Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.2.2.7 (heparinase)
 1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE). The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation. Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size. High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c. and were used to help characterize the gradient PAGE separation. Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively. The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed.
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PMID:Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis. 344 73

Prostacyclin (PGI2) is a well-known potent inhibitor of platelet aggregation. Its role has been implicated in physiological and pathological states of hemostasis. Heparin blocks the prostacyclin-mediated antiaggregatory activity on platelets. Prior treatment of heparin with heparinase as well as with protamine destroyed heparin's ability to neutralize PGI2. Studies on the mechanism of heparin blocking of PGI2 activity suggested that heparin interacted directly with PGI2, as shown by the loss of PGI2 mobility on thin layer chromatography concomitant with the loss of PGI2-mediated inhibition of platelet aggregation. PGI2 in this combination with heparin, nevertheless, retained its time-dependent ability to be hydrolyzed to 6-keto-PGF1 alpha. Findings of these studies may have implications in thrombosis and hemostasis, particularly in heparin-mediated abnormalities of circulating platelets.
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PMID:Heparin-mediated neutralization of platelet antiaggregatory activity of prostacyclin (PGI2): studies on mechanism. 389 23

The regulation of cell growth in the kidney glomerulus plays a key role in many physiologic and pathologic processes. In this communication, the authors examine the possible role of heparin-like species as inhibitors of mesangial-cell proliferation. Heparin profoundly inhibited the growth of cultured mesangial cells in a dose-dependent manner, with an ED50 = 5-10 micrograms/ml. The antiproliferative activity of heparin was reversible and specific for mesangial cells as the target cell in the glomerulus. Heparin was much more effective than other glycosaminoglycans. Cultured glomerular epithelial cells were found to secrete both stimulators and inhibitors of mesangial-cell growth. Approximately half of the inhibitory activity was destroyed by a highly purified heparinase; the other half was sensitive to trypsin. Approximately 80% of the mitogenic activity was protease-sensitive. These results suggest that heparin and glomerular epithelial cells may participate in mesangial-cell growth regulation.
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PMID:Heparin and glomerular epithelial cell-secreted heparin-like species inhibit mesangial-cell proliferation. 403 68

Heparin has been enzymatically depolymerized with heparinase (heparin lyase (EC 4.2.2.7)) and then separated into di-, tetra-, hexa-, octa-, and decasaccharide mixtures by low-pressure gel-permeation chromatography (GPC). These sized mixtures were resolved by strong anion-exchange (SAX) HPLC into multiple components. The fractions from the SAX-HPLC were collected and characterized for size by GPC-HPLC and sulfate content by ion chromatography. This study provides detailed methodology for the separation of larger and more highly sulfated oligosaccharides than previously reported. It describes the first use of ion chromatography for the accurate determination of the sulfate content of heparin oligosaccharides, a method which can also be applied to heparin and other glycosaminoglycans.
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PMID:High-performance liquid chromatographic separation of heparin-derived oligosaccharides. 409 Dec 60

Heparin of an average molecular weight of 13,000 was fractionated on the basis of size into five fractions of different weight-average molecular weight ranging from 8500 to 20,000. The heparin was also degraded using microbial heparinase resulting in products ranging from a disaccharide of molecular weight 500 to an oligosaccharide of molecular weight 3100. These products were also size fractionated. The individual heparin fractions and products were tested for metachromatic activity with Azure A. The metachromatic activity of the heparin fractions was independent of molecular weight, while the metachromatic activity of the products was dependent on molecular weight. Metachromatic activity was found in a fragment as small as a tetrasaccharide. Anticoagulant activity was found in fragments of tetrasaccharide or larger by a Factor Xa clotting assay and in fragments of hexasaccharide or larger by a Factor Xa amidolytic chromogenic assay.
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PMID:Metachromatic activity of heparin and heparin fragments. 620 32

Comparison of the [35S]mucopolysaccharides extracted after in vitro incubation of skin biopsy specimens from nonlesional and lesional sites of a patient with mastocytosis showed that lesional sites incorporated sulfate into heparin. After in vitro incorporation of the [35S]sulfate, the tissues were extracted sequentially by a 3-step procedure which utilized high salt concentrations, enzymatic digestion and base hydrolysis to liberate essentially all the counts. The extracted [35S]mucopolysaccharides were separated from free [35S]sulfate, histamine, protein, and hyaluronic acid by ion-exchange chromatography utilizing Dowex 1. The [35S]mucopolysaccharide extracts of the nonlesional skin were completely degraded by treatment with chondroitinase ABC, as they age predominantly dermatan sulfate with small amounts of chondroitin sulfates. The absolute quantity of sulfated mucopolysaccharides after Dowex 1 chromatography in micrograms of uronic acid per mg wet weight of starting tissue was higher in the lesional than the nonlesional specimen, while the specific incorporation of [35S]sulfate per microgram of uronic acid was the same. Approximately one-half of the [35S]mucopolysaccharides obtained in the 3 sequential extracts of lesional tissue was resistant to degradation by chondroitinase ABC as determined by gel filtration before and after enzyme treatment, indicating the presence of sulfated mucopolysaccharides in addition to chondroitin and dermatan sulfates. Heparinase treatment of the chondroitinase ABC-resistant [35S]mucopolysaccharides followed by gel filtration revealed an equal distribution of label between heparin and heparinase-resistant material presumed to be heparan sulfate. Heparin was also directly demonstrated in extracts of lesional mastocytosis skin by chemical and functional criteria.
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PMID:Identification of sulfated mucopolysaccharides including heparin in the lesional skin of a patient with mastocytosis. 644 88

Anticoagulation with heparin is required during extracorporeal circulation for hemodialysis and cardiopulmonary bypass as well as during vascular surgery. Reversal of anticoagulation with protamine may be associated with hypotension and rebound anticoagulation and requires stoichiometric doses. Heparinase from Flavobacterium heparinum catalytically degrades heparin and reverses its anticoagulant effect. Heparin was administered to New Zealand White rabbits and plasma levels were assayed with the APTT anticoagulant assay and the azure A chemical assay. Heparinase actively degraded heparin both in vitro in rabbit plasma and in vivo in rabbit blood as determined by both the anticoagulant and chemical assays when compared to control heparin disappearance curves. Antibodies to heparinase were demonstrated by the ELISA technique in rabbits receiving i.v. heparinase. These antibodies, however, did not effect the activity of the enzyme in vitro or in vivo. No toxic effects of heparinase were noted in observations of the animals or in blood and histologic studies. Heparinase, either free or immobilized, may be a useful heparin-reversing agent without the drawbacks of protamine.
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PMID:Heparinase: in vivo activity and immunogenicity in rabbits. 663 Nov 73

Heparin of an average molecular weight of 13,000 with known polydispersity was degraded using microbial heparinase. The kinetics of this degradation were followed by four assays which measured the anticoagulant activity of the heparin digestion products. Both clotting and amidolytic chromogenic assays were used to measure heparin-potentiated inhibition of both thrombin and Factor Xa. These assays showed different profiles throughout the digestion and were related to the average molecular weight of the digestion products.l The final products of this enzymatic digestion were fractionated on the basis of size and their anticoagulant activities were measured. Fragments causing Factor Xa inhibition but not thrombin inhibition were isolated. Anticoagulant activity was found in a fragment as small as a tetrasaccharide.
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PMID:Differential anticoagulant activity of heparin fragments prepared using microbial heparinase. 708 27

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 mug/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.
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PMID:Cultured endothelial cells produce a heparinlike inhibitor of smooth muscle cell growth. 728 12

Migration of capillary endothelial cells is an important component of angiogenesis in vivo. Increased numbers of mast cells have been associated with several types of angiogenesis. We have used a quantitative assay in vitro to demonstrate that mast cells release a factor that significantly increases bovine capillary endothelial cell migration. The factor is present in medium conditioned by mast cells as well as lysates of mast cells. The stimulatory effect of mast cells on migration is specific for capillary endothelial cells. Furthermore, mast cells have no mitogenic activity for capillary endothelial cells. Of all the secretory products of mast cells tested, only heparin stimulated capillary endothelial cell migration in vitro. Heparin preparations from a variety of sources stimulated capillary endothelial cell migration to the same degree but did not stimulate migration of several other cell types. The migration activity of heparin and mast cell conditioned medium was blocked by specific antagonists of heparin (protamine and heparinase), but not by chondroitinase ABC. The migration activity of mast cell conditioned medium was resistant to heat (100 degrees C) and incubation with proteolytic enzymes. These results suggest that the role of mast cells in angiogenesis may be to enhance migration of the endothelial cells of growing capillaries.
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PMID:Mast cell heparin stimulates migration of capillary endothelial cells in vitro. 742 25


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