cDNA cloning reveals that the major group rhinovirus receptor on HeLa cells is intercellular adhesion molecule 1.

A 90-kDa surface glycoprotein was previously isolated and shown to be required for infection by the "major" group of human rhinovirus (HRV) serotypes. In the present work, the amino acid sequence of the receptor protein was obtained from CNBr and tryptic peptides. Using degenerate oligonucleotides predicted from the peptide sequences, we identified four cDNA clones that encode a 3-kilobase mRNA. The clones were ligated, subcloned in a simian virus 40 expression vector, and used to transfect receptor-negative Vero (monkey) cells. Results showed that transfected cells expressed receptor molecules capable of binding HRV and a monoclonal antibody which recognizes the major group HRV receptor. The cloned receptor cDNA encoded a protein with a sequence nearly identical to that of the intercellular adhesion molecule 1 (ICAM-1), indicating that the two surface proteins are one and the same. Both proteins have identical mass, carbohydrate composition, and tissue distribution. In addition, major group receptors on HeLa cells could be induced with various cytokines in a manner similar to the ICAM-1 ligand. A similar induction of the HRV "minor" group receptor was not observed.

Chemical synthesis and enzymatic activity of a 99-residue peptide with a sequence proposed for the human immunodeficiency virus protease.

Retroviral proteins, including those from the human immunodeficiency virus (HIV), are synthesized as polyprotein precursors that require proteolytic cleavage to yield the mature viral proteins. A 99-residue polypeptide, encoded by the 5' end of the pol gene, has been proposed as the processing protease of HIV. The chemical synthesis of the 99-residue peptide was carried out by the solid-phase method, and the isolated product was found to exhibit specific proteolytic activity upon folding under reducing conditions. Upon size-exclusion chromatography, enzymatic activity was eluted at a point consistent with a dimeric molecular size. Specificity was demonstrated by the cleavage of the natural substrate HIV gag p55 into gag p24 and gag p17, as well as cleavage of small peptide substrates representing processing sites of HIV fusion proteins. The proteolytic action of the synthetic product could be inhibited by pepstatin, an aspartic protease inhibitor.

Human cytotoxic lymphocyte tryptase. Its purification from granules and the characterization of inhibitor and substrate specificity.

A trypsin-like enzyme (tryptase) has been purified to homogeneity from the granules of a human cytolytic lymphocyte (CTL) line, Q31, by a three-step procedure. By including 0.3% (v/v) Triton X-100 and 1 mg/ml heparin in purification buffers, near total yields of tryptase activity were obtained during the purification. The enzyme, referred to as Q31 tryptase, migrated in polyacrylamide gels with sodium dodecyl sulfate at a position corresponding to 28 kDa with and to 45 kDa without 2-mercaptoethanol. It had an amino-terminal sequence identical to a previously reported human CTL tryptase at 20 of 22 positions identified. It hydrolyzed N alpha-carbobenzyloxy-L-lysyl-thiobenzyl ester (BLT), and this BLT esterase activity was most efficient at slightly alkaline pH and was relatively more active near neutral pH than mouse CTL tryptase. Human alpha 1-protease inhibitor, human antithrombin III, phenylmethanesulfonyl fluoride, and p-aminobenzamidine inhibited the Q31 tryptase. The inhibition by human antithrombin III was rapid enough to be of physiological significance. A survey of oligopeptide p-nitroanilides found that the best substrate for human Q31 tryptase is H-D-(epsilon-carbobenzyloxy)Lys-L-Pro-L-Arg-p-nitroanilide. The Q31 tryptase appears to have broad specificity for amino acid residues at P2 and P3, i.e. at 2 and 3 residues amino-terminal to the scissile bond.

Rat alkaline phosphatase. II. Structural similarities between the osteosarcoma, bone, kidney, and placenta isoenzymes.

A mouse monoclonal antibody raised against rat osteosarcoma alkaline phosphatase (AP) was covalently coupled to protein A-Sepharose and used to purify this enzyme from preparations of rat osteosarcoma, calvaria, kidney, and placenta in a single-step procedure. The tissue-specific isoenzymes purified in this manner showed identity in the immunodiffusion reaction with a polyclonal anti-AP antibody, but differed in apparent molecular weight and degree of polydispersity on sodium dodecyl sulfate-polyacrylamide gels. Treatment with N-glycanase abolished these differences, yielding proteins with an apparent molecular weight of 52,000 Da and identical V8 protease digestion patterns. Alkaline phosphatase from these tissues showed no significant difference in amino acid composition and identity in the first 20 N-terminal amino acids. These findings provide structural evidence which supports the hypothesis that the tissue-specific alkaline phosphatase isoenzymes share a common protein sequence subject to different glycosylation pattern.

Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein.

We have determined the nucleotide sequence of a cDNA clone, pDTD55, complementary to rat liver quinone reductase mRNA (Williams, J.B., Lu, A.Y.H., Cameron, R.G., and Pickett, C.B. (1986) J. Biol. Chem. 261, 5524-5528). The cDNA clone contains an open reading frame of 759 nucleotides encoding a polypeptide comprised of 253 amino acids with a Mr = 28,564. To verify the predicted amino acid sequence of quinone reductase, we have been able to align the amino acid sequences of a cyanogen bromide digest of the purified enzyme to the sequence deduced from the cDNA clone. A comparison of the quinone reductase sequence with other known flavoenzymes did not reveal a significant degree of amino acid sequence homology. These data suggest that the quinone reductase gene has evolved independently from genes encoding other flavoenzymes.

Rat liver glutathione S-transferases. DNA sequence analysis of a Yb2 cDNA clone and regulation of the Yb1 and Yb2 mRNAs by phenobarbital.

We have constructed a cDNA clone, pGTA/C48, which is complementary to the rat liver glutathione S-transferase Yb2 mRNA. Recombinant clone pGTA/C48 contains a cDNA insert of 845 base pairs which overlaps nucleotides 108-952 of the Yb1 cDNA clone, pGTA/C44, described previously by our laboratory (Ding, G. J.-F., Lu, A. Y. H., and Pickett, C. B. (1985) J. Biol. Chem. 260, 13268-13271). Over the protein coding region of the Yb1 and Yb2 cDNA clones there is an 84% nucleotide sequence homology, whereas the 3' untranslated regions are only 32% homologous. The complete amino acid sequence of the Yb2 subunit has been determined from a combination of DNA sequence analysis of pGTA/C48 and conventional protein sequence analysis of the glutathione S-transferase Yb1 Yb2 heterodimer. The Yb2 subunit is comprised of 218 amino acids with a molecular weight of 25,705 and has an amino acid sequence which is 79% homologous to the sequence of the Yb1 subunit. We have utilized the divergent 3' untranslated regions of three rat liver glutathione S-transferase cDNA clones as specific probes to determine the effect of phenobarbital on the level of Yb1, Yb2, and Yc mRNAs. Our results clearly show that the Yb1 and Yb2 mRNAs are elevated approximately 5-6-fold by phenobarbital administration; whereas the Yc mRNA is only modestly elevated by this xenobiotic. Finally, our data suggest that the Yb2 subunit is encoded by a gene(s) which is distinct from the Yb1 gene(s) and provides direct evidence for the existence of multiple glutathione S-transferase Yb genes in the rat.

Rat liver glutathione S-transferases. Construction of a cDNA clone complementary to a Yc mRNA and prediction of the complete amino acid sequence of a Yc subunit.

Using polysomal immunoselected rat liver glutathione S-transferase mRNAs, we have constructed cDNA clones using DNA polymerase I, RNase H, and Escherichia coli ligase (NAD+)-mediated second strand cDNA synthesis as described by Gubler and Hoffman (Gubler, U., and Hoffman, B. S. (1983) Gene 25, 263-269). Recombinant clone, pGTB42, contained a cDNA insert of 900 base pairs whose 3' end showed specificity for the Yc mRNA in hybrid-select translation experiments. The nucleotide sequence of pGTB42 has been determined, and the complete amino acid sequence of a Yc subunit has been deduced. The cDNA clone contains an open reading frame of 663 nucleotides encoding a polypeptide comprising 221 amino acids with a molecular weight of 25,322. The NH2-terminal sequence deduced from pGTB42 is in agreement with the first 39 amino acids determined for a Ya-Yc heterodimer by conventional protein-sequencing techniques. A comparison of the nucleotide sequence of pGTB42 with the sequence of a Ya clone, pGTB38, described previously by our laboratory (Pickett, C. B., Telakowski-Hopkins, C. A., Ding, G. J.-F., Argenbright, L., and Lu, A.Y.H. (1984) J. Biol. Chem. 259, 5182-5188) reveals a sequence homology of 66% over the same regions of both clones; however, the 5'- and 3'-untranslated regions of the Ya and Yc mRNAs are totally divergent in their sequences. The overall amino acid sequence homology between the Ya and Yc subunits is 68%, however, the NH2-terminal domain is more highly conserved than the middle or carboxyl-terminal domains. Our data suggest that the Ya and Yc subunits of the rat liver glutathione S-transferases are products of two different mRNAs which are derived from two related yet different genes.

Purification and properties of chicken growth hormone and the development of a homologous radioimmunoassay.

Highly purified growth hormone (GH) has been isolated from pituitary glands of chicken (Gallus domesticus), and a specific homologous radioimmunoassay (RIA) has also been developed. The purified chicken GH was active in the rat tibia bioassay and it gave a dose-dependent response which paralleled that of the bovine GH standard. High pressure liquid chromatography revealed that the purified chicken GH was homogenous. Chicken GH had an Rf value of 0.2 in disc electrophoresis, and a MW of 26,000 from sodium dodecyl sulfate-gel electrophoresis. The isoelectric point was estimated to be 7.6 by gel isoelectric focusing. The amino acid composition of chicken GH was found to be similar to that of mammalian GH, and the NH2-terminal amino acid was threonine. Partial sequencing (114 amino acids) of the chicken GH showed 79% homology with bovine GH. An antiserum was developed to the purified chicken GH in a rabbit, and it was used to develop a homologous RIA using 125I-labeled chicken GH as the ligand. The purified chicken GH was iodinated via the lactoperoxidase method to a specific activity of approximately 100 microCi/micrograms. Plasma from chickens, medium from incubation of pituitary glands, and homogenates of pituitary glands gave parallel dilution-response curves with the chicken GH standard. Mammalian GH, prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) showed no cross-reaction with the 125I-labeled chicken GH. Purified turkey GH showed parallel dose response with the chicken GH, but purified turkey PRL did not cross-react. Chicken FSH and LH also showed no inhibition of binding. The minimum detectable concentration of the assay was 0.93 ng/tube, and the intraassay and interassay coefficients of variation were 9 and 16%, respectively. The specific binding of 125I-labeled chicken GH to a microsomal fraction isolated from chicken liver was identified, and the specific binding was generally low (1-4%). Turkey PRL, and chicken LH and FSH showed no inhibition of the 125I-labeled chicken GH hepatic binding and the ontogeny of the hepatic GH receptor binding sites in male and female chickens was examined.

Mouse submaxillary gland renin. Purification and properties of minor forms, which include several differently processed forms of the major gene product and a second gene product.

Three minor forms of renin from the submaxillary glands of male mice called D1, D2, and E have been purified to homogeneity. Their amino acid compositions are identical to the principal form of mouse submaxillary gland renin (renin A), except for 1, 1, and 2 extra arginine residues, respectively. The electrophoretic mobility of renin D2 does not change upon reduction, indicating that its heavy and light chains are linked by more than a disulfide bond. The light chain of renin D1 has an electrophoretic mobility different from the light chain of renin A. Renin D2 is proposed to be renin A with an arginine-arginine dipeptide connecting the carboxyl terminus of the heavy chain to the NH2 terminus of the light chain, with the light chain missing the carboxyl-terminal arginine of renin A. Renin D1 is suggested to be renin D2 with the peptide bond between an arginine and the NH2 terminus of the light chain cleaved. Renin E is proposed to be renin D1 plus the carboxyl-terminal arginine of the light chain. A fourth minor form of male mouse submaxillary renin, called renin B/A, has been purified to homogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in isoelectric focusing. Renin B/A seems to be a second renin gene product which is difficult to separate from renin A. Renin B/A has an amino acid composition significantly different from renin A, and all three preparations of B/A had compositions significantly different from one another. For renin B/A, the light chain sequence and the first 53 NH2-terminal residues of its heavy chain sequence were identical to renin A.

Isolation and sequence determination of peptide components of atrial natriuretic factor.

The independent isolation and sequence determination in our laboratories of three closely related Atrial Natriuretic Factor peptides from rat atria confirm the sequences of ANF peptides reported by Seidah et al and synthesized by Nutt et al [Proc. Natl. Acad. Sci., (1984) in press] and contain the sequences reported by Flynn et al [Biochem. Biophys. Res. Commun. (1983) 117: 859-865] and by Currie et al [Science (1984) 223: 67-69]. In addition, we provide proof for a C-terminal tyrosine rather than tyrosine amide in our isolated peptides.

Identification of the active-site residue of gamma-cystathionase labeled by the suicide inactivator beta, beta, beta-trifluoroalanine.

Inactivation of gamma-cystathionase by beta, beta, beta-trifluoroalanine, a suicide inactivator of the enzyme, results in covalent labeling of an amino group of the protein [Silverman, R. B., & Abeles, R. H. (1977) Biochemistry 16, 5515-5520]. We have established that this modified amino function is the epsilon-NH2 group of a lysine residue. A heptapeptide which includes this modified lysine residue was isolated, and its sequence was found to be Cys-Ser-Ala-Thr-Lys-Tyr-Met. The amino acid sequence was the same as that determined for peptides containing the active-site lysine residue which forms a Schiff base with pyridoxal phosphate. Therefore the epsilon-NH2 group of the active-site lysine which binds pyridoxal phosphate is capable of interacting with the beta carbon of trifluoroalanine, and presumably the beta carbon of normal substrates. We therefore propose that this lysine residue may function as a proton-transfer agent in the reactions catalyzed by gamma-cystathionase.

Isolation and characterization of somatostatin from pigeon pancreas.

Most of the somatostatin-like activity from pigeon pancreas was found to correspond to small species with an apparent molecular weight of 1500--2500. This species was isolated under conditions minimizing intermolecular interactions and protease activities. The isolated product was characterized by two somatostatin radioimmunoassays, a bioassay, endgroup determination, and amino acid analysis. The structure of the isolated compound was determined to be H-Ala-Gly-cyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Additionally, small amounts of des-Ala1-somatostatin, a possible degradation product of pancreatic somatostatin, and a large somatostatin-like species with an apparent molecular weight of 11,000--12,500 were detected. It is concluded that the main somatostatin-like polypeptide isolated from pigeon pancreas is identical to the mammalian hypothalamic tetradecapeptide somatostatin.

Conversion of rat pre-proalbumin to proalbumin in vitro by ascites membranes. Demonstration by NH2-TERMINAL SEQUENCE ANALYSIS.

Rat liver poly(A)-containing RNA was translated in an ascites cell-free system. Labeled protein precipitable by antibody directed against rat serum albumin was identified as pre-proalbumin based on its size and partial NH2-terminal sequence. However, when an ascites membrane fraction was added to the translation reaction, the albumin antibody-precipitable material was smaller than pre-proalbumin. Partial NH2-terminal sequence analysis of this protein revealed that it was proalbumin. Conversion of pre-proalbumin to proalbumin by the ascites membrane fraction was complete and precise--i.e. no serum albumin was observed. Reconstitution in vitro of the processing of pre-proalbumin to its stable intracellular form, proalbumin, provides a method for studying the initial proteloytic event involved in secretion of rat serum albumin.

Dihydrofolate reductase: the amino acid sequence of the enzyme from a methotrexate-resistant mutant of Escherichia coli.

The determination of the amino acid sequence of the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) from a mutant of Escherichia coli B is described. The 159 residues were positioned by automatic Edman degradation of the whole protein, of the reduced and alkylated cyanogen bromide fragments, and of selected tryptic, chymotryptic, and thermolytic digestion products. An N-bromosuccinimide produced fragment of the largest cyanogen bromide peptide was also used in the sequence determination.

Rat liver preproalbumin: in vitro synthesis and partial amino acid sequence.

Rat liver poly(A)-containing RNA greatly stimulated incorporation of radioactive amino acids into protein when added to a wheat germ in vitro translation system. Approximately 7% of the labeled synthetic product was precipitated following indirect immunoprecipitation with antisera to rat serum albumin. Analysis of this material, and of the cyanogen bromide fragments derived from it, by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that it contained an NH2-terminal extension of about 2500 daltons when compared to rat serum albumin. Automated sequence determination of purified cell-free product labeled with various radioactive amino acids revealed the presence of 18 additional amino acids NH2-terminal to the sequence of rat proalbumin. The partial sequence of this extension was found to be: Met-X-X-X-X-Phe-Leu-Leu-Leu-Leu-Phe-X-X-X-X-X-Phe-X-proalbumin. On the basis of this evidence, the immunoprecipitable cell-free product was designated preproalbumin.