Protease from Courgette (Luffa Acutangula L (Roxb)): Isolation, Purification, and Some Characteristics

The Courgette or oyong (Luffa acutangula L. (Roxb)) is member of Cucurbitaceae mainly used as vegetable. Beside used as vegetables, courgette aslo used as keratolytic agent. This fact is supposed that this vegetables contain protease. This research is succeed to purified courgette’s protease by four step. That was precipitate by 70% ammonium sulphate saturation, purification using DEAE cellulose ion exchange chromatography and gel filtration chromatography using sephadex G-100 and G-75. Purified courgette’s protease had 81,922 U/mg for specific activity and 34 kDa molecular weight. This enzyme had the characteristic such as activated optimally at 37oC, pH 7 and 10 minute duration time. This enzyme activity can decrease by PMSF and H2O2, its remarkable that courgette protease is serine protease and had the thiol group in its structure. The ability to digest food proteins materials like boiled meat and boiled white egg by courgette protease proves that the courgette protease enzyme is could be used in enzyme replacement therapy in mild digestion problem.

Partial Purifi cation, Stability Analysis, and Preservation of Xylanase from Xylanolytic Alkalophylic Bacteria

A xylanase, which produces xylose from oat spelt xylans, was isolated from the culture medium of xylanolytic alkalophylic bacteria mutant. The enzyme was purifi ed by ammonium sulphate with level 30, 40, 50, 60, 70, 80, and 90%. The purify of the fi nal preparation was demonstrated by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. The molecular masses of the purifi ed xylanase were 137.61 and 165.34 kDa. Result of ammonium sulphate saturation with the highest activity was used as standart for saturation for enzyme production and preservation, using corn, tapioca, soy bean meal and gaplek fl our as carriers. Addition of 60% ammonium sulphate showed the highest xylanase activity (62.03 U/g), and produced 89.40% enzyme recovery. Tapioca, as a carrier, produced the highest xylanase activity. Key words: preservation, purifi cation, stability analysis, xylanase.

Partial Characterization of a Fibrinolytic Inhibitor Produced by Cultured Endothelial Cells Derived from Human Umbilical Vein

SummaryA preliminary characterization of a fibrinolytic inhibitor released by human umbilical vein endothelial cells in primary culture is reported. This molecule of Mr comprised between 2 × 105 and 106 and of μ2 mobility precipitates at 43% ammonium sulphate saturation and is totally adsorbed on Concanavalin A Sepharose 4 B. A possible relationship with a macroglobulins is discussed.

Competing addition and hydrolysis of the cytidylylcytidylyladenosine terminal residues of transfer ribonucleic acid isolated from the non-lactating bovine mammary gland

1. The enzyme fraction obtained from the pH5 enzyme of non-lactating bovine mammary gland between 40 and 100% ammonium sulphate saturation markedly inhibited the AMP-incorporating activity of rat liver nucleotide-incorporating enzyme. This inhibitory effect has been attributed to high nuclease activity which can be partially removed by adsorption of the enzyme fraction on to calcium phosphate gel. 2. The degradation action of the calcium phosphate-purified enzyme is confined mainly to the terminal trinucleotide sequence -pCpCpA of tRNA, its effect being analogous to that of venom phosphodiesterase. This enzyme is heat labile and very readily loses its degradative activity. 3. Treatment of the enzyme fraction with Macaloid results in complete removal of the phosphodiesterase, leaving an enzyme capable of incorporating AMP into tRNA. 4. Transfer RNA extracted from non-lactating bovine mammary gland in the presence of polyvinyl sulphate and Macaloid is able to accept amino acids with an efficiency 30% of that shown by lactating bovine mammary-gland tRNA isolated under identical conditions.

A Normal Inhibitor of the Blood Coagulation Contact Reaction Product

A method is described for studying and measuring the activity of a normally occurring inhibitor of the blood coagulation contact reaction product (activated PTA). The inhibitor, stable on storage at -20 C. was inactivated by heating plasma to 56 C. for 30 minutes. The inhibitor was stable between pH 5 and 9. Inhibitory activity was increased by aluminum hydroxide adsorption and not apparently affected by celite exhaustion of plasma. The inhibitor was present in the fraction of plasma precipitated between 55 and 65 per cent ammonium sulphate saturation and migrated with the alpha globulins electrophoretically. The action of the inhibitor was prevented by soy bean trypsin inhibitor. Inhibitory activity was present in serum and in all normal plasma samples examined as well as in plasma from patients deficient in Hageman factor, PTA factor or factors VIII or IX. The physiologic and pathologic significance of this inhibitor remains to be determined.

EFFECTS OF INTRAVENOUS INJECTIONS OF THE ANTICOAGULANT FRACTION OF INCUBATED FIBRINOGEN ON BLOOD COAGULATION

AFIF (Anticoagulant Fraction of Incubated Fibrinogen precipitated between 25 and 50% ammonium sulphate saturation) prepared from Armour's incubated bovine fibrinogen was injected in a dosage of 46–106 mg (mean: 66.5 mg) tyrosine per kilogram body weight in the external jugular vein of 10 rabbits. Blood samples were withdrawn at [Formula: see text]- or 1-hour intervals until the aspirated blood began showing signs of coagulation. The anticoagulant effect was manifested immediately and lasted for [Formula: see text] to [Formula: see text] hours in the animal. Although the blood aspirated during this time remained unclotted even after 24 hours, the surgical wound in the neck of the animal did not bleed and autopsies revealed no sign of internal haemorrhage. The bleeding time, however, was found prolonged when tested by cutting the marginal vein of the ear. The level of the coagulation factors was determined both in oxalated specimens and in specimens with no anticoagulant added. Both kinds of sample showed: (1) clottable fibrinogen content normal, thus excluding fibrinolysis as the cause of the anticoagulant effect; (2) thrombin clotting time of infinity; (3) one-stage prothrombin time longer than 60 seconds; (4) no correction of the infinite thrombin clotting time of oxalated specimens following the addition of 0.25 mg protamine per ml. This makes unlikely any appreciable release of heparin by the animal. However, oxalated and non-oxalated specimens differed in the following respects: (1) prothrombin time determined after adsorption and mixing of the eluate with adsorbed plasma was found to be normal for the oxalated blood but variably increased for the non-oxalated specimen (10–34.5 seconds); (2) the plasma precursors of plasma thromboplastin were normal in oxalated but very low in native specimens. The serum precursors of plasma thromboplastin were normal.

EFFECTS OF INTRAVENOUS INJECTIONS OF THE ANTICOAGULANT FRACTION OF INCUBATED FIBRINOGEN ON BLOOD COAGULATION

AFIF (Anticoagulant Fraction of Incubated Fibrinogen precipitated between 25 and 50% ammonium sulphate saturation) prepared from Armour's incubated bovine fibrinogen was injected in a dosage of 46–106 mg (mean: 66.5 mg) tyrosine per kilogram body weight in the external jugular vein of 10 rabbits. Blood samples were withdrawn at [Formula: see text]- or 1-hour intervals until the aspirated blood began showing signs of coagulation. The anticoagulant effect was manifested immediately and lasted for [Formula: see text] to [Formula: see text] hours in the animal. Although the blood aspirated during this time remained unclotted even after 24 hours, the surgical wound in the neck of the animal did not bleed and autopsies revealed no sign of internal haemorrhage. The bleeding time, however, was found prolonged when tested by cutting the marginal vein of the ear. The level of the coagulation factors was determined both in oxalated specimens and in specimens with no anticoagulant added. Both kinds of sample showed: (1) clottable fibrinogen content normal, thus excluding fibrinolysis as the cause of the anticoagulant effect; (2) thrombin clotting time of infinity; (3) one-stage prothrombin time longer than 60 seconds; (4) no correction of the infinite thrombin clotting time of oxalated specimens following the addition of 0.25 mg protamine per ml. This makes unlikely any appreciable release of heparin by the animal. However, oxalated and non-oxalated specimens differed in the following respects: (1) prothrombin time determined after adsorption and mixing of the eluate with adsorbed plasma was found to be normal for the oxalated blood but variably increased for the non-oxalated specimen (10–34.5 seconds); (2) the plasma precursors of plasma thromboplastin were normal in oxalated but very low in native specimens. The serum precursors of plasma thromboplastin were normal.

ANTICOAGULANT EFFECT OF INCUBATED FIBRINOGEN

Sterile fibrinogen rendered non-clottable by incubation was mixed with fresh plasma and the thrombin time determined. An appreciable prolongation was observed. The incubated fibrinogen was then fractionally precipitated with ammonium sulphate. The material precipitated between 25 and 50% ammonium sulphate saturation, when added to freshly drawn but still unclotted blood, or native plasma, prevented its coagulation. This action could be reversed by an approximately fivefold dilution with distilled water and addition of calcium chloride and thrombin, thus excluding fibrinolysis as the cause of the anticoagulant effect. Determinations of the respective coagulation factors showed that no decrease occurred in prothrombin, factor VII, plasma thromboplastin component, and fibrinogen. On the other hand a statistically significant decrease in factor V was observed when calcium was present.

ANTICOAGULANT EFFECT OF INCUBATED FIBRINOGEN

Sterile fibrinogen rendered non-clottable by incubation was mixed with fresh plasma and the thrombin time determined. An appreciable prolongation was observed. The incubated fibrinogen was then fractionally precipitated with ammonium sulphate. The material precipitated between 25 and 50% ammonium sulphate saturation, when added to freshly drawn but still unclotted blood, or native plasma, prevented its coagulation. This action could be reversed by an approximately fivefold dilution with distilled water and addition of calcium chloride and thrombin, thus excluding fibrinolysis as the cause of the anticoagulant effect. Determinations of the respective coagulation factors showed that no decrease occurred in prothrombin, factor VII, plasma thromboplastin component, and fibrinogen. On the other hand a statistically significant decrease in factor V was observed when calcium was present.