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J W Goding

Publications and source records attributed to J W Goding.

At least 19 recordsLinked to original sources
PubMed1997

Molecular cloning and chromosomal localization of PD-Ibeta (PDNP3), a new member of the human phosphodiesterase I genes.

Phosphodiesterase I (EC 3.1.4.1)/nucleotide pyrophosphatase (EC 3.6.1.9) enzymes are a family of type II transmembrane proteins that catalyze the cleavage of phosphodiester and phosphosulfate bonds of a variety of molecules, including deoxynucleotides, NAD, and nucleotide sugars. The human genes for two members of this family have been cloned and designated PC-1 (PDNP1) and PD-Ialpha/autotaxin (PDNP2). We have now cloned the third member of this family from a human prostate cDNA library and designated it human phosphodiesterase-Ibeta (PD-Ibeta). The PD-Ibeta cDNA contains a 2625-bp-long open reading frame which encodes an 875-amino-acid protein. COS-7 cells transfected with an expression vector, pBK-CMV, containing PD-Ibeta cDNA had high phosphodiesterase I activity compared to the mock-transfected cells. By using in situ hybridization to human metaphase chromosomes, we have assigned the locus for the PD-Ibeta (PDNP3) gene to the q22 region of human chromosome 6.

Amino Acid Sequence↗
PubMed1997

Biochemical and molecular identification of distinct forms of alkaline phosphodiesterase I expressed on the apical and basolateral plasma membrane surfaces of rat hepatocytes.

We have identified B10, a plasma membrane protein previously defined by a monoclonal antibody, as an alkaline phosphodiesterase I (APDE) expressed in the plasma membrane of rat hepatocytes and enterocytes, with a restricted apical distribution. B10 complementary DNA (cDNA) was cloned from a rat intestinal library screened with a polyclonal antibody directed to the hepatic protein. Two distinct B10 clones with an open reading frame of 2,625 bp were obtained that differed only by 12 bases in the coding region. One B10 clone had a single base difference with gp130RB13-6 cDNA, which was recently cloned in rat fetal brain. B10/gp130RB13-6 had 50% identity at the amino acid level with the plasma cell antigen PC-1, an APDE cloned in the mouse and in human. Anti-B10 antibodies immunoprecipitated 34% of the APDE activity in liver plasma membranes and over 95% of the APDE activity in intestinal cells. Most of the remaining activity in hepatocytes (44%) could be immunoprecipitated by antibodies directed to PC-1. APDE activity immunoprecipitated with anti-B10 antibodies was found in the apical rat liver plasma membrane fractions on a sucrose gradient whereas most of the remaining APDE activity was associated with the basolateral fractions, which contained PC-1. By immunofluorescence, B10 was localized to the apical surfaces of hepatocytes and enterocytes whereas PC-1 was present on the basolateral surfaces of hepatocytes. B10/gp130RB13-6 and rat PC-1 are a unique example of distinct molecules having similar enzymatic activity but different apical/basolateral location, and possibly different functions.

Amino Acid Sequence↗
PubMed1997

Expression of the putative inhibitor of the insulin receptor tyrosine kinase PC-1 in dermal fibroblasts from patients with syndromes of severe insulin resistance.

OBJECTIVE: To date, mutations in the insulin receptor gene are the only clearly defined cause of extreme insulin resistance in man. Recently, however, some patients with severe insulin resistance have been reported to have marked over-expression of the transmembrane glycoprotein PC-1 in their cultured fibroblasts. This protein appears to act as an endogenous inhibitor of the insulin receptor tyrosine kinase which suggests that primary over-expression of PC-1 may play an aetiological role in some forms of insulin resistance. One sub-type of extreme insulin resistance in which the insulin receptor gene has been reported to be normal is pseudo-acromegalic insulin resistance. The main aim of this study was to determine whether overexpression of PC-1 might contribute to the severe insulin resistance exhibited by some patients with pseudo-acromegaly. DESIGN AND PATIENTS PC-1 phosphodiesterase activity and PC-1 protein and mRNA content were measured in cultured dermal fibroblast from three severely insulin resistant pseudo-acromegalic patients. These were compared with fibroblasts from normoinsulinaemic normoglycaemic controls and from subjects with known genetic defects in the insulin receptor or IRS-1. RESULTS: In the fibroblasts from pseudo-acromegalic insulin resistant subjects PC-1 activity and PC-1 protein and mRNA levels were indistinguishable from the normoinsulinaemic controls. Consistent with this observation, insulin receptor tyrosine kinase activity was similar in extracts from fibroblasts of pseudo-acromegalic subjects and normal controls. Surprisingly, subjects with insulin receptor or IRS-1 mutations had a profound reduction in PC-1 activity (p < or = 0.005), protein (p < or = 0.05) and mRNA levels (P < or = 0.005). CONCLUSIONS: The results indicate that PC-1 over-expression does not appear to contribute to the insulin resistant state of pseudo-acromegalic patients. The finding of normal insulin receptor tyrosine kinase activity in these subjects suggests that the site of defective insulin signalling is likely to be distal to the receptor. The unexpected finding that PC-1 activity, protein and mRNA were all dramatically reduced in patients with lesions early in the insulin signalling cascade provides further evidence for a link, albeit as yet poorly understood, between cellular insulin action and the expression of PC-1.

Cells, Cultured↗
PubMed1996

Expression of the nucleoside triphosphate pyrophosphohydrolase PC-1 is induced by basic fibroblast growth factor (bFGF) and modulated by activation of the protein kinase A and C pathways in osteoblast-like osteosarcoma cells.

The closely related cytokines bFGF and aFGF regulate the function of bone cells and mineralization. Osteoblasts express PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH)/nucleotide phosphodiesterase I activity. bFGF and aFGF (10 ng/ml) up-regulated NTPPPH in human SaOS-2 and U2OS osteosarcoma cells, which express osteoblast-like features in culture. The induction was selective as alkaline phosphatase activity was down-regulated and specific as insulin-like growth factor-1 (IGF-1) and interleukin-1 beta (IL-1 beta) were not active. Furthermore, IL-1 beta but not IGF-1 inhibited bFGF-induced up-regulation of NTPPPH. The induced NTPPPH remained predominantly associated with cells. bFGF can induce signaling through pathways including protein kinase A (PKA) and protein kinase C (PKC)-mediated transduction. An activator of the PKA pathway (8-bromo cyclic adenosine monophosphate [cAMP]) induced NTPPPH. Furthermore, pretreatment with the PKC activator phorbol myristate acetate (PMA) (80 nM) markedly increased subsequent NTPPPH induction by both bFGF and cAMP. The PMA effect was associated with morphologic changes characterized by long, thin intercellular extensions. PKC desensitization also potentially contributed to this effect because the PKC inhibitors staurosporine and H-7 enhanced bFGF-induced and cAMP-induced NTPPPH expression in the absence of morphologic changes. We observed that bFGF induced expression of PC-1, a member of the NTPPPH gene family. The majority of NTPPPH activity was depleted by immunoadsorption using a monoclonal antibody to native human PC-1. bFGF- and aFGF-induced production of PC-1/NTPPPH in osteoblastoid cells may contribute to the effects of FGFs on bone metabolism.

Cyclic AMP-Dependent Protein Kinases↗
PubMed1995

Autophosphorylation of PC-1 (alkaline phosphodiesterase I/nucleotide pyrophosphatase) and analysis of the active site.

PC-1 is an ecto-enzyme possessing alkaline phosphodiesterase I (EC 3.1.4.1) and nucleotide pyrophosphatase (EC 3.6.1.9) activities. It has also been proposed to be an ecto-protein kinase capable of phosphorylating itself as well as exogenous proteins. We have investigated the phosphorylation capability of PC-1 and have developed a novel method for its detection and characterization based on autophosphorylation, which allows detection without the use of antibodies. When cells expressing membrane PC-1 were held on ice with [gamma-32P]ATP, SDS/PAGE of whole cell lysates showed a single band which was PC-1; this band was absent in cells not expressing PC-1. Immunoprecipitates of soluble PC-1 isolated from culture supernatants of cells expressing PC-1 were also capable of autophosphorylation, and the size of the labeled protein was the same as previously reported for soluble PC-1. PC-1 was also labeled with [alpha-32P]ATP and [35S]dATP[alpha S]. We found no evidence that PC-1 was capable of phosphorylating proteins other than itself, and conclude that it is not a true kinase, and that the observed labeling with [gamma-32P]ATP, [alpha-32P]ATP and [35S]dATP[alpha S] reflect transient covalent adducts that are part of the catalytic cycle of phosphodiesterase/pyrophosphatase activity rather than intrinsic kinase activity. Mutation of the active-site threonine to tyrosine, serine or alanine reduced the 5'-nucleotide phosphodiesterase activity of PC-1 and its ability to autophosphorylate to undetectable levels. Together, these data suggest that both activities depend on the same site.

Adenosine Triphosphate↗
PubMed1994

Biochemical characterization of human PC-1, an enzyme possessing alkaline phosphodiesterase I and nucleotide pyrophosphatase activities.

PC-1 is an ecto-enzyme possessing alkaline phosphodiesterase I and nucleotide pyrophosphatase activities. In this paper, we demonstrate the expression, biochemical characterization and biosynthesis of human PC-1. Previously, there has been uncertainty concerning which of two methionine residues is the initiator. It is now shown that expression of PC-1 is much greater if the first methionine residue is present, and that the sequence between the two methionine residues is translated in both human and mouse, in both transfected cells and cells naturally expressing PC-1. The first methionine residue is therefore the initiator. Human PC-1 is capable of autophosphorylation, and conditions are described in which PC-1 is the only labelled phosphoprotein on the plasma membranes of intact cells, allowing the demonstration that the mature membrane form of human PC-1 is approximately 10 kDa larger than that of the mouse form. Pulse-chase biosynthetic studies and treatment with two different endoglycosidases show that most of this difference is due to N-linked oligosaccharides. The polypeptide backbone of human PC-1 is 20 amino acids longer than that of the mouse PC-1, with most of the difference in polypeptide chain length being in the cytoplasmic domain. The revised cytoplasmic domain of human PC-1 has 76 amino acids, while the mouse cytoplasmic domain has 58 amino acids. Optimal alignment of mouse and human cytoplasmic domains reveals areas of sequence conservation in which the third bases vary. It is suggested that these regions of conservation may point to functionally important sequences in the cytoplasmic domain.

Amidohydrolases↗
PubMed1994

Divalent cations stabilize the conformation of plasma cell membrane glycoprotein PC-1 (alkaline phosphodiesterase I).

The plasma cell-membrane glycoprotein PC-1 is an ectoenzyme with alkaline phosphodiesterase I/5'-nucleotide phosphodiesterase (EC 3.1.4.1) and nucleotide pyrophosphatase (EC 3.6.1.9) activities. It contains sequence motifs which closely match the consensus EF-hand (helix-loop-helix) Ca(2+)-binding regions of parvalbumin, troponin-C and calmodulin, and its enzymic activity is increased in the presence of divalent cations and decreased in the presence of chelating agents. We have undertaken experiments to determine whether divalent cations affect the conformation of the PC-1 protein, as assessed by their effect on thermal stability, resistance to proteolysis and binding of polyclonal antibodies to the whole native protein and monoclonal antibodies to a putative Ca(2+)-binding region. Divalent cations were found to protect solubilized PC-1 against thermal denaturation and proteolysis. They also stabilized PC-1 on intact cells; this form was much more resistant to proteolysis than Triton X-100 solubilized PC-1. Ca2+, Mg2+ and Zn2+ ions were equally effective. Monoclonal antibodies to the bacterially expressed C-terminal EF-hand homology region only bound to mammalian PC-1 in the absence of Ca2+. In contrast, the great majority of polyclonal antibodies to native PC-1 bound regardless of whether Ca2+ was present or not, but with increased binding when Ca2+ was present. These results provide evidence that divalent cations bind to PC-1 and stabilize its conformation.

Amino Acid Sequence↗
PubMed1994

Anti-galactosyl antibodies that react with unmodified agarose: a potential source of artifacts in immunoaffinity chromatography.

Rabbits were immunized with glycolipid and membrane glycoprotein extracts of the promastigotes of Leishmania major partially purified by two-phase extraction with Triton X-114. The resulting antibodies were affinity purified on agarose immunoadsorbents to which a recombinant DNA-produced L. major polypeptide had been coupled. In addition to the expected reaction with the polypeptide, it was found that the affinity-purified antibodies reacted strongly with lipophosphoglycan of L. major amastigotes and to a lesser extent with the lipophosphoglycan of promastigotes. Antibodies with this reactivity could be affinity purified on unmodified agarose and were probably directed against beta-1,3-galactosyl determinants shared between the agarose matrix and Leishmania. In this and other special situations the properties of the support matrix can be exploited to obtain antibodies of defined specificity which may be useful probes for the identification and characterization of carbohydrate structures. This work also points to a potential source of artifacts in affinity chromatography, in which the bead matrix is usually considered as inert. Carbohydrate antigens are ubiquitous, particularly in micro-organisms, and the presence of such antibodies may be more common than previously recognized.

Animals↗
PubMed1993

Identification and characterization of a soluble form of the plasma cell membrane glycoprotein PC-1 (5'-nucleotide phosphodiesterase).

PC-1 is a membrane glycoprotein, found on the surface of plasma cells and a few types of nonlymphoid cells, which has recently been found to have 5'-nucleotide phosphodiesterase activity. In this paper, we demonstrate the existence of enzymically active water-soluble forms of PC-1 in ascites from plasmacytoma-bearing mice, normal mouse serum, and in supernatants of cultured mouse plasmacytoma cells and mouse L cells transfected with a cDNA encoding the membrane form of PC-1. The water-soluble enzyme activity can be specifically immunoprecipitated by a monoclonal antibody to an allotypic determinant on the membrane form of PC-1, and resides on a slightly smaller polypeptide than membrane PC-1. Biosynthetic studies revealed a single, monomeric, endoglycosidase-H-sensitive membrane PC-1 precursor, which was gradually converted to a disulphide-bonded, endoglycosidase-H-resistant form over a period of about 2 h. Soluble PC-1 was first detectable in the supernatant after about 2 h. A distinct intracellular form of soluble PC-1 was not seen. The soluble form of PC-1 does not appear to arise by proteolytic cleavage from the cell surface, although cleavage inside the cell remains a possibility. When taken together with the structure of the relevant portions of PC-1 gene exons, the data suggest that the most likely site of cleavage is between Pro152 and Ala153.

Amino Acid Sequence↗
PubMed1993

Identification, characterisation and genomic cloning of a O-linked N-acetylglucosamine-containing cytoplasmic Leishmania glycoprotein.

Antibodies against Leishmania major wheat germ agglutinin-binding glycoproteins were used to select from a genomic lambda gt11 expression library a clone coding for a L. major glycoprotein. The partial DNA sequence indicated the presence of a mosaic of repetitive sequences. Southern blot hybridisation on genomic DNA using the cloned gene as a probe at high stringency suggested a single gene, which was localised to chromosome band 18. Northern blot analysis of L. major mRNA detected a major transcript of 7.5 kb and a minor 4.0-kb transcript. Antibodies affinity-purified on the fusion protein identified a complex of two water-soluble cytoplasmic polypeptides of approximately 96 kDa and 92 kDa in L. major promastigotes and amastigotes. They also recognised polypeptides in other Leishmania species, in Crithidia lucilliae and very weakly in Leptomonas. The apparent molecular weight of these polypeptides, while conserved within each species, varied between species. A peptide map of the two polypeptides from L. major generated an identical pattern suggesting a close relatedness at the protein level. This protein complex was not hydrolysed by N-glycanase and was not affected by tunicamycin, but treatment with anhydrous hydrogen fluoride suggested that it is O-glycosylated. The glycan moiety appears to be N-acetylglucosamine, and N-acetylglucosamine beta-1,4-galactosyltransferase was capable of adding [3H]galactose to it. This was susceptible to beta elimination and beta-galactosidase treatment. Taken together, the data indicates that gp96/92 belongs to the newly described class of cytoplasmic and nuclear glycoproteins containing O-linked N-acetylglucosamine.

Acetylglucosamine↗
PubMed1990

Plasma cell membrane glycoprotein PC-1. cDNA cloning of the human molecule, amino acid sequence, and chromosomal location.

The murine cell membrane glycoprotein PC-1 is a homodimer with restricted tissue distribution, being first characterized in plasma cells. We now describe the isolation of cDNA clones encoding the human homolog of the murine PC-1 protein, its complete amino acid sequence, and its chromosomal location. Overall, the amino acid sequence of the human protein is about 80% identical to the murine protein, although the extent of homology varies in different domains. It had not been possible to assign a definitive amino terminus to the murine protein. Comparison of the murine and human sequence necessitates reassignment of the amino terminus, resulting in a cytoplasmic tail of 24 amino acids rather than 58 amino acids as previously published for the mouse. The sequence of several independently obtained cDNA clones indicates that the 3' end of the mRNA is subject to alternative splicing. Southern blots suggest a single copy gene. In situ chromosomal hybridization localizes the gene for human PC-1 to chromosome 6q22-q23, a common site for deletions in human lymphoid neoplasia.

Amino Acid Sequence↗
PubMed1989

Assessment of a PstI polymorphism of the apolipoprotein-AI gene in Australian patients with coronary artery disease.

In 1986 Ordovas et al., reported that a polymorphism in the 3' flanking region of the apolipoprotein AI gene was strongly associated with premature coronary artery disease. This polymorphism affects a restriction site for the endonuclease PstI, resulting in the identification of a 3.3 kb band, rather than the more common 2.2 kb band, when genomic blots of PstI digested human DNA are probed with an apolipoprotein AI gene probe. In a study population of 88 patients with severe coronary artery disease before the age of 60, 28 (32%) carried the 3.3 kb allele, which was found in only five (4%) of 123 randomly chosen control subjects. In the present study, we have assessed the prevalence of this polymorphism in coronary artery disease patients and outpatients with abnormal lipid levels at the Alfred Hospital, Melbourne, and in normal volunteers. The 3.3 kb allele was present in 7-12% of subjects in these populations, and showed no association with coronary artery disease.

Aged↗
PubMed1988

Preparation of bacteriophage lambda DNA using the TL-100 ultracentrifuge.

A procedure for the preparation of DNA from bacteriophage lambda is described, using the Beckman TL-100 bench-top ultracentrifuge. The procedure involves growth of phage in agar plates, precipitation with polyethylene glycol, and a single centrifugation in cesium chloride under conditions that disrupt the phage coat. The method avoids the use of enzymes, ion exchange resins, and phenol. It can be completed in less than a day. The resulting DNA is of good purity and is easily cuttable by restriction enzymes.

Bacteriophage lambda↗
PubMed1988

Distribution of the murine plasma cell antigen PC-1 in non-lymphoid tissues.

PC-1 is an alloantigen of murine plasma cells. Its close association with secretory function in lymphoid cells previously raised the question of whether PC-1 was part of the secretory apparatus. In addition to its expression on lymphocytes, PC-1 had been known to be present in liver, brain, and kidney, although the data were derived almost entirely from bulk absorption studies of polyclonal alloantisera, and virtually nothing was known about the nature of the cells expressing PC-1 in these organs. If PC-1 was functionally involved in the secretory process. it might be expected to be present at secretory sites within these and other organs. We now report the results of an immunohistochemical survey of the distribution of PC-1 in a variety of non-lymphoid organs, using a mAb. The PC-1 Ag was found in a small number of highly discrete locations that were mostly, but not exclusively, associated with epithelia. Sites of strong expression included the distal convoluted tubule of the kidney, ducts of the salivary glands, epididymis, proximal part of the vas deferens, and chondrocytes. The PC-1 glycoprotein was also found in the capillaries of the brain, but did not appear to be present in capillaries elsewhere, a pattern that is strikingly similar to that of the receptor for the iron transport protein, transferrin. Negative sites included the thyroid, pancreas, choroid plexus, smooth and striated muscle, stomach, small and large intestine, gall bladder, renal glomeruli, testis, and seminal vesicles. These results are not consistent with a generalized role for PC-1 in secretion, but are compatible with a role in a specialized subset of macromolecular transport events.

Animals↗
PubMed1987

Plasma cell membrane glycoprotein PC-1. Primary structure deduced from cDNA clones.

The PC-1 protein is a membrane glycoprotein that is selectively expressed on the surface of antibody-secreting cells. Previous work has shown that it consists of two apparently identical disulfide-bonded polypeptides, each of molecular weight approximately 120,000. We now describe the sequence of PC-1 mRNA and protein. The PC-1 protein is shown to consist of 905 amino acids and to have an uncommon transmembrane orientation. The NH2-terminal 58 residues are intracellular and the COOH-terminal 826 residues are extracellular. A cysteine-rich region of 85 amino acids lies adjacent to the extracellular surface of the membrane and appears to have arisen by exon duplication. In common with other membrane glycoproteins with this orientation, there is no obvious signal sequence other than the transmembrane segment. The PC-1 protein therefore has an overall structure and membrane orientation that resembles those of the transferrin receptor, the asialoglycoprotein receptor, and the Ia invariant chain.

Amino Acid Sequence↗
PubMed1987

Leishmania major: a very sensitive dot-blot ELISA for detection of parasites in cutaneous lesions.

There is a need for accurate, rapid and early diagnosis of leishmaniasis, which would distinguish between the benign and severe forms of the disease. We have used a monoclonal antibody directed to a polymorphic, species-specific antigen in diagnostic assays for leishmaniasis. The dot-blot enzyme-linked immunosorbent assay described here can detect as few as 300 culture promastigotes and 20,000 amastigotes of Leishmania major with no cross-reaction with other species and no background from skin macrophages or other cells. This level of sensitivity is sufficient to detect parasite antigen aspirated in a few microliters of liquid from small lesions in mice. This assay could form the basis for a sensitive, rapid and inexpensive dip-stick test for large-scale use for the diagnosis and epidemiology of cutaneous leishmaniasis.

Animals↗
PubMed1986

Use of microbore high-performance liquid chromatography for purifying subnanomole levels of polypeptides for microsequencing. Structural studies on the murine plasma cell antigen PC-1.

A procedure for the purification of subnanomole levels of polypeptides has been developed. Reversed-phase high performance liquid chromatography on short (10 cm or less) microbore (1-2 mm internal diameter) columns has been used to fractionate and purify a number of tryptic peptides generated from approximately 600 pmol of purified murine plasma cell antigen PC-1, a major membrane glycoprotein on all cells secreting immunoglobulins. The use of reversed-phase microbore columns permits the recovery of subnanomole amounts of polypeptides from large volumes in high yield (greater than 90%) and in small eluent volumes (40-60 microL) which can be loaded directly onto the gas-phase sequencer without further concentration. This procedure avoids the severe sample loss which frequently occurs with other concentration procedures such as lyophilization and evaporation. The use of a photodiode-array detector for identifying tryptophan-containing peptides from on-the-fly, ultraviolet spectra is described. This procedure permits the selection of tryptophan-containing peptides from complex tryptic digests for use as candidate peptides for oligonucleotide probe construction. Automated Edman degradation was performed on seven tryptic peptides, yielding 110 unique assignments; this corresponds to approximately 11% of the molecule.

Amino Acid Sequence↗