The kinetics of 3a-hydroxysteroid : NAD oxidoreductase (EC 1.1.1.50) from Pseudomonas testosteroni (ATCC 11 996) have been investigated. The kinetic analysis based on initial activity measurements and product inhibition studies, indicates that the addition of substrate to the enzyme and the release of products from it, follows an obligatory order (ordered bi bi mechanism). The ability of the enzyme to utilize the thionicotinamide analogue of NAD (sNAD) as cofactor has been investigated using various 3a-hydroxysteroids from both the C19, CZl and CZ4 series. The results show that the reaction velocity with sNAD as the cofactor is generally lower than with NAD. The decrease, however, varies considerably, being negligible with some steroids such as litocholic acid and deoxycholic acid and very pronounced with other such as tetrahydrocortisol and tetrahydrocortisone. The introduction of an 11P-hydroxy or an 1 l-0x0 group into the steroid molecule significantly reduces the ability of the enzyme to attack the 3a-hydroxy group. No such effect could be seen when the ll-hydroxy group was in the a-position. The results also indicate that, whereas NAD can serve as cofactor for both the monomeric and the dimeric forms of the enzyme, sNAD only acts as cofactor for the monomeric form. Thus sNAD is a valuable tool for the study of the reversible, concentration-dependent monomeric-dimeric transition of the 3a-hydroxysteroid deh ydrogenase.The NAD-linked, steroid-induced 3a-hydroxysteroid dehydrogenase from Pseudomonas testosteroni reversibly oxidizes 3a-hydroxysteroids from the CI9, CZ1 and CZ4 series [1,2]. The enzyme has been purified and studied by several authors [3 -61. 3a-Hydroxysteroid dehydrogenase is of considerable practical importance for the quantitisation of bile acids in Abbreviation.sNAD, thionicotinamide-adenine dinucleotide.Trivial names. Androsterone, 3u-hydroxy-Su-androstane-17-one; 1 1/?-hydroxyandrosterone, 3u,ll/l-dihydroxy-Su-androstane-17-one; 1 lu-hydroxyandrosterone, 3u,1 la-dihydroxy-5a-androstane-17-one; 1 I-oxoandrosterone, 3u-hydroxyxy-Sa-androstane-ll,17-dione ; etiocholanolone, 3a-hydroxy-S/?-androstane-17-one; tetrahydrocortisone, 3u,17u,21-trihydroxy-5~-pregnane-11,20-dione ; tetrahydrocortisol, 3a,-11 ~,17~,21-tetrahydroxy-5~-pregnane-2O-one; pregnandiol, 3~,20fl-dihydroxy-5fl-pregnane; litocholate, 3a-hydroxy-Sflcholanic acid; deoxycholate, 3u,l2u-dihydroxy-5/?-cholanic acid; chendeoxycholate, 3~,7u-dihydroxy-5~-cholanic acid; cholate, 3u,7u, 12u-trihydroxy-5~-cholanic acid.Enzyme. 3a-Hydroxysteroid dehydrogenase or 3a-hydroxysteroid : NAD oxidoreductase (EC 1
SummaryThe ADP-induced platelet adhesiveness has been investigated under experimental various conditions by the in vitro method of Hellem. The platelet, adhesiveness was found to be proportional to the logarithm of the ADP concentrations between 0.025—0.2 μg/ml in citrated and heparinized plasma. The effect of ADP on platelets decreased with increasing concentrations of citrate. In contrast, increasing amounts of heparin did not alter the platelet adhesiveness significantly, except when extremely high concentrations were used. Variations in the temperature highly influenced the result of the estimations when suboptimal concentrations of ADP were used. The reason for this probably inactivation of ADP by plasma enzymes, which have an optimal activity at about 37° C. This may explain why estimation at 20° C gives higher values of the platelet adhesiveness than at 37° C. If too high concentrations of ADP are used, this effect is not detectable. This may be the reason why O’Brien (1962) did not find any inactivating property of platelet poor plasma. Washing of platelets in buffered saline leads to a marked release of ADP, indicating that washed platelets are damaged. Repeated resuspension of platelets even in their own plasma gives release of ADP. When studying the mechanism of the ADP-induced reaction all the above variations must be taken into account.
SummaryIn a group of 25 patients with insulin-treated diabetes mellitus a marked increase in the ADP-induced platelet adhesiveness was demonstrated. This phenomenon was due to a plasmatic factor. In vitro-plasma from these patients restored the decreased ADP-induced platelet adhesiveness in von Willebrand’s disease.The factor was also effective in vivo, since transfusion of 450 ml diabetic plasma to a patient with von Willebrand’s disease normalized the decreased platelet adhesiveness and shortened the prolonged bleeding time. The role of this factor in the ADP platelet reaction as a cofactor together with calcium is stressed.
A crude 7alpha-hydroxysteroid dehydrogenase isolate from P. testosteroni requires NADP specifically and provides a simple and specific means for the determination of 7alpha-hydroxy bile acids and their conjugates. A specific and reliable enzymatic-fluorimetric method for the quantitative measurement of the primary bile acids in serum is described. Overall recovery from normal and jaundiced serum averaged 85%. The mean concentration of serum 7alpha-hydroxy bile acids in fasting, healthy subjects was 2.5 mumoles/1 (S.D. 1.45 AND RANGE 1.2-6.3), averaging 73% (S.D. 11.5) of the total, measured as the 3alpha-hydroxy bile acid concentration. In parenchymatous liver disease and cholestatic hepatobiliary disorders, the proportion of 7alpha-hydroxy bile acids was significantly increased, mean 92.5% (S.D. 6.6)(p less than 0.001).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.