been isolated by immunoprecipitation of in vitro and in vivo synthesized products. Rat alpha 1AT is synthesized in a rabbit reticulocyte system as a 45,000-Da preprotein with a 23-amino acid signal sequence. The majority of the amino acids in the signal sequence have been identified and resemble the signal peptides of other secretory proteins with respect to the abundance and positions of hydrophobic amino acids. Evidence from the translation of rat liver RNA in the presence of dog pancreas microsomes, from the translation of rat liver polysomes, and from tunicamycin-treated rat hepatocytes established that cleavage of the signal peptide of pre-alpha 1AT results in the formation of a 42,000-Da protein, the polypeptide backbone of mature alpha 1AT. A 50,000-Da glycoprotein is immunoprecipitated from translations programmed with rat liver microsomes or with rat liver mRNA and dog pancreas microsomes. Cotranslational glycosylation of alpha 1AT appears to occur in a stepwise fashion since three glycosylated forms of alpha 1AT (approximately 45,000, 47,000, and 50,000 Da) can be detected in polysome translations. These proteins are susceptible to cleavage by endo-beta-N-acetylglucosaminidase H and are digested to the same product, indicating that they have identical polypeptide chains. Two intracellular forms of alpha 1AT were detected in cultured rat hepatocytes, a 50,000- and a 52,000-Da protein; only the larger protein was immunoprecipitated from the medium of these cells. Digestion with endo-beta-N-acetylglucosaminidase H indicated that the 50,000-Da protein is a core glycosylated processing intermediate, whereas the 52,000-Da protein, which comigrated with purified serum alpha 1AT, appears to contain complex carbohydrate sidechains. When glycosylation was inhibited by incubation of hepatocytes with tunicamycin, a nonglycosylated 42,000-Da protein was immunoprecipitated from the cells and the culture medium, indicating that glycosylation of alpha 1AT is not essential for its secretion.     

Nucleotide sequence reveals overlap between T4 phage genes encoding dihydrofolate reductase and thymidylate synthase

We have determined the nucleotide sequence of a 1075-base-pair HindIII fragment of the T4 phage genome. This fragment contains the structural gene (frd) for dihydrofolate reductase and part of the gene (td) encoding thymidylate synthase. The fragment contains a 579-base-pair open reading frame, encoding a 193-residue polypeptide with a calculated mass of 21,603 Da, in agreement with our reported subunit molecular mass of 23,000. The deduced amino acid sequence shows partial homology with other dihydrofolate reductases, with most of the identities lying in regions known to be involved in substrate binding and catalysis. The 3' end of the coding strand overlaps the coding region for thymidylate synthase; the sequence - ATGA -includes an opal terminator for the frd gene and an initiating triplet for the td gene. The deduced amino acid sequence from this initiating ATG is identical, for the first 20 residues, with the NH2-terminal 20 residues reported for the td protein (M. Belfort , A. Moelleken , G. F. Maley , and F. Maley (1983) J. Biol. Chem. 258, 2045-2051). The sequenced HindIII fragment was transferred into a high expression plasmid vector for large scale production of homogeneous T4 dihydrofolate reductase. The experimentally determined sequence of 20 residues at the NH2-terminus of this protein is identical with that deduced from the nucleotide sequence for T4 dihydrofolate reductase.     

Fe2+ oxidation of alpha-crystallin produces a 43,000 Da aggregate composed of A and B chains cross-linked by non-reducible covalent bonds

The structure of a 43,000 Da aggregate generated from bovine lens alpha-crystallin polypeptides of 20,000 Da using Fe2+ catalyzed oxidation, was studied by sequence analysis of a 30,000 Da proteolytic fragment. Three polypeptide components were simultaneously sequenced in the electroblotted 30,000 Da fragment, corresponding to Phe114-Ser130... of the alpha A chain, and His 111-Ser135... and Ser 35-Leu49... of the alpha B chain. The relative proportions of the components suggests that the three polypeptides are present in equimolar amounts. It is concluded that the 30,000 Da fragment and, therefore, the 43,000 Da aggregate is constructed of both the A and B polypeptide chains covalently cross-linked with non-reducible bonds. At least one of these cross-links is present towards the carboxy-terminus of the A and B chains after Phe114 and His111, respectively.     

COMP (cartilage oligomeric matrix protein) is structurally related to the thrombospondins.

Cloning and sequence analysis of cartilage oligomeric matrix protein (COMP) cDNA, representing a cartilage pentameric protein, revealed a protein of 755 amino acid residues with a calculated molecular mass of 82,700 Da. Expression of the cDNA in COS cells showed that COMP is a homopolymer composed of five identical disulfide-linked subunits. COMP is homologous to the carboxyl-terminal half of thrombospondin, and the homologies include 89% and 54% of the residues in COMP and thrombospondin, respectively. The similarities are most pronounced in the carboxyl-terminal domains and in the calcium binding type 3 repeat domains in which about 60% of the amino acid residues are identical. In the type 2/epidermal growth factor repeat domains the two proteins contain 41% identical residues. The sequence of the amino-terminal 84-amino acid residues is unique for COMP. Comparison of the amino acid sequences in the type 2 and type 3 repeat domains of COMP and the thrombospondins shows that COMP is the product of a unique gene and not the result of an alternatively spliced thrombospondin gene.     

Characterization of transducin from bovine retinal rod outer segments. II. Evidence for distinct binding sites and conformational changes revealed by limited proteolysis with trypsin

The first stage of amplification in the cyclic GMP cascade in bovine retinal rod is carried out by transducin, a guanine nucleotide regulatory protein consisting of two functional subunits, T alpha (Mr approximately 39,000) and T beta gamma (Mr approximately 36,000 and approximately 10,000). Limited trypsin digestion of the T beta gamma subunit converted the beta polypeptide to two stable fragments (Mr approximately 26,000 and approximately 14,000). The GTPase and Gpp(NH)p binding activities were not significantly affected by the cleavage. Trypsin digestion of the T alpha subunit initially removed a small segment from the polypeptide terminus and resulted in the formation of a single 38,000-Da fragment. When this fragment was recombined with the intact T beta gamma subunit in the presence of membranes containing photolyzed rhodopsin, the reconstituted transducin exhibited greatly reduced GTPase and Gpp(NH)p binding activities. The loss in activities was due to the inability of the cleaved T alpha to bind to the photolyzed rhodopsin. Prolonged digestion converted the 38,000-Da fragment to a transient 32,000-Da fragment and then to two stable 23,000-Da and 12,000-Da fragments. The cleavage of the 32,000-Da fragment, however, can be blocked by bound Gpp(NH)p. The 32,000-Da fragment contains the Gpp(NH)p binding site and retains the ability to activate phosphodiesterase. These results indicate that the guanine nucleotide binding and rhodopsin binding sites are located in topologically distinct regions of the T alpha subunit and proved evidence that a large conformational transition of the molecule occurs upon the conversion of the bound GDP to GTP.