Bovine cartilage types VI and IX collagens. Characterization of their forms in vivo.

1. Collagens were extracted from bovine cartilage by 4 M-guanidinium chloride in the presence of proteinase inhibitors and identified by immunoblotting with specific anti-collagen sera. 2. The collagens retained their native conformations (shown by the resistance of their triple-helical domains to pepsin digestion), and the molecular masses of their component alpha-chains indicated that the chains were intact. 3. Type VI collagen was extracted as a large-molecular-mass disulphide-bonded aggregate composed of components of molecular mass 140 kDa and 200-240 kDa, and was therefore similar to type VI collagen identified in noncartilaginous tissues. Immunoblotting established the 200-240 kDa components as intact forms of the alpha 3(VI) chain. 4. Type IX collagen consisted of three clearly separable components of molecular mass 84 kDa, 72 kDa and 66 kDa, which were assigned to the alpha 1(IX)-, alpha 3(IX)- and alpha 2(IX)-chains respectively, and a large proportion of this collagen had no covalently bound glycosaminoglycan attached to the alpha 2(IX)-chain. 5. Differences between the type IX collagen extracted from bovine cartilage and that identified in biosynthetic studies on chick cartilage are discussed.     

Type VI collagen is composed of a 200 kd subunit and two 140 kd subunits.

We have isolated type VI collagen, a transformation-sensitive glycoprotein of the extracellular matrix, in an intact, disulfide-bonded form. The protein contains a 200 kd subunit and two different 140 kd subunits in a stoichiometric ratio. Based on the amount of hydroxyproline and hydroxylysine, the sensitivity to bacterial collagenase and the cross-reactivity with antibodies to pepsin-extracted type VI collagen, we have identified the 200 kd subunit as the alpha 3(VI) chain and the two 140 kd subunits as the alpha 1(VI) and alpha 2(VI) chains. The alpha 3(VI) chain is synthesized by cells in culture as a precursor of 260 kd, while no precursor form of the other two chains could be detected.     

The polymerase subunit of DNA polymerase III of Escherichia coli. II. Purification of the alpha subunit, devoid of nuclease activities

The alpha subunit (140 kDa) of DNA polymerase III (pol III) holoenzyme has been purified to near-homogeneity from a plasmid-carrying Escherichia coli strain which overproduced the alpha subunit about 20-fold. Pol III core (containing only the alpha, epsilon, and theta subunits), produced at twice the normal level, was also purified in good yield. The isolated alpha subunit has DNA polymerase activity, which is completely inhibited by 10 mM N-ethylmaleimide or 150 mM KCl as observed in the pol III core or holoenzyme. The alpha subunit has an apparent turnover number of 7.7 nucleotides polymerized per s, compared to 20 for pol III core, and is more thermolabile. The alpha subunit lacks the 3'----5' exonuclease (proofreading) activity of pol III core; neither alpha subunit nor core (nor holoenzyme) possesses any of the previously reported 5'----3' exonuclease activity. Thus, the alpha polypeptide is the polymerase subunit and epsilon (27 kDa) is the proofreading subunit (Scheuermann, R. H., and Echols, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 7747-7751). Together with the theta polypeptide (10 kDa), of unknown function, they form a pol III core with greater stability and catalytic efficiency.     

C-terminal truncated forms of Met, the hepatocyte growth factor receptor.

The MET proto-oncogene encodes a transmembrane tyrosine kinase of 190 kDa (p190MET), which has recently been identified as the receptor for hepatocyte growth factor/scatter factor. p190MET is a heterodimer composed of two disulfide-linked chains of 50 kDa (p50 alpha) and 145 kDa (p145 beta). We have produced four different monoclonal antibodies that are specific for the extracellular domain of the Met receptor. These antibodies immunoprecipitate with p190MET two additional Met proteins of 140 and 130 kDa. The first protein (p140MET) is membrane bound and is composed of an alpha chain (p50 alpha) and an 85-kDa C-terminal truncated beta chain (p85 beta). The second protein (p130MET) is released in the culture supernatant and consists of an alpha chain (p50 alpha) and a 75-kDa C-terminal truncated beta chain (p75 beta). Both truncated forms lack the tyrosine kinase domain. p140MET and p130MET are consistently detected in vivo, together with p190MET, in different cell lines or their culture supernatants. p140MET is preferentially localized at the cell surface, where it is present in roughly half the amount of p190MET. The two C-terminal truncated forms of the Met receptor are also found in stable transfectants expressing the full-length MET cDNA, thus showing that they originate from posttranslational proteolysis. This process is regulated by protein kinase C activation. Together, these data suggest that the production of the C-terminal truncated Met forms may have a physiological role in modulating the Met receptor function.     

Identification of a novel 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide in calcium channel preparations

A 1,4-dihydropyridine- and phenylalkylamine-binding polypeptide has been identified by photoaffinity labeling of purified rabbit and guinea pig skeletal muscle calcium channel preparations. The arylazide ligands (-)-[3H]azidopine and (-)-5-[(3-azidophenethyl)[N-methyl-3H]methylamino]-2-(3,4,5- trimethoxyphenyl)-2-isopropylvaleronitrile [( N-methyl-3H]LU 49888) were used to label 1,4-dihydropyridine- and phenylalkylamine-binding sites, respectively. A single, 155 to 170-kDa polypeptide was specifically labeled by both ligands in rabbit and guinea pig preparations provided that the skeletal muscle membranes used for purification were derived from fresh and not previously frozen and thawed tissue. The photoaffinity labeled polypeptide (termed here alpha 1) is different from the previously described alpha subunit in that it has the identical electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels irrespective of pretreatment either with N-ethylmaleimide or with dithiothreitol. The use of transverse tubular membranes isolated from previously frozen and thawed skeletal muscle results in a purified calcium channel preparation devoid of the alpha 1 subunit. In these preparations proteolytic degradation products of alpha 1 are labeled with both (-)-[3H]azidopine and [N-methyl-3H]LU 49888. Another large molecular weight polypeptide (termed here alpha 2) was also present in every purified calcium channel preparation studied. alpha 2 is distinct from alpha 1 in that reduction with dithiothreitol changes its apparent mass from 160-190 to 130-150 kDa. The alpha 2 subunit is not photoaffinity labeled either with (-)-[3H]azidopine or [N-methyl-3H]LU 49888. These data suggest that two distinct high molecular weight polypeptides (termed alpha 1 and alpha 2) are putative subunits of skeletal muscle calcium channels. Only the alpha 1 subunit contains both 1,4-dihydropyridine and phenylalkylamine receptors. alpha 2 is the same as the previously described alpha subunit (Curtis, B. M., and Catterall, W. A. (1984) Biochemistry 23, 2113-2118), but is neither a 1,4-dihydropyridine- nor a phenylalkylamine-binding protein.     

The soluble IL-6 receptors: serum levels and biological function.

IL-6 exerts its biological activities through interaction with specific receptors expressed on the surface of target cells. IL-6 binds first to a low-affinity (10(-9) M) subunit, a 80 kDa glycoprotein also called gp80 or IL-6R alpha. The IL-6/IL-6R alpha complex recruits the signal-transducing b subunit, a 130 kDa glycoprotein called gp130. The association of gp130 with IL-6 and IL-6R alpha leads to the formation of the high-affinity IL-6 receptor complex, to the linkage of two gp130 subunits and to signal transduction. Soluble forms of both receptors have been described and found in biological fluids. Soluble cytokine receptors are generated by either proteolytic cleavage of their membrane moiety or by alternative splicing. Both mechanisms have been described for sIL-6R and sgp130 formation. Interestingly, the association of IL-6 with the soluble form of IL-6R alpha is capable of eliciting a biological response in cells that express only the membrane gp130. This type of activation, called "trans-signalling", renders virtually all cells capable of responding to IL-6/sIL-6R alpha complexes, making for a large new spectrum of IL-6 activities, ranging from the control of the immune response to involvement in pathological states. In this review the biological activities of IL-6 will be considered in the light of new knowledge concerning the association of IL-6 and the soluble IL-6 receptors.     

Human type VI collagen: isolation and characterization of alpha 1 and alpha 2 chains by two-dimensional preparative gel electrophoresis

Two 140 kDa collagenous glycoproteins were isolated from 5 M guanidinium chloride extracts of human uterine leiomyoma by two-dimensional preparative gel electrophoresis. The glycoproteins represented the major concanavalin A binding fraction of the extract and were also present in adult human skin. On two-dimensional gel electrophoresis the glycoproteins appeared as elongated spots, indicating variations of their isoelectric points from 5 to 6. These glycoproteins were disulfide-bonded components of high molecular mass protein and, after reduction, became sensitive to collagenase treatment that generated peptides corresponding in size to those of the noncollagenous domains of type VI collagen. Antisera raised against these purified glycoproteins reacted with either pepsin-derived alpha 1(VI) or pepsin-derived alpha 2(VI) chains but not with alpha 3(VI) chain of human type VI collagen. Reciprocally, these glycoproteins reacted with monoclonal antibodies against type VI collagen. These results indicate that the glycoproteins represent the integral alpha 1 and alpha 2 chains of type VI collagen. The globular domains of alpha 1(VI) and alpha 2(VI) chains remaining after collagenase treatment appeared on two-dimensional gel electrophoresis as elongated spots, suggesting that the noncollagenous portions determine the well known microheterogeneity of the molecule. The differences in isoelectric points between and within alpha chains may facilitate the formation of microfibrillar network.     

Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant alpha subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, alpha subunit kinase activity declined rapidly. Addition of the beta subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 microM), whereas the alpha subunit alone had a Km of 240 microM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.     

Resolution of distinct membrane-bound enzymes from Enterobacter cloacae SLD1a-1 that are responsible for selective reduction of nitrate and selenate oxyanions

Enterobacter cloacae SLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett. 228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with alpha and beta subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (alphabetagamma) complex with an apparent molecular mass of approximately 600 kDa. The individual subunit sizes are approximately 100 kDa (alpha), approximately 55 kDa (beta), and approximately 36 kDa (gamma), with a predicted overall subunit composition of alpha3beta3gamma3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of a b-type cytochrome in the active complex. The apparent Km for selenate was determined to be approximately 2 mM, with an observed Vmax of 500 nmol SeO4(2-) min(-1) mg(-1) (kcat, approximately 5.0 s(-1)). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.     

Integrin alpha3beta1 expressed by human colon cancer cells is a major carrier of oncodevelopmental carbohydrate epitopes

Oncodevelopmental carbohydrate epitopes are a common feature of human colorectal carcinoma, yet their biological significance remains unclear. We have shown previously that monoclonal antibody (MAb) 3A7, which recognizes a determinant on type 2 chain blood group A and B oligosaccharides, detects oncodevelopmental changes in azoxymethane-induced rat colon tumors and some human colon cancer cell lines. (Laferté S et al. [19951 J. Cell. Biochem. 57:101-119). In this study, we set out to purify gp140, the major glycoprotein carrier of the 3A7 epitope expressed by human colon cancer cells, as a first step towards elucidating the contribution of the 3A7 epitope and its major glycoprotein carrier to colon cancer progression. To this end, gp140 was purified from HT29 cells and used for the preparation of polypeptide-specific monoclonal antibodies. Five monoclonal antibodies (7A8, 7B11, 8C7, 8H7, and 11D4) immunoprecipitated a 3A7-immunoreactive glycoprotein complex of 140 kDa which was subsequently identified by partial protein sequencing as alpha3beta1 integrin. Flow cytometric analysis of Chinese hamster ovary (CHO) cells expressing different human integrin chains revealed that MAbs 7A8 and 7B11 detect the alpha3 integrin subunit whereas MAbs 8C7 and 8H7 detect the beta1 integrin subunit. MAb 11D4, which did not bind to any of the CHO transfectants, detected type 2 chain blood group A determinant. Flow cytometric analysis of a panel of human colon carcinoma cell lines obtained from blood group A, AB, or B individuals revealed a direct correlation between cell-surface expression of the 3A7 epitope and alpha3 integrin subunit, suggesting that alpha3beta1 integrin is a preferred target of the 3A7 epitope in colon cancer cells. Using lectins and glycosidases to examine further the carbohydrate structure of alpha3beta1 integrin, we demonstrated that it is a sialoglycoprotein containing both N- and O-linked oligosaccharides. In addition, both alpha3 and beta1 integrin subunits express beta1-6 branched Asn-linked oligosaccharides and short poly-N-acetyllactosamine units (Galbeta1-4GlcNAc-R; n < or = 3), glycans previously implicated in cancer metastasis.Thus, alpha3beta1 integrin expressed by human colon carcinoma cells is a major carrier of oncodevelopmental carbohydrate epitopes whose presence may modulate tumor cell adhesion, migration, and/or invasion.     

Identification and characterization of a novel high-molecular-weight form of the integrin alpha 3 subunit.

The integrin alpha 3 beta 1 is a multiligand extracellular matrix receptor found on many cell types. Immunoprecipitations of 125I-surface-labeled prostate carcinoma cell lines, DU145 and PC-3, with the anti-alpha 3 integrin monoclonal antibodies J143 or PIB5, resulted in the coimmunoprecipitation, along with the expected alpha 3 beta 1 heterodimer, of a polypeptide with a molecular mass of 225 kDa. This protein could also be copurified with the 155-kDa alpha 3 and 115-kDa beta 1 subunits upon affinity chromatography of 125I-surface-labeled cell extracts on anti-alpha 3 antibody-Sepharose columns. Upon reduction, this 225-kDa protein generated 130- and 95-kDa polypeptides, while the 155-kDa alpha 3 subunit generated 130- and 25-kDa polypeptides. The 225-kDa protein did not generate a 25-kDa polypeptide. Deglycosylation and reduction of the 225-kDa protein resulted in the generation of 110- and 95-kDa polypeptides, while deglycosylation and reduction of the 155-kDa alpha 3 resulted in a 110-kDa polypeptide identical in size to the 110-kDa polypeptide generated from the 225-kDa protein. Peptide maps generated from the 110-kDa components of the 225-kDa polypeptide and the 155-kDa alpha 3 integrin subunit were identical, as were their N-terminal amino acid sequences. An antibody directed against the cytoplasmic domain of the alpha 3 subunit immunoprecipitated the 225-kDa polypeptide in addition to the 155-kDa alpha 3 subunit. Furthermore, Northern blot analysis of RNA from DU145 and PC-3 cells with a human alpha 3 cDNA probe identified an mRNA species of 6.2 kb in addition to a major mRNA species of 4.3 kb. The larger mRNA species, which is of an appropriate size for encoding a polypeptide of approximately 220-kDa, was not detectable in cells which did not express the 225-kDa protein. These data demonstrate that the 225-kDa polypeptide represents a novel integrin alpha 3 subunit consisting of the alpha 3 integrin heavy chain disulfide-bonded to a 95-kDa polypeptide which may represent an alternative "light" chain to the 25-kDa light chain of the alpha 3 subunit.     

Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ring-like structures and binds beta1 integrins, collagens and fibronectin.

Human Mac-2 binding protein (M2BP) was prepared in recombinant form from the culture medium of 293 kidney cells and consisted of a 92 kDa subunit. The protein was obtained in a native state as indicated by CD spectroscopy, demonstrating alpha-helical and beta-type structure, and by protease resistance and immunological analysis. It was highly modified by N- and O-glycosylation but not by glycosaminoglycans. Ultracentrifugation showed non-covalent association into oligomers with molar masses of 1000-1500 kDa. Electron microscopy showed ring-like shapes with diameters of 30-40 nm. M2BP bound in solid-phase assays to collagens IV, V and VI, fibronectin and nidogen, but not to fibrillar collagens I and III or other basement membrane proteins. The protein also mediated adhesion of cell lines at comparable strength with laminin. Adhesion to M2BP was inhibited by antibodies to integrin beta1 subunits but not to alpha2 and alpha6 subunits, RGD peptide or lactose. This distinguishes cell adhesion of M2BP from that of laminin and excludes involvement of lactose-binding galectin-3. Immunological assays demonstrated variable secretion by cultured human cells of M2BP, which was detected in the extracellular matrix of several mouse tissues.     

The mode of anchoring and precursor forms of sucrase-isomaltase and maltase-glucoamylase in chicken intestinal brush-border membrane. Phylogenetic implications

Chicken intestinal sucrase-isomaltase and maltase-glucoamylase have been isolated in their intact form by detergent solubilization and characterized as to their subunit composition and mode of anchoring in the brush-border membrane. Both are heterodimeric enzyme complexes composed of two subunits each of approximately 140 and 130 kDa. Contrary to the mammalian sucrase-isomaltase, chicken isomaltase was identified as the smaller of the two subunits. As was shown by hydrophobic labeling, only one of the two subunits in each heterodimer is anchored in the bilayer, the smaller 130 kDa isomaltase subunit of the sucrase-isomaltase complex, and the larger 140 kDa subunit of the maltase-glucoamylase complex. Both preparations contain a high-molecular weight polypeptide of approximately 250 kDa which in the case of sucrase-isomaltase could be identified by peptide mapping as a single-chain precursor not (yet) proteolytically processed to the final heterodimer. These first data on the mode of membrane anchoring of non-mammalian glycosidases indicate that they are synthesized, inserted into the membrane, and processed in ways similar to the mammalian enzymes. The fundamental unity between avian and mammalian sucrase-isomaltases suggests that the partial gene duplication of an ancestral isomaltase gene and the subsequent mutation of one of the active sites resulting in pro-sucrase-isomaltase has occurred prior to the separation of mammals from reptiles, i.e. more than 300 million years ago.     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase.

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase     

Possible involvement of a 95-kDa protein phosphorylation in phorbol 12-myristate 13-acetate-induced suppression of zymosan phagocytosis in guinea pig macrophages

Recently, we characterized a surface antigen (Z-1) of guinea pig macrophages by monoclonal anti-Z-1 antibody. The Z-1 antigen consists of two different polypeptide chains; alpha (140 kDa) and beta (95 kDa). This antigen is closely correlated with the phagocytic activity of the cells for zymosan and presumably functions as a receptor for zymosan. In the present study, the effect of phorbol 12-myristate 13-acetate (PMA) on the function of Z-1 was examined. Incubation of ortho-[32P]phosphate-labeled macrophages with PMA greatly increased the phosphorylation of the beta subunit of Z-1 but not that of the alpha subunit. Optimal phosphorylation was observed when cells were incubated with 300 ng/ml of PMA for 60-120 min. The PMA-induced phosphorylation was markedly suppressed by treatment of the macrophages with H-7, an inhibitor of protein kinase C. A chemotactic peptide, N-formyl-Met-Leu-Phe (fMLP) also caused phosphorylation of the beta subunit. Unlike PMA, fMLP maximized the phosphorylation within 30 s. Purified Z-1 was an excellent substrate for the exogenously added protein kinase C only in the presence of both Ca2+ and phosphatidylserine. H-7 completely inhibited the in vitro phosphorylation. These data suggest that the beta subunit of Z-1 is phosphorylated by protein kinase C. The phosphorylation of Z-1 by PMA and fMLP coincided with inhibition of zymosan phagocytosis. A linear relationship was obtained between the level of phosphorylation of Z-1 and the degree of inhibition of zymosan phagocytosis induced by PMA. Thus, the results suggest that zymosan uptake is negatively regulated by protein kinase C-mediated phosphorylation of the beta subunit of Z-1.     

Cloning, expression, and chromosomal localization of the 140-kilodalton subunit of replication factor C from mice and humans.

We have isolated a full-length mouse cDNA encoding a lysine-rich protein of 1,131 amino acids with a calculated molecular mass of 126 kDa. The protein binds in a sequence-unspecific manner to DNA, is localized exclusively in the nucleus, and contains a putative ATP binding site and a stretch of 80 amino acids with homology to the carboxy terminus of prokaryotic DNA ligases. On the basis of the following facts, we conclude that the isolated cDNA encodes the 140-kDa subunit of mouse replication factor C (mRFC140). (i) The sequence around the ATP binding site shows significant homology to three small subunits of human replication factor C. (ii) Polyclonal antibodies raised against the protein encoded by this cDNA cross-react with the 140-kDa subunit of purified human replication factor C (hRFC140) and recognize in mouse cell extracts an authentic protein with an apparent molecular mass of 130 kDa. (iii) Sequence comparison with a human cDNA isolated by using tryptic peptide sequence information from purified hRFC140 revealed 83% identity of the encoded proteins. The mRFC140 gene is ubiquitously expressed, and two mRNAs approximately 5.0 and 4.5 kb long have been detected. The gene was mapped by in situ hybridization to mouse chromosome 5, and its human homolog was mapped to chromosome 4 (p13-p14).     

Identification of cell-binding sites on the Laminin alpha 5 N-terminal domain by site-directed mutagenesis

The newly discovered laminin alpha(5) chain is a multidomain, extracellular matrix protein implicated in various biological functions such as the development of blood vessels and nerves. The N-terminal globular domain of the laminin alpha chains has an important role for biological activities through interactions with cell surface receptors. In this study, we identified residues that are critical for cell binding within the laminin alpha(5) N-terminal globular domain VI (approximately 270 residues) using site-directed mutagenesis and synthetic peptides. A recombinant protein of domain VI and the first four epidermal growth factor-like repeats of domain V, generated in a mammalian expression system, was highly active for HT-1080 cell binding, while a recombinant protein consisting of only the epidermal growth factor-like repeats showed no cell binding. By competition analysis with synthetic peptides for cell binding, we identified two sequences: S2, (123)GQVFHVAYVLIKF(135) and S6, (225)RDFTKATNIRLRFLR(239), within domain VI that inhibited cell binding to domain VI. Alanine substitution mutagenesis indicated that four residues (Tyr(130), Arg(225), Lys(229), and Arg(239)) within these two sequences are crucial for cell binding. Real-time heparin-binding kinetics of the domain VI mutants analyzed by surface plasmon resonance indicated that Arg(239) of S6 was critical for both heparin and cell binding. In addition, cell binding to domain VI was inhibited by heparin/heparan sulfate, which suggests an overlap of cell and heparin-binding sites. Furthermore, inhibition studies using integrin subunit monoclonal antibodies showed that integrin alpha(3)beta(1) was a major receptor for domain VI binding. Our results provide evidence that two sites spaced about 90 residues apart within the laminin alpha(5) chain N-terminal globular domain VI are critical for cell surface receptor binding.     

Topological analysis of the pyridine nucleotide transhydrogenase of Escherichia coli using proteolytic enzymes

The pyridine nucleotide transhydrogenase of Escherichia coli has an alpha 2 beta 2 structure (alpha: Mr, 54,000; beta: Mr, 48,700). Hydropathy analysis of the amino acid sequences suggested that the 10 kDa C-terminal portion of the alpha subunit and the N-terminal 20-25 kDa region of the beta subunit are composed of transmembranous alpha-helices. The topology of these subunits in the membrane was investigated using proteolytic enzymes. Trypsin digestion of everted cytoplasmic membrane vesicles released a 43 kDa polypeptide from the alpha subunit. The beta subunit was not susceptible to trypsin digestion. However, it was digested by proteinase K in everted vesicles. Both alpha and beta subunits were not attacked by trypsin and proteinase K in right-side out membrane vesicles. The beta subunit in the solubilized enzyme was only susceptible to digestion by trypsin if the substrates NADP(H) were present. NAD(H) did not affect digestion of the beta subunit. Digestion of the beta subunit of the membrane-bound enzyme by trypsin was not induced by NADP(H) unless the membranes had been previously stripped of extrinsic proteins by detergent. It is concluded that binding of NADP(H) induces a conformational change in the transhydrogenase. The location of the trypsin cleavage sites in the sequences of the alpha and beta subunits were determined by N- and C-terminal sequencing. A model is proposed in which the N-terminal 43 kDa region of the alpha subunit and the C-terminal 30 kDa region of the beta subunit are exposed on the cytoplasmic side of the inner membrane of E. coli. Binding sites for pyridine nucleotide coenzymes in these regions were suggested by affinity chromatography on NAD-agarose columns.     

Regulation of squid visual phospholipase C by activated G-protein alpha.

Phospholipase C (PLC) is the key enzyme in the phototransduction cascade of invertebrate rhabdomeric photoreceptors. In addition to 130 kDa PLC, a 95 kDa protein recognized by antibody against the catalytic site of PLC was found in the squid retina. The PLC-like 95 kDa protein (95 kDa PLC) was produced from 130 kDa PLC by an intrinsic protease in the presence of calcium. The 130 kDa PLC was stimulated by the active form of Gq-class G-protein alpha (Gq alpha), but the 95 kDa PLC was not, although their PLC activity was similar. A 35 kDa fragment, the counterpart of 95 kDa PLC, was not recognized by antibodies against catalytic site or N-terminal site of the 130 kDa PLC, indicating that the cleavage site is on the C-terminal side beyond the catalytic site. In the presence of a large excess of the 35 kDa fragment, 95 kDa PLC was stimulated by Gq alpha to a similar extent as intact 130 kDa PLC. These results indicate that the C-terminal polypeptide of PLC is necessary for regulation of its enzyme activity by Gq alpha. The uncoupling of PLC from Gq alpha, caused by limited proteolysis, is therefore a candidate regulatory mechanism of the phototransduction cascade in rhabdomeric photoreceptors.