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Biomedical subjects

C L Cooney

Publications and source records attributed to C L Cooney.

At least 19 recordsLinked to original sources
PubMed1995

Heparinase I from Flavobacterium heparinum: the role of the cysteine residue in catalysis as probed by chemical modification and site-directed mutagenesis.

Heparinase I (heparin lyase I, EC 4.2.2.7), a heparin-degrading enzyme produced by Flavobacterium heparinum, is used to deheparinize blood following extracorporeal procedures in surgery and in other applications. The present study of mapping and characterization of the cysteines of heparinase I represents the first structural characterization of a heparinase. [3H]Iodoacetic acid labeling demonstrated that heparinase I has two free cysteines. One of the two cysteines is surface accessible and lies in a hydrophilic environment while the other is in a hydrophobic environment. Chemical modification of the cysteines, both in the presence and in the absence of heparin, suggests that the surface-accessible cysteine lies in or near the active site of heparinase I. Preferential reactivity of this cysteine with negatively charged sulfhydryl-modifying reagents and the cysteines' high reactivity to iodoacetic acid at pH 6.5 indicate that the surface-accessible cysteine is in a positively charged region. The surface-accessible cysteine (cysteine-135) was mapped as the active-site cysteine by radiolabeling with [3H]iodoacetic acid and by tryptic digestion and peptide sequencing. Site-directed mutagenesis of cysteine-135 to a serine or an alanine in r-heparinase I demonstrates that this cysteine is essential for enzymatic activity. However, replacement of the surface-inaccessible cysteine by a serine or alanine has no effect.

Base Sequence↗
PubMed1995

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Amino Acid Sequence↗
PubMed1994

A stereochemical approach to pyranose ring flexibility: its implications for the conformation of dermatan sulfate.

Glycosaminoglycans, such as heparin, heparan sulfate, and dermatan sulfate, are characterized by a disaccharide repeating unit of a uronate and a hexosamine and are increasingly understood to be important physiologically as soluble components of the extracellular matrix. The secondary structure of this class of acidic polysaccharides is believed to play a key role in determining the wide range of biological specificities. Central to the structural diversity of the glycosaminoglycans is the experimentally documented conformational flexibility of the iduronate residue. Here, we outline an approach to explore the iduronate conformational flexibility by imposing stereochemical criteria of nonbonded contact distances. By performing a complete search of all possible torsions that define the iduronate ring geometry, we eliminate any prior bias with regard to minimum energy conformers. The approach led to alternative feasible conformers for the iduronate ring that are stereochemically satisfactory and are consistent with the available physico-chemical data.

Carbohydrate Conformation↗
PubMed1994

Heparinase inhibits neovascularization.

Neovascularization is associated with the regulation of tissue development, wound healing, and tumor metastasis. A number of studies have focused on the role of heparin-like molecules in neovascularization; however, little is known about the role of heparin-degrading enzymes in neovascularization. We report here that the heparin-degrading enzymes, heparinases I and III, but not heparinase II, inhibited both neovascularization in vivo and proliferation of capillary endothelial cells mediated by basic fibroblast growth factor in vitro. We suggest that the role of heparinases in inhibition of neovascularization is through depletion of heparan sulfate receptors that are critical for growth factor-mediated endothelial cell proliferation and hence neovascularization. The differences in the effects of the three heparinases on neovascularization could be due to different substrate specificities for the enzymes, influencing the availability of specific heparin fragments that modulate heparin-binding cytokines involved in angiogenesis.

Allantois↗
PubMed1992

Fermentation monitoring.

Fermentation monitoring continues to be the focus of much research. Over the last year, important strides were made in improving bioprocess monitoring using NADH fluorescence, viscosity, affinity techniques, enzyme and microbial sensors, calorimetry, flow injection analysis and bioluminescence. Better fermentation monitoring is important for improving understanding, operation, development and control of the process. We expect progress in these areas of research to continue. In addition, we highlight some non-conventional approaches.

Biosensing Techniques↗
PubMed1991

The release of heparinase from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock.

Heparinase was released from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock procedure. The procedure involves resuspending exponentially growing cells consecutively into (1) 40% sucrose, (2) 10 mM sodium phosphate, 2 mM magnesium chloride, pH 7, and (3) 10 mM sodium phosphate, 300 mM sodium chloride, 2 mM magnesium chloride, pH 7. Typically, 50-75% of the total heparinase activity is recovered by this procedure with an observed 7-15-fold increase in purity. The majority of heparinase activity is released in the final step of the procedure allowing for resolution from cytoplasmic and nonspecific periplasmic material. F. heparinum cells can be stored in 40% sucrose at 4 degrees C for up to one week without significant losses in recovery yields.

Biotechnology↗
PubMed1990

Intracellular lytic enzyme systems and their use for disruption of Escherichia coli.

This article focusses on lytic enzyme systems available in E. coli and their potential use for cellular disruption. In the systems described here the genetic information for lysis would be carried within the microbial host, either integrated or naturally occurring on chromosomal DNA, or on extrachromosomal elements such as plasmids. Each microbe would carry complete information for endogenous enzymatic lysis, and lysis would occur in a controlled manner after being triggered by an external factor such as temperature or inducer addition. The lytic systems explored in this review include the autolytic enzymes, colicin lytic enzymes, and bacteriophage lytic enzymes from phage phiX174, T4, lambda, MS2 and Q beta. Many of the colicin lytic enzymes and all of the bacteriophage lytic enzymes described here have been cloned, and in some instances examined as cellular disruption methods. None of the E. coli autolytic enzymes have been cloned, but information pertinent for use as a disruption method is described.

Autolysis↗
PubMed1990

Specific plate assay for bacterial heparinase.

A procedure was developed for detecting heparinase activity on heparin agar plates. The method is based on the differential precipitation of heparin and heparinase-generated heparin fragments by protamine sulfate. Heparinase activity is detected by the presence of clear zones against a white background. This method can be used to screen for the expression of recombinant heparinase and to identify Flavobacterium heparinum mutants expressing heparinase constitutively.

Agar↗
PubMed1990

Optimization and simulation of continuous affinity-recycle extraction (care).

Simulation and optimization of continuous affinity recycle extraction (CARE), a protein purification unit operation based on protein adsorption to solid phase adsorbents, is described in this paper. Rather than packing conventional adsorbent particles in a fixed bed (column), solid/liquid contact is carried out in well-mixed reactors. Continuous operation is achieved by recirculation of the adsorbent particles between two or more contactors. The feasibility of this purification scheme was established with the recovery and isolation of the enzyme beta-galactosidase from E.coli, using the affinity support PABTG/Agarose. A mathematical model describing system performance was developed. The mathematical model was used to optimize several facets of the system design and operation. The base two-stage contractor design was modified by the addition of an intermediate wash stage as well as the incorporation of multiple adsorption stages. These design modifications serve to increase purification, concentration and recovery while utilizing the same amount of adsorbent. The methodology for defining and optimizing objective functions was developed and experimentally validated. Finally, optimum system start-up protocols, minimizing the time required to reach steady-state operation, were developed and experimentally validated. The impact of early introduction of adsorptive purification in a downstream processing sequence, with CARE, was evaluated and is described. Through the early introduction of a highly specific adsorptive step, significant purification is achieved simultaneously with clarification and concentration. In addition, purification performance in CARE was contrasted with that achievable in conventional column chromatography.

Chromatography, Affinity↗
PubMed1986

Removal of the anticoagulant activities of the low molecular weight heparin fractions and fragments with flavobacterial heparinase.

Recently, the development of low molecular weight heparin fractions and fragments (LMHF) as potential antithrombotic agents has gained increased attention. However, the lack of antagonists to neutralize the anticoagulant effects of these drugs may seriously exclude them from possible uses in extracorporeal therapy. This is mainly because of the concern that the high dosage of the drugs employed in extracorporeal therapy could lead to serious bleeding risks. Our earlier work has demonstrated that immobilized heparinase can remove polydisperse heparin both in vitro and in vivo. To examine whether such a system may be used as a novel approach to neutralize the anticoagulant effects of LMHF, different LMHF were tested using heparinase. In vitro data showed that both the APTT and anti-FXa activities of the LMHF including Kabi 2165, PK 10169, Cy 216 and CY 222 were nearly completely eliminated by heparinase in less than 20 min. This study suggests that an immobilized heparinase system may be an useful element for the acceptance of the LMHF for their use in extracorporeal therapy.

Anticoagulants↗
PubMed1986

Polysaccharide lyases.

Polysaccharide lyases (or eliminases) are a class of enzymes (EC 4.2.2.-) that act to cleave certain activated glycosidic linkages present in acidic polysaccharides. These enzymes act through an eliminase mechanism, rather than through hydrolysis, resulting in unsaturated oligosaccharide products. Acidic polysaccharides are ubiquitous and so are the lyases that degrade them. This review article examines lyases that act on acidic polysaccharides of plant, animal, and microbial origin. These lyases are predominantly of microbial origin and come from a wide variety of both pathogenic and nonpathogenic bacteria and fungi. The lyases discussed include alginate lyase (EC 4.2.2.3), pectin lyase (EC 4.2.2.10), pectate lyase (EC 4.2.2.2), oligogalacturonide lyase (EC 4.2.2.6), exopolygalacturonate lyase (EC 4.2.2.9), chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5), hyaluronate lyase (EC 4.2.2.1), heparin lyase (EC 4.2.2.7), heparan lyase (EC 4.2.2.8), and other unclassified lyases. This review examines the sources, regulation, purification, and properties of these polysaccharide lyases.

Animals↗
PubMed1986

Oxygen limitation on L-serine production in a hollow-fiber bioreactor.

Pseudomonas AM1 utilizes glycine and methanol to produce L-serine aerobically. The consumption of methanol in this bioconversion is stoichiometrically in excess of L-serine production. Consequently, the oxygen requirement associated with L-serine production is higher than expected for the conversion from glycine. One method of L-serine production investigated was a technique utilizing a hollow-fiber ultrafiltration cartridge as a bioreactor. Oxygen diffusion limitations appear to impede the consumption of methanol and, consequently, the production of L-serine in such a reactor. Methanol consumption data agree with predictions based on a hollow-fiber diffusion model.

Glycine↗
PubMed1986

Sulfur regulation of heparinase and sulfatases in Flavobacterium heparinum.

Sulfur regulation of heparinase synthesis and sulfatase synthesis was studied in Flavobacterium heparinum. Heparinase synthesis was strongly repressed by sulfate and L-cysteine, while the activity of this enzyme showed little or no inhibition by these compounds. Heparinase was synthesized in the absence of heparin when L-methionine was used as the sole sulfur source. The sulfatases produced by F. heparinum, which include the sulfatases involved in heparin catabolism, were also studied. At least some of the sulfatase activity was regulated by sulfur compounds in a manner similar to heparinase regulation. L-Cysteic acid and taurine were not suitable sulfur sources to support the growth of F. heparinum.

Ammonium Sulfate↗
PubMed1985

Purification and characterization of heparinase from Flavobacterium heparinum.

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.

Amino Acids↗
PubMed1984

An immobilized microbial heparinase for blood deheparinization.

A new medical application of an immobilized microbial enzyme is described. Extracorporeal devices require systemic heparin administration to prevent thrombus formation; however, the use of heparin often leads to serious hemorrhagic complications. Heparinase isolated from Flavobacterium has been immobilized and used in a fluidized bed reactor to eliminate heparin from blood passing through an extracorporeal circuit both in vitro and in vivo. This paper discusses the stepwise development of this heparinase reactor including: (1) improvements in the fermentation resulting in an inexpensive large-scale source of heparinase without the addition of the previously required inducer, heparin; (2) the use of batch processes to adapt previous purification schemes to large-scale heparinase production and the subsequent purification of heparinase to a single SDS-PAGE banding protein; (3) the immobilization of heparinase with a 91% activity recovery and good stability, (4) the design and successful testing of a fluidized bed reactor containing immobilized heparinase in the removal of clinically used quantities of heparin from both human blood in vitro and canine blood in vivo; and (5) the initiation of animal studies focusing on the toxicology of heparinase-derived heparin degradation products and the short and long term effects of exposure to these products and to heparinase.

Enzymes, Immobilized↗