Rat clusterin isolated from primary Sertoli cell-enriched culture medium is sulfated glycoprotein-2 (SGP-2)

Clusterin, a glycoprotein originally isolated from ram rete testis fluid, is a dimer composed of monomers with non-identical NH2-terminal amino acid sequences. In view of its possible role in cell-cell interactions in the seminiferous epithelium, we sought to identify such a protein in the rat. Using the bioassay developed for the ovine protein, rat clusterin was purified to apparent homogeneity by HPLC from primary Sertoli cell-enriched culture media. This protein is also a heterodimer consisting of monomers of Mr 43,000 (alpha) and Mr 35,000 (beta). NH2-Terminal amino acid sequence analysis indicated that the alpha subunit has a sequence of NH2-SLMPLSHYGPLSFHNMFQPFFDMIHQAQQA and the beta subunit, NH2-EQEFSDNELQELSTQGSRYVNKEIQNAVQG. These two subunits show marked similarity with the corresponding subunits of ram clusterin isolated from rete testis fluid. Using an antibody against the alpha subunit of rat clusterin, a cDNA clone was isolated from a rat testicular lambda gt11 cDNA library. Analyses of the amino acid sequence derived from the isolated rat clusterin cDNA and of the NH2-terminal amino acid sequences indicate that rat clusterin is identical to a Sertoli cell glycoprotein previously designated sulfated glycoprotein-2.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

Characterization of the subunits of beta-conglycinin

Four subunits of beta-conglycinin were purified from soybean cultivar CX 635-1-1-1, and were designated alpha, alpha', beta, and beta' in accordance with nomenclature proposed by Thanh and Shibasaki [(1977) Biochim. Biophys. Acta 490, 370-384]. Of these subunits, beta' has not previously been reported or characterized. Consistent with the low levels of methionine in these proteins, cyanogen bromide cleavage of alpha', alpha, and beta' subunits produced only a few fragments. The beta subunit contains no methionine and was not cleaved by cyanogen bromide. The NH2-terminal amino acid sequences of the alpha and alpha' subunits are homologous, and each has valine at its amino terminus. The beta subunit has a very different NH2-terminal sequence from those of the alpha and alpha' subunits, and has leucine at its amino terminus. The NH2-terminal sequence of the beta' subunit could not be determined, as it appeared to be blocked to Edman degradation. Although alpha and alpha' subunits have similar NH2-terminal sequences, they differ in the number of methionine residues and so yielded different numbers of cyanogen bromide fragments. Two cyanogen bromide fragments (CB-1 and CB-2) were purified from the alpha subunit. CB-1 originated from the NH2-terminal end of the subunit. The amino acid sequence of CB-2 was identical to that predicted from the nucleotide sequence of cDNA clone pB36. The insert in pB36 encoded 216 amino acids from the COOH-terminal end of the alpha subunit and contained a 138-bp trailer sequence which was followed by a poly-(A) tail. Maps showing the relative positions of methionine residues and carbohydrate moieties in the alpha and alpha' subunits were drawn, based on primary sequence data, and the size and carbohydrate content of the CNBr fragments derived from the subunits.     

Isolation and partial characterization of two different subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase

The molybdenum-iron protein of Azotobacter vinelandii nitrogenase was separated into two subunits of equal concentration by ion exchange chromatography on sulfopropyl (SP) Sephadex at pH 5.4 in 7 M urea. Better than 90% yield of each subunit was obtained on a preparative scale if the reduced carboxymethylated molybdenum-iron protein was incubated at 45 degrees C for 45 min prior to chromatography. Without the heating step low yields of the subunits were obtained. Although the amino acid compositions of the two subunits were very similar, the NH2-terminal sequences were completely different as determined by automated sequential Edman degradation. The sequence for the alpha subunit was NH2-Ser-Gln-Gln-Val-Asp-Lys-Ile-Lys-Ala-Ser-Tyr-Pro-Leu-Phe-Leu-Asp-Gln-Asp-Tyr- and for the beta subunit the sequence was NH2-Thr-Gly-Met-Ser-Arg-Glu-Glu-Val-Glu-Ser-Leu-Ile-Gln-Glu-Val-Leu-Glu-Val-Tyr-. Likewise the COOH-terminal sequences for the two subunits, as determined with carboxypeptidase Y, were tota-ly different. The sequence for the alpha subunit was -Leu-Arg-Val-COOH and that for the beta subunit was -Ile-(Phe, Glu)-Ala-Phe-COOH. Radioautographs of tryptic peptide maps were prepared for the molybdenum-iron protein and the two subunits which had been labeled at the cysteinyl residues with iodo[2-14C]acetic acid. These maps indicated that the two subunits had no cysteinyl peptides in common and that the cysteinyl residues were clustered in both subunits.     

Primary amino acid sequence of follicle-stimulating hormone from human pituitary glands. I. alpha subunit.

Follicle-stimulating hormone of a high state of physicochemical and biological purity was isolated from acetone-preserved human pituitary glands. The follicle-stimulating hormone was dissociated into alpha and beta subunits by treatment with 8 M urea and the subunits were separated by ion exchange chromatography on DEAE-Sephadex A-25. The subunits were freed of undissociated or reassociated follicle-stimulating hormone by gel filtration on Sephadex G-100. For the establishment of the primary amino acid sequence, the alpha subunit was reduced and either carboxyamidomethylated or S-aminoethylated prior to a thermolytic or a tryptic digestion. Each digest was gel filtered on a column of Sephadex G-50 to separate the glycopeptides from the peptides. The glycopeptides and the peptides were purified further by sequential gel filtration on Sephadex G-25, G-15, and Bio-Gel-P-2 and were isolated by high voltage electrophoresis at pH 6, 3.5, and 2. The purity of the isolated peptides was ascertained further by amino acid analysis. The amino acid sequences of the peptides were determined by Edman degradation followed by subtractive amino acid analysis. COOH-terminal sequences were established by digestion with carboxypeptidases A and B. The primary amino acid sequence of human follicle-stimulating hormone-alpha is identical to that of human chorionic gonadotropin-alpha and differs from that of human luteinizing hormone-alpha in having the tripeptide Ala-Pro-Asx- at the NH2-terminal end.     

Primary structure of the leukocyte function-associated molecule-1 alpha subunit: an integrin with an embedded domain defining a protein superfamily

The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.     

Purification of atrial natriuretic peptide receptor from bovine lung. Evidence for a disulfide-linked subunit structure

The receptor for atrial natriuretic peptide (ANP) was purified to apparent homogeneity from bovine lung by a combination of detergent extraction, ammonium sulfate precipitation, and affinity chromatography on ANP-Affi-Gel 10. The Mr of the purified receptor is about 140,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After reduction, the protein migrated as a single band with an Mr near 70,000. NH2-terminal sequence analysis of the purified material revealed only one sequence, indicating that the ANP receptor is composed of two probably identical subunits held together by disulfide bond(s), although it remains possible that one of the subunits is blocked at the NH2 terminus. Antibody was produced to the nonreduced Mr = 140,000 species and shown to interact with detergent-solubilized forms of the lung and kidney ANP receptor.     

Comparison of the NH2-terminal amino acid sequences of the four non-identical subunits of the NAD-linked hydrogenases from Nocardia opaca 1b and Alcaligenes eutrophus H16

The cytoplasmic, NAD-linked hydrogenase of the Gram-positive hydrogen-oxidizing bacterium Nocardia opaca 1b was compared with the analogous enzyme isolated from the Gram-negative bacterium Alcaligenes eutrophus H16. The hydrogenase of N. opaca 1b was purified by a new procedure applying chromatography on phenyl-Sepharose and DEAE-Sephacel with two columns in series. A homogeneous enzyme preparation with a specific activity of 74 mumol H2 oxidized.min-1.mg protein-1 and a yield of 32% was isolated. The A. eutrophus enzyme was purified as previously published. Both enzymes are tetrameric proteins composed of four non-identical subunits (alpha, beta, gamma, delta). The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the alpha, beta, gamma, and delta subunits of one organism were serologically related to the analogous subunits of the other organism. Among themselves, the four subunits do not have any serological relationship. The eight individual polypeptides were also compared with respect to the NH2-terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions. Strong sequence similarities exist between the analogous subunits isolated from the two bacteria. Within the established N-terminal sequences the similarities between both alpha, beta, gamma and delta subunits amount to 63%, 79%, 80% and 65%, respectively. No similarities exist between the different, non-analogous subunits alpha, beta, gamma and delta.     

The NH2-terminal sequence of the alpha and gamma subunits of eukaryotic initiation factor 2 and the phosphorylation site for the heme-regulated eIF-2 alpha kinase

Rabbit reticulocyte eukaryotic initiation factor 2 was phosphorylated with the heme-regulated alpha subunit of eukaryotic initiation factor 2 kinase, and then the individual subunits were resolved by reversed-phase high performance liquid chromatography. Phosphorylated and unphosphorylated forms of the alpha subunit also were well resolved. The NH2-terminal sequences of intact alpha and gamma subunits were determined. No sequence was obtained from the beta subunit, suggesting that it may have a blocked NH2-terminus. Overlapping tryptic and chymotryptic phosphopeptides from the NH2-terminal sequence of the alpha subunit of eukaryotic initiation factor 2 were used to establish the order of amino acids 1-52 and localized the phosphorylation site within the sequence: -Leu-Leu-Ser48-Glu-Leu-Ser51-. Subdigestion of a tryptic fragment with chymotrypsin generated only phosphopeptides that appeared to terminate at leucine 50, indicating phosphorylation at serine 48.     

Escherichia coli S-adenosylmethionine decarboxylase. Subunit structure, reductive amination, and NH2-terminal sequences

S-Adenosylmethionine decarboxylase is one of a small group of enzymes that use a pyruvoyl residue as a cofactor. Histidine decarboxylase from Lactobacillus 30a, the best studied pyruvoyl-containing enzyme, has an (alpha beta)6 subunit structure with the pyruvoyl moiety linked through an amide bond to the NH2-terminal of the larger alpha subunit (Recsei, P. A., Huynh, Q. K., and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 973-977). To examine potential structural analogies between the two enzymes, we have isolated and partially characterized S-adenosylmethionine decarboxylase. The purified enzyme comprises equimolar amounts of two subunits of Mr = 14,000 and 19,000 (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and has a native molecular weight of 136,000 (by gel filtration). Approximately 4 mol of [methyl-3H] adenosylmethionine are incorporated per mol of enzyme (Mr = 136,000) when the enzyme is inactivated with this substrate and NaCNBH3. These data suggest an (alpha beta)4 structure with 1 pyruvoyl residue for each alpha beta pair. The two subunits have been separated by reversed-phase high performance liquid chromatography after reduction and carboxymethylation. The smaller subunit (beta) has a free amino terminus. The amino terminus of the larger subunit (alpha) appears to be blocked by a pyruvoyl group; this subunit can be sequenced only after this group is converted to an alanyl residue by reduction with sodium cyanoborohydride in the presence of ammonium acetate. This work suggests that S-adenosylmethionine decarboxylase is structurally much more similar to histidine decarboxylase than previously thought.     

Molecular cloning of the rat integrin alpha 1-subunit: a receptor for laminin and collagen

Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an M-200 kD integrin alpha subunit that associates with the integrin beta 1 subunit to form a receptor for both laminin and collagen. Similarities in ligand-binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the alpha subunit of the human integrin VLA-1. The full-length rat alpha 1 cDNAs encode a protein containing a purative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin alpha chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat alpha 1 sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-1, LFA-1, p150). The rat alpha 1 and human VLA-2 apha subunits share greater than 50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin alpha 1 subunit has several unique features, including a 38-residue insert between two Ca2+/Mg2+ binding domains, and a divergent 15-residue cytoplasmic sequence, that may potentially account for unique functions of this integrin.     

Purification and partial amino acid sequence of papain-solubilized class II transplantation antigens

Papain-solubilized human class II (HLA-DR) antigens have been purified from cadaveric spleens by ion-exchange chromatography, gel chromatography, and immunosorbent purification. The isolated papain-solubilized antigens comprised two subunits with apparent molecular weights of 23 000 and 30 000, respectively. The circular dichroism spectrum for the isolated class II antigens was similar to spectra recorded for HLA-A, -B, and -C antigens, immunoglobulins, and immunoglobulin fragments. Thus, class II antigens contain a considerable amount of beta structure. The small subunit (beta chain) exhibited extensive charge heterogeneity on two-dimensional isoelectric focusing polyacrylamide gel electrophoresis, whereas the large subunit (alpha chain) was more homogeneous. The structural heterogeneity of beta chains remained after neuraminidase treatment. The NH2-terminal amino acid sequence of the beta chains displayed multiple residues in several positions in accordance with the genetic polymorphism displayed by this chain. The alpha chain also displayed multiple residues in some positions, suggesting either that some of the genetic polymorphism of the class II antigens may be endowed in this chain or that multiple loci control the expression of several alpha chains. Papain-solubilized class II antigen subunits were homologous in their amino acid sequences with HLA-DR antigens of defined antigenic specificity as well as with murine I-E/C antigens.     

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.     

The GABAA/benzodiazepine receptor is a heterotetramer of homologous alpha and beta subunits.

The GABAA receptor has been purified to homogeneity from bovine cerebral cortex. Under stringent conditions of isolation, the GABAA receptor was shown to consist only of alpha (Mr 53 000) and beta (Mr 57 000) subunits. A densitometric scan of SDS-PAGE gels under reducing conditions showed that these subunits were present in a 1:1 ratio. A model of the receptor as a heterologous tetramer alpha 2 beta 2 is proposed. Monoclonal antibodies have been raised to the purified bovine GABAA receptor. One of these antibodies, 1A6, was shown to react with both the alpha and beta subunits of the purified receptor. The subunits were still positive in immunoblots following the removal of the carbohydrate moieties of the respective polypeptides by endoglycosidase F treatment. This antibody has been employed to demonstrate antigenic cross-reactivity between the GABAA receptors of three vertebrate species. It is further proposed that there is partial amino acid sequence homology between the alpha and beta polypeptides and hence that they are derived from a single ancestral gene.     

Ovostatin: a novel proteinase inhibitor from chicken egg white. I. Purification, physicochemical properties, and tissue distribution of ovostatin

A proteinase inhibitor which has strong anti-collagenase activity was found in chicken egg white. The inhibitor (pI = 4.9) was purified by poly(ethylene glycol) (5.5-10%) precipitation and chromatography on Ultrogel AcA 34, DEAE-cellulose, and Sephacryl S-300. The final product was homogeneous on 5% polyacrylamide gel electrophoresis. Stoichiometric inhibition was observed with the inhibitor and rabbit synovial collagenase and thermolysin (1:1 molar ratio with thermolysin). The inhibitor ran on sodium dodecyl sulfate-gel electrophoresis with reduction as a single protein band of Mr = 165,000. The molecular weight of the native inhibitor was estimated to be 780,000 by sedimentation equilibrium centrifugation. Centrifugation analysis in 6 M guanidine hydrochloride and of the reduced sample gave M omega = 380,000 and M omega = 195,000, respectively, where M omega is the weight-average molecular weight determined by equilibrium ultra-centrifugation. The results indicated that the inhibitor molecule is a tetramer of identical subunits linked in pairs by disulfide bonds. Since the molecular weight and the quaternary structure of the inhibitor were similar to those of alpha 2-macroglobulin (alpha 2M) in plasma, chicken alpha 2M was isolated and compared with the inhibitor. The inhibitor was not sensitive to methylamine, whereas chicken alpha 2M was. No immunocross-reactivity was observed between the inhibitor and chicken alpha 2M. The NH2-terminal sequence of the egg white inhibitor is Lys-Glu-Pro-Glu-Pro-Gln-Tyr-Val-Leu-Met-Val-Pro-Ala. The sequence of chicken alpha 2M is Ser-Thr-Val-Thr-Glu-Pro-Gln-Tyr-Met-Val-Leu-Leu-Pro-Phe. Considerable homology was found between the two sequences and to the NH2-terminal sequence of human alpha 2M. Monospecific antibody raised against the egg white inhibitor was employed to examine the tissue distribution of the inhibitor. The inhibitor was found only in oviduct and egg white, but not in other tissues or serum of chickens.     

Heavy riboflavin synthase of Bacillus subtilis. Primary structure of the beta subunit

Heavy riboflavin synthase is a 1,000,000-Da protein catalyzing the last two reactions of riboflavin biosynthesis. The enzyme complex consists of 60 beta subunits (Mr = 16,200) and approximately three alpha subunits (Mr = 23,000). beta subunits were isolated and cleaved with cyanogen bromide. Fragments were isolated and further digested with trypsin and staphylococcal protease. Peptides were isolated by high performance liquid chromatography. Sequences were determined by automated liquid-phase Edman degradation. The complete sequence of the beta subunit (154 amino acids) was established by direct sequencing of the NH2 terminus, sequencing of overlapping peptides, and carboxypeptidase degradation of the COOH terminus. The sequence shows no detectable homologies to other proteins. A computer prediction of secondary structure elements indicates 34% alpha helix and 30% beta sheet.     

The subunit structure and active site sequence of porcine spleen deoxyribonuclease

An acid DNase (DNase II) from porcine spleen was purified by sequential chromatography over carboxymethyl-cellulose, blue dextran-Sepharose, hydroxylapatite, and sulfoxyethyl-cellulose. The purified enzyme shows two polypeptide bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at Mr 35,000 (alpha chain) and 10,000 (beta chain). The sum of the two molecular weights is that of the native enzyme (45,000). Thus, the DNase II molecule is an alpha,beta dimer. The two polypeptides are not joined by disulfide bonds, but can be cross-linked chemically with dimethyl suberimidate. They are dissociable in 8 M urea, after which they can be isolated by gel filtration on Sephadex G-100, eluting with 1 M acetic acid. Once dissociated, the two polypeptides cannot be reassociated to regenerate DNase II activity. The sum of the amino acid compositions of the two polypeptides is that of the native enzyme, and both contain carbohydrate. The beta chain is devoid of histidine, half-cystine, valine, and methionine. The NH2-terminal amino acid of the alpha chain is leucine, while that of the beta chain cannot be identified by either dansylation or Edman degradation. Alkylation of an essential histidine residue of DNase II occurs on incubation of the enzyme with [2-14C] ICH2COOH (Oshima, R. G., and Price, P. A. (1973) J. Biol. Chem. 248, 7522-7526). Radioactivity is found only in the alpha chain. After hydrolysis of the alpha chain with trypsin, chymotrypsin, and thermolysin, radioactive peptides were isolated by gel filtration on Sephadex G-25 and reversed-phase high performance liquid chromatography. Sequence analyses of the radioactive peptides show alkylation of 1 of 9 histidines in the entire amino acid sequence of DNase II. The sequence around this histidine, determined by manual microsequencing and by the release of amino acids with carboxypeptidases A and B, is Ala-Thr-Glu-Asp-His-Ser-Lys-Trp.     

Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity.

To identify structural characteristics of the closely related cell surface receptors for insulin and IGF-I that define their distinct physiological roles, we determined the complete primary structure of the human IGF-I receptor from cloned cDNA. The deduced sequence predicts a 1367 amino acid receptor precursor, including a 30-residue signal peptide, which is removed during translocation of the nascent polypeptide chain. The 1337 residue, unmodified proreceptor polypeptide has a predicted Mr of 151,869, which compares with the 180,000 Mr IGF-I receptor precursor. In analogy with the 152,784 Mr insulin receptor precursor, cleavage of the Arg-Lys-Arg-Arg sequence at position 707 of the IGF-I receptor precursor will generate alpha (80,423 Mr) and beta (70,866 Mr) subunits, which compare with approximately 135,000 Mr (alpha) and 90,000 Mr (beta) fully glycosylated subunits.     

Purification of high and low molecular weight plasminogen activator inhibitor 1 from fibrosarcoma cell-line HT 1080 conditioned medium

Functionally active (high-Mr) and inactive (low-Mr) plasminogen activator inhibitor 1 (PAI) have been purified from fibrosarcoma cell-line HT 1080 conditioned medium, containing 1% fetal calf serum. The two forms were first purified by affinity chromatography on heparin-Sepharose and then separated from each other by gel filtration on Sephadex G-150. The final purification was achieved by affinity chromatography on insolubilized monoclonal antibodies towards human PAI. Alternatively, the low-Mr form was purified by chromatography on carboxymethyl-cellulose. Low-Mr PAI purified in this way, could be almost fully reactivated by treatment with guanidinium chloride. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by immunoblotting revealed that the low-Mr form contained nothing but PAI at an Mr of about 50,000. In addition to PAI, the high-Mr form contained a component, which was not antigenically related to PAI. This compound had a molecular weight of about 75,000 and its NH2-terminal amino acid sequence corresponded to that of human vitronectin. We conclude that the high-Mr form of PAI constitutes a complex between 50,000 Mr PAI and vitronectin from fetal calf serum.     

Cloning of the murine lymphocyte function-associated molecule-1 alpha-subunit and its expression in COS cells

The lymphocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is an integrin that mediates adhesion of immune cells by interaction with two members of the Ig superfamily, ICAM-1 and ICAM-2. LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report here the isolation and expression of the murine alpha subunit cDNA (GenBank accession no. M60778). The deduced sequence comprises a 1061 amino acid extracellular domain, a 29 amino acid transmembrane region, and a 50 amino acid cytoplasmic domain. It has a 72% amino acid identity with its human counterpart and 34% identity with the murine Mac-1 alpha subunit. The murine LFA-1 alpha subunit could be expressed on the cell surface of a fibroblastoid cell line, COS, by cotransfection with either the human or murine beta subunit cDNA.