Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new method has been described for the isolation of factor VIII. The method results in a high yield of factor VIII that is homogeneous by several different criteria. The purified protein is very stable and is not dissociated in the presence of 1 M NaCl or 0.25 M CaCl2. The highly purified protein is readily activated and inactivated by various proteolytic enzymes, such as thrombin, plasmin, and trypsin. The molecular events that lead to the activation reaction, however, have not been established.
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PMID:Isolation, subunit structure, and proteolytic modification of bovine factor VIII. 12 88

The receptors for aggregated immunoglobulin G (IgG) (an Fc receptor) and for ristocetin-von Willebrand factor on human platelets were studied by means of various modifications of the platelet surface. The expression of these receptors was measured by the agglutination of platelets to ristocetin in the presence of von Willebrand factor, which is part of the factor VIII complex, and by the binding of aggregated IgG coupled to 3H-labelled diazobenzene. Treatment of platelets with chymotrypsin, trypsin, papain and pronase which removed protein and glycoprotein from the platelet under conditions where the release reaction was inhibited caused loss of the expression of the receptor for ristocetin-von Willebrand factor and an enhancement of that for aggregated IgG. Induction of membrane changes with ADP and of the release reaction with the ionophore A23187 abolished agglutination to ristocentin-von Willebrand factor but did not alter the receptor for aggregated IgC. Possible contributions of unspecific membrane changes, produced by protease treatment of platelets, to the modification of receptor expression were eliminated by the use of formaldehyde-treated platelets. Trypsin, papain and pronase destroyed the ability of these platelets to agglutinate to ristocetin-von Willebrand factor but produced no change in the binding of aggregated IgC. Therefore, the receptor for ristocetin-von Willebrand factor is truly sensitive to proteolysis while the Fc receptor is not, but is partially masked by protease-sensitive material.
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PMID:A comparative study of the effect of modification of the surface of human platelets on the receptors for aggregated immunoglobulins and for ristocentin-von Willebrand factor. 31 30

Thrombin first activates and then inactivates factor VIII and for this reason thrombin has been considered responsible for the inactivation of factor VIII which occurs during clotting. Experiments described in this paper indicated that the activity of factor VIII is not reduced in factor IX or factor X deficient sera, while on the other hand this factor becomes inactivated in blood anticoagulated with high concentrations of hirudin which inhibit thrombin activity completely. This suggests that some other factor, besides thrombin, which is generated only in trace amounts in factor IX or factor X deficient plasmas, is also able to inactivate factor VIII. Purified factor X activated with insolubilized trypsin was added to purified preparations of factor VIII, which were free of both fibrinogen and prothrombin. Factor X a was allowed to act for 5-60 minutes and then inactivated with phenylmethanesulfonyl fluoride. Depending on the duration of the action of factor X a partial or complete inactivation of factor VIII was observed. This inactivation was also observed in the presence of hirudin, thus excluding the possibility that the effect was due to contamination with trace amounts of thrombin.
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PMID:Inactivation of factor VIII by a mechanism independent of the generation of thrombin. 50 1

A simple method for the culture of human retinal capillary endothelial cells in vitro is described in this report. Through a process of gentle mechanical disruption and sieving, a sufficient yield of retinal microvessels was obtained from one or two human eyes to allow the culture of endothelial cells in abundance. The cells were factor VIII-positive and demonstrated typical vascular endothelial morphology. Pericyte contamination was prevented by using human platelet-poor serum in primary culture and passaging cells with a low concentration of trypsin. Proliferation assays revealed that the cells grew best in Iscove's modified Dulbecco's modified Eagle's medium with 15% fetal calf serum (FCS). There was no difference in induced proliferation when FCS was compared to human platelet-poor serum. The method seemed to be as good as and much simpler than other recently described techniques. The study of human retinal capillary endothelial cells cultured in this way may shed light on the earliest stages of diabetic retinopathy and other diseases of the retinal microvasculature, particularly AIDS-related retinopathy and radiation retinopathy.
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PMID:A simple method for the in vitro culture of human retinal capillary endothelial cells. 152 30

We previously reported a functional defect of von Willebrand factor (vWF) in a new variant of von Willebrand disease (vWD) tentatively named vWD "Normandy." The present work has attempted to characterize the molecular abnormality of this vWF that fails to bind factor VIII (FVIII). The immunopurified vWF from normal and patient's plasma were digested by trypsin and the resulting peptides were compared. The electrophoresis of "vWF Normandy" showed a shift in the band corresponding to a polypeptide from amino acid 1 to 272. Consequently, we performed the molecular analysis of the portion of the vWF gene of this patient encoding this amino acid sequence. Exons 18-24 were amplified by the use of polymerase chain reaction and their nucleotide sequences corresponding to 1.8 kb were determined. Our analysis showed a point mutation C to T at codon 791, resulting in the substitution of Methionine for Threonine at position 28 of the mature vWF subunit. Because this nucleotide substitution destroyed a Mae II restriction site, this mutation was conveniently sought in various individual DNAs. The patterns obtained were consistent with the homozygous and heterozygous state of this mutation in the patient and in her son, respectively, and with its absence in 28 normal individuals. We conclude that Threonine at position 28 in plasma vWF may be crucial for the conformation and FVIII-binding capacity of its cystine-rich N-terminal domain.
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PMID:The "Normandy" variant of von Willebrand disease: characterization of a point mutation in the von Willebrand factor gene. 201 34

Different methods for fixation and exposure of antigenic determinants were tested for detection of a granulocytic differentiation antigen by the monoclonal antibody L12-2, using an indirect immunoperoxidase method on semi-thin sections of undecalcified, glycolmethacrylate-embedded human bone marrow biopsies. Fixation in Bouin's solution for 3 hr gave a more intense and more homogeneous immunological staining than fixation in absolute methanol, 4% formalin, B5, or Michel's medium, and the morphological detail was excellent. Digestion by pronase or trypsin was required. Coating the glass slides with Alcian blue prevented loss of sections from the slides during the staining procedure. Bouin fixation also made possible detection of two other differentiation antigens expressed in the granulocytic series, using the monoclonal antibodies 1G10 and R1B19. Furthermore, the same technique also permitted detection of factor VIII-RAg in the megakaryocytes, as well as recognition of cells of the erythroid series by use of polyclonal rabbit antisera.
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PMID:Immunoperoxidase detection of myeloid antigens in glycolmethacrylate-embedded human bone marrow. 243 85

Proteolytic enzymes were tested for improving histochemical localization of tissue antigens. Sections, 2-4 micron in thickness, were prepared on sodium-silicate coated slides from formalin-fixed, paraffin-embedded human biopsies. A modification of the Sternberger technique (PAP) and the indirect immunofluorescence method were used for the localization of 15 various antigens: heavy chain immunoglobulins, light chain immunoglobulins, alpha 1-fetoprotein, alpha 1-antichymotrypsin, myoglobin, fibronectin, factor VIII (ass. ag), fibrinogen, lysozyme and cytokeratin. The ability of different proteolytic enzymes (trypsin, pronase, pepsin) to unmask antigen in formalin-fixed sections were tested by variation of concentration, incubation time, temperature and pH. Although proteolytic unmasking to some extent is reliable, good restoration of antigenicity is not always possible. Best results were obtained with pronase E (Serva, FRG).
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PMID:[The proteolytic pretreatment of formalin-fixed tissue in immunohistochemical diagnosis]. 245 12

Vascular endothelial cell factor VIII/von Willebrand factor antigen (FVIII/vWF Ag) of normal and disordered gastric tissues was studied with staphylococcus protein A-gold (PAG) labelling followed by photochemical silver reaction. FVIII/vWF Ag was localized clearly in the tissue fixed with various common fixatives and embedded in paraffin without enzyme treatment. The most satisfactory staining and the least nonspecific background were observed in the tissues fixed with Zamboni's and Bouin's solutions. The staining reaction could be enhanced, if the sections were pretreated with trypsin and subtilisin. Under the electron microscope, the gold particles were found over the Weibel-Palade bodies of vascular endothelial cells in the tissues fixed either in Zamboni's solution or in Zamboni's solution-osmium tetroxide, and embedded either with Lowicryl K4M or with Epon 812. It has been proved to be a better technique in investigation of FVIII/vWF Ag in vascular endothelial cells.
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PMID:Immunohistochemical localization of vascular endothelial cell factor VIII/von Willebrand factor antigen in human normal and disordered gastric tissues. 251 30

Factor IX BM Nagoya (IX Nagoya) is a natural mutant of factor IX responsible for severe hemophilia B. A patient with this mutant is characterized by a markedly prolonged ox brain prothrombin time. IX Nagoya was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that treatment of IX Nagoya with factor XIa/Ca2+ resulted in cleavage only at the Arg145-Ala146 bond. Reversed-phase high performance liquid chromatography of a trypsin digest of IX Nagoya showed an aberrant peptide, which was further digested with proteinase Asp-N. Primary structure analysis of one of the Asp-N peptides revealed that Arg180 is replaced by Trp. An essentially complete (99%) amino acid sequence of IX Nagoya was obtained by sequencing fragments derived from a lysyl endopeptidase digest in which no other substitutions in the catalytic triad or substrate binding site were found. We also found that IX Nagoya is activated by alpha-chymotrypsin or rat mast cell chymase by monitoring the rate of factor X activation using a fluorogenic peptide substrate in the presence of factor VIII, phospholipids, and Ca2+. These results indicate that the substitution of Arg180 by Trp impairs the cleavage by factor XIa required for activation of this zymogen and that the substitution causes hemophilia BM.
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PMID:Blood clotting factor IX BM Nagoya. Substitution of arginine 180 by tryptophan and its activation by alpha-chymotrypsin and rat mast cell chymase. 259 73

The factors responsible for the lability of factor VIII coagulant activity (VIII:C) and factor VIII coagulant antigen (VIII:CAg) are poorly understood. In this study the VIII:C and VIII:CAg are studied after incubation with plasmin, trypsin or alpha-chymotrypsin. Both isolated human VIII:CAg and VIII:CAg associated with factor VIII-related antigen (VIII R:Ag) are evaluated. The antigenic sites of the VIII:CAg are somewhat more stable to the action of these enzymes than the functional activity, although both follow a generally parallel degradation. A biphasic decay curve is seen in the initial time points. No stabilization of the functional or antigenic reactivity is observed in the presence of the VIII R:Ag. Lower concentrations of each enzyme cause an initial rise in the factor VIII:C in the presence of VIII R:Ag, but not in the isolated VIII:CAg. Higher concentrations of alpha-chymotrypsin cause activation of VIII:C and a slight decrease in the VIII:CAg values in both preparations. These enzymes may play a modulating role in the coagulation cascade through the activation and degradation of VIII:C and VIII:CAg.
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PMID:Degradation of factor VIII coagulant antigen by proteolytic enzymes. 293 56


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