Local thrombus formation in the site of intravenous injection of chlorpromazine: possible colloid-osmotic lysis of the local endothelial cells.

Since amphiphilic drugs are known to interact with biomembranes, we investigated local vessel damage and thrombosis which might be brought about by intravenous dosing using chlorpromazine (CPZ) as a representative compound. CPZ-induced hemolysis was suppressed by an increase in sucrose concentration in the medium, characterizing this hemolysis to be colloid-osmotic lysis, which includes the enhancement of membrane phospholipid fluidity and consequent small pore formation in the membranes. This was supported by the observation that hemolysis by filipin, not featuring the stage of small pore formation, was not affected by sucrose. [14C]Glucose-entrapping liposomes were degraded by CPZ, and this degradation was enhanced by an increase in the intravesicle glucose concentration. These results indicated that the compound could induce colloid-osmotic lysis in erythrocytes and artificial membrane vesicles. CPZ also injured cultured porcine aortic endothelial cells (PAEC), as evidenced by lactate dehydrogenase (LDH) leakage. This injury was also suppressed by increase in sucrose concentration in the medium, suggesting that colloid-osmotic lysis again occurred. When rats were intravenously injected with CPZ, local endothelial cell (EC) injury and associated thrombus formation were observed, suggesting that CPZ's action was also evident in vivo. To our knowledge, this is the first finding which suggests that an intravenously dosed amphiphilic drug can injure local ECs based on a colloid-osmotic lysis mechanism leading to thrombosis.

Isolation and characterization of Taka-amylase A apoprotein deglycosylated by digestion with almond glycopeptidase immobilized on Sepharose.

Taka-amylase A (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1), which contains a single asparagine-linked oligosaccharide unit, was digested with almond glycopeptidase immobilized on Sepharose 6B at 20 degrees C for 4 h. A maximum of 10% of the parent protein was isolated as apoprotein by column chromatography on Con-A Sepharose. The characteristics of the apoprotein were compared to those of the native Taka-amylase A. The removal of the sugar chain from Taka-amylase. A caused no change in the pH-activity profile or in kinetic parameters of the hydrolysis of soluble starch. The stability of the apoprotein toward changing pH and digestion by proteases did not show any appreciable difference from that of the native Taka-amylase. These results suggest that the carbohydrate moiety of Taka-amylase A is not an essential participant in the catalysis.

The release of carbohydrate moieties from human fibrinogen by almond glycopeptidase without alteration in fibrinogen clottability.

The possible noninvolvement of the carbohydrate moiety of human fibrinogen in the clotting mechanism was examined by eliminating the neutral sugar chains from desialylated fibrinogen by almond glycopeptidase digestion. 40% of the total neutral sugars was removed from the desialylated fibrinogen. The neutral sugars from both the beta- and gamma-polypeptide chains were released equally. The protein moiety of the glycopeptidase-digested fibrinogen was found to be intact. No significant change was observed in the thrombin time(fibrinogen clottability) of the resultant fibrinogen. The results suggest that the carbohydrate moiety of fibrinogen is not involved in the clotting mechanism. Oligosaccharide was detected in the glyopeptidase digest of desialylated fibrinogen by thin-layer chromatography (TLC), and was found to be identical with those released quantitatively from the peptic digests of beta- and gamma-polypeptide chains. The structure of the sugar chain was identified tentatively as Gal2-GlcNAc2-Man3-GlcNAc2, by sequential exoglycosidase digestion and quantitative analysis of carbohydrate components.

Almond glycopeptidase acting on aspartylglycosylamine linkages. Multiplicity and substrate specificity.

The glycopeptidase preparation that has been isolated from almond emulsin and acts on beta-aspartylglycosylamine linkages in glycopeptides was separated into three active fractions by DEAE-cellulose column chromatography. The three discrete species of glycopeptidase (Groups A, B and C) have been purified 30-, 136-, and 99-fold, respectively. The optimum pH value of Group A was 6.0 and those of Groups B and C, 5.0. Isoelectric points of Groups A, B and C were pH 7.7, 8.6 and 8.7, respectively. All three glycopeptidases hydrolyzed quantitatively glycopeptides with 3-11 amino acid residues prepared from stem bromelain, ovalbumin and ovotransferrin. Group C preferred glycopeptides with shorter peptide chains, whereas Groups A and B preferred those with longer chains. Glycopeptidase Group A also hydrolyzed intact glycoproteins such as stem bromelain, ovalbumin, Taka-amylase A and desialylated human transferrin.

The assay of factor V in plasma using a synthetic chromogenic substrate.

An analytical procedure using a synthetic substrate was devised for the determination of factor V in plasma. Factor V was activated by the reaction of immunobilized thrombin and incubated with an optimal concentration of a factor Xa, phospholipid, calcium and prothrombin. The thrombin generated was determined with the chromogenic substrate (Testzym S-2238). The initial rate of hydrolysis of the substrate was directly proportional to the factor V concentration. Effectors in plasma, such as prothrombin, fibrinogen, antithrombins and bilirubin, were controlled to minimize their effects. The normal value of factor V was from 3.3 to 5.5 units.

Some characteristics of a new glycopeptidase acting on aspartylglycosylamine linkages.

A new type of glycopeptidase hydrolyzing beta-aspartylglycosylamine linkages was partially purified from almond emulsin by chromatography on Sephadex G-200 and DE 52. The enzyme degraded stem bromelain glycopeptide, Asn-Asn(Man3,Xyl1,Fuc1,GlcNAc2)-Glu-Ser-Ser, to yield equimolar amounts of intact oligosaccharide, peptide (Asn-Asp-Glu-Ser-Ser), and ammonia. The Km value for the stem bromelain glycopeptide was 4 mM, and the optimum pH was 5.2. The enzyme was markedly inhibited by 10 mM Cu2+, Fe3+, and Zn2+. Thiol inhibitors and actinomycete protease inhibitors had no effect. The glycopeptides used as substrates were prepared from stem bromelain, ovalbumin or ovotransferrin. The enzyme hydrolyzed glycopeptides with 3-11 amino acid residues, whereas it did not hydrolyze glycopeptides with 1-2 amino acid residues. Furthermore, Asn-oligosaccharide was not inhibitory to the enzyme.

Ultramicromethod for the determination of human arginase in the presence of urea.

A technique for arginase determination in body fluids in the presence of urea is described. [14C]Arginine is hydrolysed by arginase to [14C]urea and ornithine. [14C]Urea is separated with paper chromatography and measured in a liquid scintillation counter. The experimental conditions including the pH, substrate concentration, activator, solvent for chromatography, urea inhibition, and arginase in hemolysates, are discussed.

A case of sideroblastic anaemia associated with marked elevation of erythrocytic arginase activity.

Abnormal erythrocytic arginase activity about 30 times higher than normal controls, was found in a 63-year-old male with sideroblastic anaemia. We suggest that an abnormal concentration of arginase in the erythroblasts, in this case reduces the arginine concentration and results in decreased haemoglobin synthesis.