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J W Fox

Publications and source records attributed to J W Fox.

At least 73 records · Page 4Linked to original sources
PubMed1995

Characterization of a collagenolytic serine proteinase from the Atlantic cod (Gadus morhua).

A collagenolytic proteinase was purified from the intestines of Atlantic cod by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The proteinase has an estimated molecular weight of 24.1 (+/- 0.5) kDa as determined by SDS-PAGE and belongs to the chymotrypsin family of serine proteinases. The enzyme cleaves native collagen types I, III, IV and V, and also readily hydrolyzes succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin, as well as succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, a reported elastase substrate, but had no detectable activity towards several other substrates of these proteinases or of trypsin. The pH optimum of the enzyme was between pH 8.0 and 9.5 and it was unstable at pH values below 7. Maximal activity of the enzyme when assayed against sAAPFpna was centered between 45 and 50 degrees C. Calcium binding stabilized the cod collagenase against thermal inactivation, but even in the presence of calcium, the enzyme was unstable at temperatures above 30 degrees C.

Amino Acid Sequence↗
PubMed1994

Structural interaction of natural and synthetic inhibitors with the venom metalloproteinase, atrolysin C (form d).

The structure of the metalloproteinase and hemorrhagic toxin atrolysin C form d (EC 3.4.24.42), from the venom of the western diamondback rattlesnake Crotalus atrox, has been determined to atomic resolution by x-ray crystallographic methods. This study illuminates the nature of inhibitor binding with natural (< Glu-Asn-Trp, where < Glu is pyroglutamic acid) and synthetic (SCH 47890) ligands. The primary specificity pocket is exceptionally deep; the nature of inhibitor and productive substrate binding is discussed. Insights gained from the study of these complexes facilitate the design of potential drugs to treat diseases where matrix metalloproteinases have been implicated, e.g., arthritis and tumor metastasis.

Amides↗
PubMed1994

Two non-contiguous regions contribute to nidogen binding to a single EGF-like motif of the laminin gamma 1 chain.

High affinity binding of nidogen to laminin is mediated by an EGF-like repeat gamma 1III4 of the mouse laminin gamma 1 chain and has now been restricted to two short noncontiguous regions of its 56 residue sequence by use of synthetic peptides and recombinant mutants. Disulfide loop a,b of the repeat and a modified loop a,c could completely inhibit binding, with a 5000-fold or 300-fold reduced affinity respectively. Synthetic loops c and d lacked inhibitory activity. Some binding contribution of Tyr819 in loop c was, however, shown by mutation and side chain modification. Together with studies of loop chimeras, this indicated a distinct cooperativity between the two binding sites. The major binding site of loop a was localized to the heptapeptide NIDPNAV (position 798-804). A change of Asp800 to Asn or Ala803 to Val caused a strong reduction in binding activity, while only small effects were observed for the changes Pro801 to Gln and Ile799 to Val. The latter replacement corresponds to the single substitution found in the same region of the Drosophila laminin gamma 1 chain. However, the changes Asn802 to Ser or Val804 to Ser, both known to exist in the laminin gamma 2 chain, were deleterious mutations. This demonstrated conservation of binding structures in laminins of distantly related species, but not between homologous chains of laminin isoforms.

Amino Acid Sequence↗
PubMed1994

cDNA sequences for four snake venom metalloproteinases: structure, classification, and their relationship to mammalian reproductive proteins.

Presented here are four new cDNA sequences for hemorrhagic metalloproteinases from Crotalus atrox venom, hemorrhagic toxins a, b, c, and d. Comparison of the translated open reading frames to the mature protein sequences gives evidence for post-translational processing at both the amino and carboxyl termini. This comparison is also the basis for a new classification system for these precursors, based on their different sizes. Protein sequences in the zymogen region support the hypothesis of a cysteine-switch type mechanism of maintaining latency. The coordination geometry around the active site zinc ion is discussed. The relationship between these venom metalloproteinases and a family of mammalian reproductive proteins is also supported by these sequences. The cysteine pattern of the carboxyl-terminal domain of the largest proteinase, hemorrhagic toxin a, is compared to other venom proteinases and to the mammalian proteins, showing both striking similarities and subtle differences. It would appear that these hemorrhagic toxins have resulted from deletions and subsequent divergence from a larger ancestor, one they may have shared with the aforementioned mammalian reproductive proteins.

Amino Acid Sequence↗
PubMed1994

Hemorrhagic metalloproteinases from snake venoms.

One of the more significant consequences of crotalid envenomation is hemorrhage. Over the past 50 years of investigation, it is clear that the primary factors responsible for hemorrhage are metalloproteinases present in the venom of these snakes. The biochemical basis for their activity is the proteolytic destruction of basement membrane and extracellular matrix surrounding capillaries and small vessels. These proteinase toxins may also interfere with coagulation, thus complementing loss of blood from the vasculature. Structural studies have shown that these proteinases are synthesized as zymogens and are processed at both the amino and carboxy termini to give the mature protein. The variety of hemorrhagic toxins found in snake venoms is due to the presence of structurally related proteins composed of various domains. The type of domains found in each toxin plays an important role in the hemorrhagic potency of the protein. Recently, structural homologs to the venom hemorrhagic metalloproteinases have been identified in several mammalian reproductive systems. The functional significance of the reproductive proteins is not clear, but in light of the presence of similar domains shared with the venom metalloproteinases, their basic biochemical activities may be similar but with very different consequences. This review discusses the history of hemorrhagic toxin research with emphasis on the Crotalus atrox proteinases. The structural similarities observed among the hemorrhagic toxins are outlined, and the structural relationships of the toxins to the mammalian reproductive proteins are described.

Amino Acid Sequence↗
PubMed1994

Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src.

The phosphorylation of protein tyrosine kinases (PTKs) on tyrosine residues is a critical regulatory event that modulates catalytic activity and triggers the physical association of PTKs with Src homology 2 (SH2)-containing proteins. The integrin-linked focal adhesion kinase, pp125FAK, exhibits extracellular matrix-dependent phosphorylation on tyrosine and physically associates with two nonreceptor PTKs, pp60src and pp59fyn, via their SH2 domains. Herein, we identify Tyr-397 as the major site of tyrosine phosphorylation on pp125FAK both in vivo and in vitro. Tyrosine 397 is located at the juncture of the N-terminal and catalytic domains, a novel site for PTK autophosphorylation. Mutation of Tyr-397 to a nonphosphorylatable residue dramatically impairs the phosphorylation of pp125FAK on tyrosine in vivo and in vitro. The mutation of Tyr-397 to Phe also inhibits the formation of stable complexes with pp60src in cells expressing Src and FAK397F, suggesting that autophosphorylation of pp125FAK may regulate the association of pp125FAK with Src family kinases in vivo. The identification of Tyr-397 as a major site for FAK autophosphorylation provides one of the first examples of a cellular protein containing a high-affinity binding site for a Src family kinase SH2 domain. This finding has implications for models describing the mechanisms of action of pp125FAK, the regulation of the Src family of PTKs, and signal transduction through the integrins.

Amino Acid Sequence↗
PubMed1993

Mapping of nidogen binding sites for collagen type IV, heparan sulfate proteoglycan, and zinc.

Recombinant nidogen fragments comprising the globular domains G1 plus G2, the rod-like domain, and the rod connected to the globe G3 were prepared from the culture media of transfected human cell clones. In addition, domains G1 and G2 were separated from each other after cleavage with chymotrypsin. The purified fragments were characterized by N-terminal sequences, electrophoresis, electron microscopy, and radioimmunoassays and the cell clones by Northern hybridization. Transfection with a construct comprising a large part of domain G3 showed high mRNA levels but no secreted protein, indicating a protein folding problem. All these fragments were used as soluble and/or immobilized ligands in binding assays. This demonstrated major binding sites on domain G2 for collagen IV and heparan sulfate proteoglycan. Affinity chromatography on zinc- and cobalt-loaded columns showed binding of domains G2 and G3 and the rod. Protein binding, but not metal binding, was abolished by reduction and alkylation of nidogen. This allowed for the isolation of several zinc-binding tryptic peptides, four from G2, two from the rod, and one from the G3 domain. Most of these short peptides contained several histidines that are likely to mediate binding. Zinc inhibited efficiently G3-mediated nidogen binding to laminin at 4 degrees C (IC50 approximately 5 microM) but less at higher temperatures. Similarly, zinc inhibited binding to collagen IV and proteoglycan at low temperatures but not at high (37 degrees C) temperatures. This indicates a complex modulation of nidogen binding to other basement membrane proteins by some, but not all, transition metals. Whether the particularly striking effects shown for zinc are of biological relevance remains to be established.

Amino Acid Sequence↗
PubMed1993

Decorin, versican, and biglycan gene expression by keloid and normal dermal fibroblasts: differential regulation by basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) is a multipotential heparin-binding factor that belongs to the fibroblast growth factor (FGF) family. The FGFs demonstrate a wide spectrum of biologic activities in vivo and in vitro. In this study, we investigated the potential of bFGF to regulate the expression of various dermal extracellular matrix proteoglycans and type I collagen mRNAs in cultured human fibroblasts from keloid, which is a prototype of dermal fibrosis, and normal skin tissue. We report that bFGF upregulates the expression of the decorin gene in normal and keloid fibroblasts. In contrast, the expression of biglycan is downregulated by bFGF. The mRNA steady-state level of versican, a large proteoglycan, is not altered by bFGF. Type I collagen gene expression is downregulated substantially in keloid and normal fibroblasts by bFGF. The results suggest that the expression of the proteoglycan genes are uncoordinately regulated and that the gene expression of type I collagen and biglycan is coordinately downregulated. The results also demonstrate that keloid fibroblasts respond similarly as do normal fibroblasts to bFGF in the regulation of proteoglycan and collagen expression.

Biglycan↗
PubMed1993

Isolation and characterization of two forms of beta-D-glucosidase from Aspergillus niger.

beta-D-glucosidase purified from commercial preparations of clarified culture broth of Aspergillus niger (Novo SP188) was shown to elute as two distinct species during analytical anion-exchange chromatography (AEC). However, the two enzyme forms behaved identically on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), high-performance size-exclusion chromatography (HPSEC), and isoelectric focusing. Also, the N-terminal amino acid sequence, amino acid composition, fingerprint of tryptic-digest peptides, circular dichroism spectra, and reaction kinetics appear identical for these forms. This feature of the A. niger enzyme is distinctly different from beta-D-glucosidase isozymes reported from other sources, where multiple forms tend to differ in molecular weight and/or isoelectric pH. Michaelis-Menten kinetic analysis also gave comparable results for the two forms. The distinct behavior on AEC was explained by considering the differences in N-linked carbohydrates liberated from both species following treatment with endoglycosidase H or F.

Amino Acid Sequence↗
PubMed1993

Nidogen mediates the formation of ternary complexes of basement membrane components.

Using a recombinant nidogen we have probed the calcium binding potential of various nidogen domains, examined the binding of nidogen to various basement membrane proteins and assessed the ability of nidogen to mediate the formation of ternary complexes between laminin and heparan sulfate proteoglycan and collagen IV and laminin. The results of these experiments indicate that the Ca+2 binding is on the rod-like domain with additional binding observed on the N-terminal G1 domain. With regard to the role of nidogen in mediating complex formation among basement membrane components it was demonstrated that nidogen effectively promotes the formation of a ternary complex between laminin and collagen IV, with both of these components interacting independently with nidogen. Similarly, nidogen mediates a ternary complex formation between laminin and proteoglycan. Interestingly, the interaction between proteoglycan and nidogen is through the protein core of the proteoglycan. We have localized the major interaction sites on nidogen with the proteoglycan core and collagen IV to a region on the globular G2 domain while the C-terminal globe G3 binds to laminin. Ca+2 binding does not appear to be important in either of the binary or ternary complex formations. The data reported allow us to hypothesize that, via the multiple interactions of nidogen with other basement membrane components, nidogen plays a crucial structural role in basement membrane organization and stabilization.

Animals↗
PubMed1993

Acidic and basic fibroblast growth factors down-regulate collagen gene expression in keloid fibroblasts.

The effects of acidic and basic fibroblast growth factors (FGFs) on collagen expression by keloid fibroblasts were examined in the absence and presence of heparin. Collagen biosynthesis and gene expression of type I collagen were down-regulated by the FGFs in the presence of heparin. Acidic FGF, in a concentration range of 0.4 to 50 ng/ml, had little or no effect on collagen synthesis after a 4-day incubation. However, in the presence of heparin (100 micrograms/ml) acidic FGF, in concentrations ranging from 2 to 50 ng/ml, decreased [3H]hydroxyproline synthesis by 44 to 68%, compared with untreated control cultures. Total [3H]hydroxyproline synthesis was similar between control and heparin-treated cultures. Basic FGF (2.0 to 50 ng/ml) was effective in suppressing [3H]hydroxyproline synthesis by 50 to 90% after a 4-day incubation without heparin in keloid and normal fibroblast cultures. The steady-state levels of type I collagen messenger RNA were significantly decreased by acidic FGF in the presence of heparin, as well as by basic FGF without heparin. The data suggest that the FGFs are effective in down-regulating excess collagen production by keloid fibroblasts and that this inhibitory effect is apparently associated with pretranslational events. Moreover, acidic FGF is apparently dependent on heparin, whereas basic FGF is not, for potentiation of the down-regulatory effects of the FGFs.

Collagen↗
PubMed1992

Sequence of a cDNA clone encoding the zinc metalloproteinase hemorrhagic toxin e from Crotalus atrox: evidence for signal, zymogen, and disintegrin-like structures.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

A new family of proteinases is defined by several snake venom metalloproteinases.

Recently, the complete amino acid sequences have been determined for several snake venom metalloproteinases from the genera Crotalus, Trimeresurus and Lachesis of the Crotalidae family. Among these are both hemorrhagic and nonhemorrhagic metalloproteinases. Despite differences in their molecular weights and activities, they all appear to be related through a single ancestral gene, as observed by the comparison of their amino acid sequences. None of these proteins bear significant similarity to any other known protein, with the exception of the zinc binding site demonstrated in thermolysin and other metalloproteinases. Thus we propose that these proteins define a new family of zinc metalloproteinases which is likely to consist of (but is not necessarily limited to) many of the zinc metalloproteinases found in snake venoms.

Amino Acid Sequence↗
PubMed1991

Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.

Animals↗
PubMed1991

Mothers' influence on their adolescents' tendency to seek medical care.

Mothers' influence on their adolescent's tendency to seek medical care is examined in a national sample of 12- to 17-year-old children. A similar measurement model fits mother's and adolescent's tendency to seek medical care for the symptoms of a sore throat, stomachache, and vomiting. Maternal influence persists as the predominant effect on adolescents' tendency to seek medical care when sex, age, race, birth order, socioeconomic status, and intellectual ability are controlled. Older and more intellectually capable adolescents have a lower tendency to seek medical care. An adolescent's tendency to seek medical care is not influenced by gender and/or birth-order differences. Socioeconomic status variables indirectly influence adolescents' tendency to seek medical care through their primary influence on adolescent's intellectual ability.

Adolescent↗
PubMed1991

Proteolytic digestion of non-collagenous basement membrane proteins by the hemorrhagic metalloproteinase Ht-e from Crotalus atrox venom.

Hemorrhagic toxin e (Ht-e), a metalloproteinase isolated from the venom of the Western Diamondback rattlesnake Crotalus atrox, digests laminin and nidogen, both in their isolated forms and when present in a purified soluble complex. The only common site of cleavage by Ht-e of isolated nidogen and nidogen when complexed with laminin is at amino acid residue 336 in the amino terminal domain. Additionally, nidogen in complex with laminin is also cleaved at sites 322, 351 and 840 as determined by sequence analysis and site 953 as proposed from the molecular mass of a digestion product. Isolated nidogen, on the other hand, was cleaved at amino acid residues 75, 336, 402, and 920, as determined by sequence determinations and approximately at residues 296, 478, 625 and 702 as proposed from the molecular mass values of the generated polypeptide chains. Products from the proteolytic cleavage of the A and B2 chains of laminin were observed with the sites of cleavage determined to be at position 2666 in the laminin A chain and position 1238 in the laminin B2 chain. The laminin digestion products were identical regardless of whether nidogen was present in a complex with the laminin chains.

Amino Acid Sequence↗
PubMed1991

Synthetic and endogenous inhibitors of snake venom metalloproteinases.

The venoms of most Crotalidae snakes contain metalloproteinases which are the agents responsible for the production of venom-induced hemorrhage via proteolytic destruction of capillary basement membranes. Prevention of hemorrhage by administration of antiserum is generally not totally effective against damage at the site of envenomation. Therefore, we have investigated alternate methods for the alleviation of hemorrhage by inhibition of the proteolytic activity of the hemorrhagic toxins. The first approach involves the synthesis of carboxyalkyl peptide inhibitors in which the peptide moiety is modeled on the substrate specificity of the toxins. With this approach we have determined that the carboxypentyl group for interaction with the active site Zn+ +ion is most effective. Also, longer peptide moieties enhance the inhibitors' activity giving Ki's in a range of 10(-6) M. Our second approach to hemorrhagic toxin inhibition was to search for the presence of endogenous inhibitors against the toxins in the venom. From the crude venom we have isolated several pyro-glutamate containing peptides, two of which are relatively good inhibitors of the toxins. The isolation and characterization of the endogenous toxin inhibitors as well as the synthetic inhibitors may ultimately serve as a basis for new, effective treatments against venom-induced hemorrhage.

Amino Acid Sequence↗