ATP-Dependent human erythrocyte glutathione-conjugate transporter. I. Purification, photoaffinity labeling, and kinetic characteristics of ATPase activity

Dinitrophenyl S-glutathione (DNP-SG) ATPase is a 38 kDa membrane protein expressed in erythrocytes and other tissues. Although stimulation of ATP hydrolysis catalyzed by DNP-SG ATPase has been demonstrated in the presence of several structurally unrelated amphiphilic ions, structural and functional properties of this protein have not been well-defined. In the present study, we have developed an improved protocol for the purification of DNP-SG ATPase and investigated its kinetic and substrate-binding properties. The purification procedure was based on highly specific elution of the 38 kDa protein from DNP-SG affinity resin in the presence of ATP. The protein could not be eluted using either ADP or adenosine-5'-[beta,gamma-methylene]triphosphate (methylene-ATP), a nonhydrolyzable analogue of ATP. Doxorubicin (DOX), a weakly basic anthracycline chemotherapy agent, was found to be the preferred activator for stimulation of ATP hydrolysis by the enzyme. ATP binding to the enzyme was demonstrated using 8-azido-ATP photoaffinity labeling and binding of trinitrophenyl (TNP)-ATP, a fluorescent analogue of ATP. The photoaffinity labeling of DNP-SG ATPase (38 kDa) was saturable with respect to 8-azido ATP (Kd = 2 microM), indicating that the enzyme was capable of specific and saturable binding to ATP. DNP-SG binding was evident from the purification procedure itself and was also demonstrable by quenching of tryptophan fluorescence. Results of quenching of tryptophan fluorescence as well as radioactive isotope-binding studies indicated that DOX was bound to the purified protein as well.     

Purification and characterization of an NAD-malic enzyme from Bradyrhizobium japonicum A1017

An NAD-malic enzyme was purified to homogeneity from Bradyrhizobium japonicum A1017, and its molecular characteristics were surveyed. The enzyme exhibited native and subunit molecular masses of 388 and 85 kDa, respectively, suggesting that it exists as a homotetramer, and was activated by metabolic intermediates in glycolysis. The role of the enzyme in bacteroids' carbon metabolism is discussed.     

Purification and characterization of the acid soluble 26-kDa polypeptide from soybean seeds

Whey proteins from soybean seeds of Japanese varieties were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Among 11 varieties of soybean, three green and one black soybeans lacked a 26-kDa band that was found in all yellow soybeans. In this paper, the 26-kDa protein was named AS26k (acid soluble 26-kDa protein) temporarily. The AS26k protein was purified from Glycine max cv. Nattosyoryu, which is yellow soybean, through four purification steps: 30-35% saturated ammonium sulfate fractionation, ion exchange chromatography on S Sepharose Fast Flow, gel filtration on Sephadex G-100, and hydrophobic chromatography on phenyl Sepharose CL-4B. Purified AS26k was cleaved with V8 proteinase from Staphylococcus aureus or CNBr. The cleaved polypeptide contained two typical dehydrin motif sequences: DEYGNPV and (M)DKIKEKLPG, and a 19 amino acids sequence similar to a pea dehydrin. Native AS26k had a molecular mass of 32 kDa on gel filtration and a pl of 7.2 on two-dimensional PAGE. Similarly to other dehydrins and late embryogenesis abundant (LEA) proteins, AS26k was rich in hydrophilic amino acids, and highly heat stable. These results showed that AS26k was a dehydrin, a group II LEA protein in soybean seeds.     

Purification and biochemical analysis of WprA, a 52-kDa serine protease secreted by B. subtilis as an active complex with its 23-kDa propeptide

The Gram-positive bacterium Bacillus subtilis produces numerous proteases that are secreted to the extracellular milieu, and as strains are generated which lack the more prominent proteases, minor ones become detectable. We have isolated a 52-kDa secreted protease from the protease-deficient strain WB600. It is encoded by the wprA gene which encompasses a signal sequence, a 46-kDa propeptide further processed to 23 kDa, and the 52-kDa mature protease. The 52-kDa and 23-kDa polypeptides were previously detected in cell-wall preparations of a wild-type strain. We have co-purified these proteins from culture supernatant, and confirmed the same N-termini and molecular weights as the membrane-bound species. The WprA protease domain has 28.5% identity to subtilisin A, and like other subtilisins, it displays a broad substrate specificity. WprA and subtilisin A have similar pH profiles, showing optimal activity near pH 7.5 for substrates with Met, Gln, or Lys residues at P1. Using a substrate with Asp at P1, another peak of activity was observed for WprA at pH 5 and at pH 6 for subtilisin A. The pH dependence of some bacterial proteases in their interaction with substrates and inhibitors may be biologically relevant.     

Molecular characterization of the 77-kDa echinoderm microtubule-associated protein. Homology to the beta-transducin family

The major microtubule-associated protein (MAP) of sea urchins and several other echinoderms is a polypeptide of M(r) 77,000. The echinoderm MAP (EMAP) is abundant in embryonic and differentiated cells, as well as in mitotic and interphase microtubule arrays. To characterize the molecular structure and function of the EMAP, we isolated a full-length cDNA clone, which has one open reading frame that predicts a polypeptide of 686 amino acids with a calculated M(r) of 75,488. On the basis of charge distribution, EMAP can be divided into two distinctive domains: The NH2-terminal basic region (amino acids 1-137, pI = 10.0) and a slightly acidic, COOH-terminal region (amino acids 138-686, pI = 5.8). This charge distribution is typical of many microtubule-binding proteins, but no significant sequence homology has been detected with any known MAPs. The EMAP, however, does show significant sequence similarity with the beta-subunit of the heterotrimeric G-protein, transducin. The homology lies in a series of 10 imperfect, 43-amino acid repeats (WD-40 repeats) that have been found in many proteins of diverse functions, including beta-transducins, Drosophila Enhancer of split, the yeast STE4, CDC4, CDC20, PRP4, and Tup1 gene products, and the dTAFII80 subunit of Drosophila TFIID. The function of these repeats still remains unknown. It is possible that these repeats are involved in protein-protein interactions, perhaps with the tetratricopeptide repeat-containing protein family. Alternatively, the EMAP may be an important link between signal transduction events and a change in microtubule organization during the cell cycle.     

Purification and activity of a wheat 9-kDa lipid transfer protein expressed in Escherichia coli as a fusion with the maltose binding protein

The cDNA encoding a wheat (Triticum durum) lipid transfer protein of 9 kDa was inserted into an Escherichia coli expression vector, pIH902, and expressed in the bacteria as a fusion with the maltose binding protein. The fusion protein was then purified to homogeneity and subjected to factor Xa cleavage. Although complete cleavage of the fusion protein was obtained, the expected lipid transfer protein was not recovered; it appears to be degraded during protease digestion. However, a fluorescent lipid transfer assay demonstrated that the fusion protein has an activity identical to that of the wheat-purified lipid transfer protein. Thus, this expression system should allow further understanding of the structure/function relationships of wheat lipid transfer proteins.     

Purification and characterization of chondroitin 4-sulfotransferase from the culture medium of a rat chondrosarcoma cell line

Chondroitin 4-sulfotransferase, which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 4 of N-acetylgalactosamine in chondroitin, was purified 1900-fold to apparent homogeneity with 6.1% yield from the serum-free culture medium of rat chondrosarcoma cells by affinity chromatography on heparin-Sepharose CL-6B, Matrex gel red A-agarose, 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands. Molecular masses of these protein were 60 and 64 kDa under reducing conditions and 50 and 54 kDa under nonreducing conditions. Both the protein bands coeluted with chondroitin 4-sulfotransferase activity from Toyopearl HW-55 around the position of 50 kDa, indicating that the active form of chondroitin 4-sulfotransferase is a monomer. Dithiothreitol activated the purified chondroitin 4-sulfotransferase. The purified enzyme transferred sulfate to chondroitin and desulfated dermatan sulfate. Chondroitin sulfate A and chondroitin sulfate C were poor acceptors. Chondroitin sulfate E from squid cartilage, dermatan sulfate, heparan sulfate, and completely desulfated N-resulfated heparin hardly served as acceptors of the sulfotransferase. The transfer of sulfate to the desulfated dermatan sulfate occurred preferentially at position 4 of the N-acetylgalactosamine residues flanked with glucuronic acid residues on both reducing and nonreducing sides.     

Purification of a 38-kDa protein from rabbit reticulocyte lysate which promotes protein renaturation by heat shock protein 70 and its identification as delta-aminolevulinic acid dehydratase and as a putative DnaJ protein

We reported recently that a rabbit reticulocyte 66-kDa protein (termed RF-hsp 70 by our laboratory and p60 and hop by others) functions as a hsp 70 recycling protein and markedly enhances the renaturation of luciferase by hsp 70 (Gross, M., and Hessefort, S. (1996) J. Biol. Chem. 271, 16833-16841). In this report, we confirm that the ability of RF-hsp 70 to promote the conversion of hsp 70. ADP to hsp 70.ATP, thus enhancing the protein folding activity of hsp 70, is caused by the purified 66-kDa protein and not by a trace DnaJ/hsp 40 protein contaminant. To determine the relationship between RF-hsp 70 and the DnaJ/hsp 40 heat shock protein family, which also enhances protein renaturation by hsp 70, we purified a 38-kDa protein from rabbit reticulocyte lysate based upon its ability to stimulate renaturation of luciferase by hsp 70. Partial amino acid sequencing of this 38-kDa protein has indicated, unexpectedly, that it is the enzyme delta-aminolevulinic acid dehydratase (ALA-D) and that it does not contain detectable sequences corresponding to the DnaJ/hsp 40 protein family. In addition, immunoblot analysis with a polyclonal antibody made to HeLa cell hsp 40 (from StressGen) confirms that our purified ALA-D contains no hsp 40, although hsp 40 is present in relatively crude rabbit reticulocyte protein fractions. Rabbit reticulocyte ALA-D is about as active in converting delta-aminolevulinic acid to porphobilinogen and as Zn2+-dependent as ALA-D purified from other sources. Rabbit reticulocyte ALA-D stimulates the renaturation of luciferase by hsp 70 up to 10-fold at concentrations that are the same as or less than that of hsp 70, and it has no renaturation activity in the absence of hsp 70. The renaturation effect of ALA-D is additive with that of RF-hsp 70 at limiting or saturating concentrations of each, and, unlike RF-hsp 70, ALA-D does not promote the dissociation of hsp 70.ADP in the presence of ATP. The renaturation-enhancing effect of ALA-D may be caused by a region near its carboxyl terminus which has sequence homology to the highly conserved domain of the DnaJ protein family, which is similar to the sequence homology between this domain and a carboxyl-terminal region in auxilin, a DnaJ-like protein that requires this region for its hsp 70-dependent function (Ungewickell, E., Ungewickell, H., Holstein, S. E. H., Lindner, R., Prasad, K., Barouch, W., Martin, B., Greene, L. E., and Eisenberg, E. (1995) Nature 378, 632-635).     

Design, synthesis and utility of novel benzophenone-containing calcitonin analogs for photoaffinity labeling the calcitonin receptor

Calcitonin (CT) is a 32-amino-acid calciotropic peptide hormone which acts on target cells via a G protein-coupled seven-transmembrane receptor (CTR). In this study, we report the design, synthesis and characterization of four potent bioactive and photoreactive CT analogs, each of which contains a single benzophenone moiety inserted at different and discrete locations within the CT molecule. Replacement of all Lys residues in salmon CT (sCT) with Arg, followed by replacement of hydrophobic residues with a Lys(epsilon-p-benzoylbenzoyl) residue [Lys(epsilon-pBz2)] was found to preserve high biological activity. We substituted Val8, Leu16 and Leu19 by Lys(epsilon-pBz2), and acylated the N-terminus by a pBz2 moiety, thus distributing the photoaffinity moiety in the different analogs across a large portion of the CT sequence. With both transfected and endogenous CTRs from several species, all four benzophenone-containing analogs were shown to be virtually indistinguishable from the parent sCT analog in both receptor binding properties and stimulation of cAMP accumulation. Upon photolysis, in the presence of CTR, the radioiodinated photoreactive CT analog ([Arg11,18,Lys19(epsilon-pBz2)]sCT (K19)) covalently labels a membrane component of approximately 70 kDa. Receptor cross-linking is inhibited specifically in the presence of excess sCT. We also examined the interaction of these CT analogs with a hemagglutinin (HA) epitope-tagged CTR. The HA-CTR displayed CT binding and CT-dependent cAMP stimulation identical with native CTR. Both K19 and another bioactive analog (-Arg11,18, Lys8(epsilon-pBz2)]sCT (K8)) specifically photoaffinity cross-link to the HA-CTR. These benzophenone-containing CT analogs should facilitate studies of hormone-receptor interactions and allow the direct identification of a CT binding domain(s) within the receptor by the analysis of photochemically cross-linked conjugates.     

Purification and characterization of a novel stress protein, the 150-kDa oxygen-regulated protein (ORP150), from cultured rat astrocytes and its expression in ischemic mouse brain

As the most abundant cell type in the central nervous system, astrocytes are positioned to nurture and sustain neurons, especially in response to cellular stresses, which occur in ischemic cerebrovascular disease. In a previous study (Hori, O., Matsumoto, M., Kuwabara, K., Maeda, M., Ueda, H., Ohtsuki, T., Kinoshita, T., Ogawa, S., Kamada, T., and Stern, D. (1996) J. Neurochem., in press), we identified five polypeptide bands on SDS-polyacrylamide gel electrophoresis, corresponding to molecular masses of about 28, 33, 78, 94, and 150 kDa, whose expression was induced/enhanced in astrocytes exposed to hypoxia or hypoxia followed by replacement into the ambient atmosphere (reoxygenation). In the current study, the approximately 150-kDa polypeptide has been characterized. Chromatography of lysates from cultured rat astrocytes on fast protein liquid chromatography Mono Q followed by preparative SDS-polyacrylamide gel electrophoresis led to isolation of a approximately 150-kDa band only observed in hypoxic cells and which had a unique N-terminal sequence of 15 amino acids. Antisera raised to either the purified approximately 150-kDa band in polyacrylamide gels or to a synthetic peptide comprising the N-terminal sequence detected the same polypeptide in extracts of cultured rat astrocytes exposed to hypoxia; expression was not observed in normoxia but was induced by hypoxia within 24 h, augmented further during early reoxygenation, and thereafter decreased to the base line by 24 h in normoxia. ORP150 expression in hypoxic astrocytes resulted from de novo protein synthesis, as shown by inhibition in the presence of cycloheximide. In contrast to hypoxia-mediated induction of the approximately 150-kDa polypeptide, neither heat shock nor a range of other stimuli, including hydrogen peroxide, cobalt chloride, 2-deoxyglucose, or tunicamycin, led to its expression, suggesting selectivity for production of ORP150 in response to oxygen deprivation, i.e. it was an oxygen-regulated protein (ORP150). Northern and nuclear run-off analysis confirmed the apparent selectivity for ORP150 mRNA induction in hypoxia. Subcellular localization studies showed ORP150 to be present intracellularly within endoplasmic reticulum and only in hypoxic astrocytes, not cultured microglia, endothelial cells, or neurons subject to hypoxia. Consistent with these in vitro results, induction of cerebral ischemia in mice resulted in expression of ORP150 (the latter was not observed in normoxic brain). These data suggest that astroglia respond to oxygen deprivation by redirection of protein synthesis with the appearance of a novel stress protein, ORP150. This polypeptide, selectively expressed by astrocytes, may contribute to their adaptive response to ischemic stress, thereby ultimately contributing to enhanced survival of neurons.     

Competitive enzyme-liked immunoassay with use of soluble enzyme/antibody immune complexes for labeling. I. Measurement of human choriogonadotropin

We described the principle of a new enzyme-immunoassay, competitive enzyme-liked immunoassay (CELIA), for quantitative measurement of soluble antigens and haptens. In the assay, binding of antibody to antigen-immunosorbent is competitively inhibited by the free antigen to be measured. The amount of first antibody bound to the immunosorbent is measured by an enzymatic technique in which a heterologous bridging antibody and a soluble antibody/enzyme immune complex are applied in sequence. The soluble complex we used was rabbit antiperoxidase/horseradish peroxidase. Peroxidase activity is inversely proportional to the concentration in the original sample of the substance to be assayed. The enzyme-linked reagents are potentially widely applicable to any substance to be measured. To demonstrate the feasibility of CELIA, we report a preliminary study of its application to the measurement of human chloriogonadotropin in serum and urine. The assay described for this hormone has a working range of 1 to 50 int. units per milliliter of sample. The technique obviates the disadvantages associated with measurement and handling of radioisotopes in radioimmunoassays and the only major instrumentation required is a centrifuge and a conventional spectrophotometer.     

Modulator protein as a Ca2+-dependent activator of rabbit skeletal myosin light-chain kinase. Purification and characterization

The Ca2+-dependent activator protein of myosin light-chain kinase, which was identified as the bovine brain modulator protein of cyclic nucleotide phosphodiesterase, was isolated from rabbit skeletal muscle. The purified protein binds about 2 Ca2+ per mol in a medium containing 5 mM MgCl2, 10 micron CaCl2, and 0.1 M KCl at pH 6.8. The Ca2+ binding caused a conformational change of the activator protein which was measured by difference ultraviolet absorption spectroscopy. In the same Ca2+ concentration range as that causing the conformational change, activation of myosin light-chain kinase was observed.     

Purification and characterization of a new enzyme, N-alkylglycine oxidase from Cladosporium sp. G-10

A new enzyme, N-alkylglycine oxidase, was isolated from a soil mold, Cladosporium sp. G-10. This protein, which was purified to near homogeneity by ammonium sulfate precipitation followed by successive column chromatography on phenyl-Sepharose, DEAE-Sepharose and Sephadex G-200, was a single polypeptide with a molecular mass of 52,000. In the presence of O2 and H2O, this enzyme acted on some N-alkylglycine derivatives, such as N epsilon-carboxymethyllysine, N-carboxymethyl-6-aminocaproic acid, sarcosine and N-ethylglycine, and produced corresponding N-alkylamine, glyoxylic acid and H2O2. This enzyme had optimum activity at 30 degrees C, pH 8-10, and was most inhibited by ZnSO4, pCMB, iodoacetic acid, and SDS.     

Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor

Attachment of Sindbis virus to the cell surface glycosaminoglycan heparan sulfate (HS) and the selection of this phenotype by cell culture adaptation were investigated. Virus (TR339) was derived from a cDNA clone representing the consensus sequence of strain AR339 (K. L. McKnight, D. A. Simpson, S. C. Lin, T. A. Knott, J. M. Polo, D. F. Pence, D. B. Johannsen, H. W. Heidner, N. L. Davis, and R. E. Johnston, J. Virol. 70:1981-1989, 1996) and from mutant clones containing either one or two dominant cell culture adaptations in the E2 structural glycoprotein (Arg instead of Ser at E2 position 1 [designated TRSB]) or this mutation plus Arg for Ser at E2 114 [designated TRSB-R114]). The consensus virus, TR339, bound to baby hamster kidney (BHK) cells very poorly. The mutation in TRSB increased binding 10- to 50-fold, and the additional mutation in TRSB-R114 increased binding 3- to 5-fold over TRSB. The magnitude of binding was positively correlated with the degree of cell culture adaptation and with attenuation of these viruses in neonatal mice. HS was identified as the attachment receptor for the mutant viruses by the following experimental results. (i) Low concentrations of soluble heparin inhibited plaque formation on and binding of mutant viruses to BHK cells by >95%. In contrast, TR339 showed minimal inhibition at high concentrations. (ii) Binding and infectivity of TRSB-R114 was sensitive to digestion of cell surface HS with heparinase III, and TRSB was sensitive to both heparinase I and heparinase III. TR339 infectivity was only slightly affected by either digestion. (iii) Radiolabeled TRSB and TRSB-R114 attached efficiently to heparin-agarose beads in binding assays, while TR339 showed virtually no binding. (iv) Binding and infectivity of TRSB and TRSB-R114, but not TR339, were greatly reduced on Chinese hamster ovary cells deficient in HS specifically or all glycosaminoglycans. (v) High-multiplicity-of-infection passage of TR339 on BHK cell cultures resulted in rapid coselection of high-affinity binding to BHK cells and attachment to heparin-agarose beads. Sequencing of the passaged virus population revealed a mutation from Glu to Lys at E2 70, a mutation common to many laboratory strains of Sindbis virus. These results suggest that TR339, the most virulent virus tested, attaches to cells through a low-affinity, primarily HS-independent mechanism. Adaptive mutations, selected during cell culture growth of Sindbis virus, enhance binding and infectivity by allowing the virus to attach by an alternative mechanism that is dependent on the presence of cell surface HS.     

Purification of a meningeal cell-derived chondroitin sulphate proteoglycan with neurotrophic activity for brain neurons and its identification as biglycan

Serum-free cultures of meningeal fibroblasts synthesize and release a chondroitin sulphate proteoglycan (CSPG) that markedly enhances survival but not adhesion of embryonic rat (embryonic day 15) neocortical neurons in vitro. The active molecule was purified from conditioned medium (meningeal cell-conditioned medium, MCM) in three steps by means of fast-performance liquid chromatography fractionation combined with a quantitative microphotometric bioassay: (i) preparative Q-Sepharose anion exchange chromatography under native conditions; (ii) rechromatography of biologically active Q-Sepharose fractions on a MonoQ column in the presence of 8 M urea; and (iii) final gel filtration of active MonoQ fractions on Superose 6 in the presence of 4 M guanidinium hydrochloride. Analytical sodium dodecyl sulphate-polyacrylamide gradient gel electrophoresis of active Superose 6 fractions revealed a single broad glycoprotein band with a molecular mass in the range of 220-340 kDa. Further characterization of the purified molecule with glycosaminoglycan:lyases revealed a core protein of 50 kDa and the nearly complete loss of neurotrophic activity after chondroitinase digestion, whereas heparitinase treatment changed neither electrophoretic mobility nor biological activity. Amino-terminal sequencing of the purified CSPG core protein revealed identity with the amino acid sequence of rat biglycan. Biglycan purified from bovine cartilage supported neuron survival with virtually the same activity as the CSPG purified from MCM (half-maximal activity approximate to 10(-8) M). In conclusion, we isolated a neurotrophic CSPG from meningeal cells with strong survival-enhancing activity for brain neurons that was identified as biglycan, a molecule not previously related to neural functions.     

Biosynthesis of the escherichia coli K4 capsule polysaccharide. A parallel system for studies of glycosyltransferases in chondroitin formation

Escherichia coli K4 bacteria synthesize a capsule polysaccharide (GalNAc-GlcA(fructose))n with the carbohydrate backbone identical to chondroitin. GlcA- and GalNAc-transferase activities from the bacterial membrane were assayed with acceptors derived from the capsule polysaccharide and radiolabeled UDP-[14C]GlcA and UDP-[3H]GalNAc, respectively. It was shown that defructosylated oligosaccharides (chondroitin) could serve as substrates for both the GlcA- and the GalNAc-transferases. The radiolabeled products were completely degraded with chondroitinase AC; the [14C]GlcA unit could be removed by beta-D-glucuronidase, and the [3H]GalNAc could be removed by beta-N-acetylhexosaminidase. A fructosylated oligosaccharide acceptor tested for GlcA-transferase activity was found to be inactive. These results indicate that the chain elongation reaction of the K4 polysaccharide proceeds in the same way as the polymerization of the chondroitin chain, by the addition of the monosaccharide units one by one to the nonreducing end of the polymer. This makes the biosynthesis of the K4 polysaccharide an interesting parallel system for studies of chondroitin sulfate biosynthesis. In the biosynthesis of capsule polysaccharides from E. coli, a similar mechanism has earlier been demonstrated for polysialic acid (NeuNAc)n (Rohr, T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342) and for the K5 polysaccharide (GlcAbeta1-4GlcNAcalpha1-4)n (Lidholt, K., Fjelstad, M., Jann, K., and Lindahl, U. (1994) Carbohydr. Res. 255, 87-101). In contrast, chain elongation of hyaluronan (GlcAbeta1-3GlcNAcbeta1-4)n is claimed to occur at the reducing end (Prehm, P. (1983) Biochem. J. 211, 181-189).     

Molecular cloning and structural characterization of the Arg-gingipain proteinase of Porphyromonas gingivalis. Biosynthesis as a proteinase-adhesin polyprotein

The identification of proteinases of Porphyromonas gingivalis that act as virulence factors in periodontal disease has important implications in the study of host-pathogen interactions as well as in the discovery of potential therapeutic and immunoprophylactic agents. We have cloned and characterized a gene that encodes the 50-kDa cysteine proteinase gingipain or Arg-gingipain-1 (RGP-1) described previously (Chen, Z., Potempa, J., Polanowski, A., Wikstrom, M., and Travis, J. (1992) J. Biol. Chem. 267, 18896-18901). Analysis of the amino acid sequence of RGP-1 deduced from the cloned DNA sequence showed that the biosynthesis of this proteinase involves processing of a polyprotein that contains multiple adhesin molecules located at its carboxyl terminus. This finding corroborates previous evidence (Pike R., McGraw, W., Potempa, J., and Travis, J. (1994) J. Biol. Chem. 269, 406-411) that RGP-1 is closely associated with adhesin molecules, and that high molecular weight forms of the proteinase are involved in the binding of erythrocytes.     

Purification and characterization of a Ca(2+)-binding 450-kDa protein (MCBP-450) in the plasma membrane-enriched fraction from a molluscan smooth muscle

In accordance with physiological and electronmicroscopic evidence that, in the anterior byssal retractor muscle (ABRM) of a common mussel Mytilus edulis, Ca2+ activating the contractile system is accumulated at the inner surface of the plasma membrane and at the membrane of sarcoplasmic reticulum (Ebashi, S. and Endo, M. (1968) Prog. Biophys. Mol. Biol. 18., 123-183; Suzuki, S. and Sugi, H. (1982) in The role of calcium in biological systems, Vol. I (Anghileri, L.J. and Tuffet-Anghileri, A.M., eds.), pp. 201-207, CRC Press, Boca Raton), we have found a high-molecular-mass (450 kDa) Ca(2+)-binding protein (MCBP-450) in the membrane fractions of the ABRM by 45Ca autoradiography of proteins transferred to nitrocellulose membrane (Rüegg, J. C. (1971) Physiol. Rev. 51, 201-248). MCBP-450, purified to electrophoretic homogeneity, exhibited Ca(2+)-dependent changes in mobility, tryptophan fluorescence, UV absorption and CD spectrum, indicating its Ca(2+)-dependent conformational changes. MCBP-450 has a high content of aspartic and glutamic acid (23.8%) and a high content of basic residues (27%). It has a high capacity Ca(2+)-binding site, which binds about 38 mol of Ca2+ per mol with an adissociation constant of 10(4) M-1, and a low-capacity Ca(2+)-binding site, which binds about 7 mol of Ca2+ per mol with an association constant of 10(5) M-1. These characteristics of MCBP-450 are consistent with the view that it is actually involved in regulating the contraction-relaxation cycle in the ABRM.     

Validation of collagenous protein synthesis as an index for angiogenesis with the use of morphological methods

Wnt genes are involved in mouse mammary cancer, but their role in human cancer is unknown. Human Wnt5a was cloned from a placental cDNA library and used to assess expression by ribonuclease protection and in situ hybridization in human breast cell lines and in normal, benign, and malignant breast tissues. Human Wnt5a shows over 99% homology at amino acid level with mouse Wnt5a, and 90% with Xenopus Wnt5a. It was expressed only at low levels in breast cell lines and normal breast tissue. Benign proliferations and invasive cancer respectively showed 10-fold and 4-fold higher Wnt5a than normal breast tissues. The greater up-regulation in benign conditions suggests a role in aberrant differentiation. In situ hybridization localized the signal to the epithelial component. Wnt5a is the first member of the Wnt family to demonstrate overexpression in human breast cancer. It was not associated with factors known to affect breast cancer prognosis such as lymph node status or epidermal growth factor receptor status.