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I A MacDonald

Publications and source records attributed to I A MacDonald.

68 records · Page 4Linked to original sources
PubMed1979

Bile salt 3 alpha- and 12 alpha-hydroxysteroid dehydrogenases from Eubacterium lentum and related organisms.

Thirty-two strains of Eubacterium lentum and phenotypically similar anaerobic gram-positive bacilli were screened for intracellular bile salt 3alpha- and 12alpha-hydroxysteroid dehydrogenase (HSDHase) activities. These organisms were categorized into four groups: (A) those containing 12alpha-HSDHase only (10 strains), (B) those containing 3alpha- and 12alpha-HSDHase (13 strains), (C) those containing 3alpha-HSDHase only (2 strains), and (D) those devoid of any measurable HSDHase activity (7 strains). Of the respective four groups, 9/10, 13/13, 0/2, and 0/7 were like the neotype strain of E. lentum (ATCC 25559) in that they produced H(2)S in a triple sugar iron agar butt, reduced nitrate to nitrite, and weakly decomposed hydrogen peroxide. The other strains were variable for nitrate reduction and activity on hydrogen peroxide, but all the organisms in the first three categories (with one exception) were H(2)S producers (triple sugar iron agar butt) and all (with one exception) were designated E. lentum, whereas the organisms of category B were non-H(2)S producers (triple sugar iron agar butt). Five of these seven were not stimulated by arginine and are designated "phenotypically similar organisms." Thin-layer chromatography of extracted spent bacterial medium of four representative strains from each group grown in the presence of cholate revealed the presence of (A) 12-oxo product, (B) 12-oxo and 3-oxo products, (C) 3-oxo product, and (D) the absence of any of these products. The 12alpha-HSDHase of category B organisms was unstable unless 10(-3) M dithioerythritol was added to the buffer. With the exception of 3 out of 32 strains, there was a positive correlation between the production of measurable amounts of 12alpha-HSDHase and H(2)S production. Growth curves and the effect of arginine on growth and the production of 3alpha- and 12alpha-HSDHase were examined in representative strains of categories A, B, and C. Both enzymes were shown to bind onto a nicotinamide adenine dinucleotide-Sepharose column and could be eluted by high-ionic-strength buffer, resulting in approximately 25-fold and 18-fold purification, respectively. Molecular weight estimations by Sephadex G-200 gave values of 205,000 and 125,000 for the 3alpha- and 12alpha-HSDHase, respectively. Purified 12alpha-HSDHase was investigated with respect to pH requirement, substrate specificity, and enzyme kinetics.

3-Hydroxysteroid Dehydrogenases↗
PubMed1977

NAD-dependent 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Eubacterium lentum ATCC no. 25559.

Eubacterium lentum (ATCC No. 25559) was shown to contain 3alpha-and 12alpha-hydroxysteroid dehydrogenases both of which were NAD-dependent and active against conjugated and unconjugated bile salts. In addition, the 3alpha-hydroxysteroid dehydrogenase was active against members of the Androstan series containing a 3alpha-hydroxyl group regardless of the stereo-orientation of the 5-H-. No measurable activity against 7alpha-, 7beta-, 11beta-, or 17beta-hydroxyl groups was demonstrated. The growth of E. lentum and the production of 3alpha- and 12alpha-hydroxysteroid dehydrogenases were greatly enhanced by the addition of L-, D- or DL-arginine to the medium. Yields of hydroxysteroid dehydrogenase were optimal in the range of 0.50-0.75% arginine; however, the growth of the organisms was further enhanced at arginine concentrations greater than 0.75%. The 12alpha-hydroxysteroid dehydrogenase was heat labile and could be selectively inactivated by heating at 50 degrees C for 45 min. Both the heated enzyme preparation (containing only 3alpha-hydroxysteroid dehydrogenase) and the unheated enzyme preparation (containing 3alpha- and 12alpha-hydroxysteroid dehydrogenases) were useful in the spectrophotometric quantification of bile salts. The optimal pH values for 3alpha- and 12alpha-hydroxysteroid dehydrogenases were 11.3 and 10.2, respectively. Kinetic studies have Km estimates of 2.10(-5) M and 1.0.10(-4) M with 3alpha,7alpha-dihydroxy-5beta-cholanoyl glycine and 7alpha,12alpha-dihydroxy-5beta-cholanoate for the two respective enzymes.

3-Hydroxysteroid Dehydrogenases↗
PubMed1977

Increased lithogenicity of bile on fasting in normal subjects.

The effect of fasting on bile lithogenicity was studied in 19 normal men and 22 normal women. The molar percentages of bile acid, phospholipid, and cholesterol, determined after random overnight fasts of 9, 12, and 16 hr, were plotted on triangular coordinates. The molar percentage of cholesterol increased in both men and women. Analysis using Admirand and Small's criteria for cholesterol saturation revealed that 4.50% of normal women were lithogenic at 9.1 hr and 54.5% at 16.5 hr (P less than 0.005). A similar trend in men was not significant. The mean values for both sexes were lithogenic at 16 hr only. Lithogenic bile was present in 4 men and 1 woman at 9 hr fasting and became more lighogenic with longer fasting. Analysis using the criteria of Hegardt and Dam revealed an increased proportion of both sexes moving into the metastable-labile and supersaturated zones on fasting, again with significant changes for women (P less than 0.01). The duration of fasting is important in interpreting the presence of lithogenic bile; although more pronounced in women, both sexes showed increased cholesterol saturation in bile with fasting.

Adult↗
PubMed1976

Enzymic analysis of bile acids.

1. The use of enzymes and thin-layer chromatography (TLC) in quantifying individual bile acids from bile or serum is reviewed. 2. Both 3alpha-hydroxysteroid dehydrogenase (commercially available P. testosteroni enzyme) and 7alpha-hydroxysteroid dehydrogenase (E. coli) can be used for spectrophotometric or fluorometric measurement of individual bile acids in TLC eluates and to estimate pool size and half-life of serially labelled samples.

Bile Acids and Salts↗
PubMed1975

Differentiation of fluorides-stimulated and non-fluoride-stimulated components of beef brain cortex adenylate cyclase cy calcium ions, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid and Triton X-100.

Beef brain cortex adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity is 84--88% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the absence of F- but only 50--60% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the presence of F-. In either case, further increase in EGTA concentration did not alter the degree of inhibition. The inhibition can be completely reversed in both cases by addition of 3 - 10(-5) M Ca2+, (yielding a [free Ca2+] of approximately 2 - 10(-6) M) and 5 - 10(-5) M Mn2+ or Co2+ and partially by 5 - 10(-5) M Sr2+ but not by addition of 5 - 10(-5) M Ba2+, Zn2+, Ni2+ or Fe2+. A [free Ca2+] of 7.2 - 10(-5) M markedly inhibited cyclase activity in the presence of F-. Solubilization by 1.8% Triton X-100 resulted in an enzyme preparation no longer stimulated by NaF and 100% inhibited by the addition of 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid either in the absence or presence of NaF. However, in contrast to ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-TETRAACETIC ACID, EDTA had no measurable effect on adenylate cyclase either in the presence or absence of NaF and ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid did not affect ATPase or phosphodiesterase activities. The data is rationalized by the postulation of two independent enzyme components in brain cortex: one component is about six-fold activated by NaF and the NaF effect is enhanced by low concentrations of Ca2+ and Mg2+. A second component is totally Ca2+ dependent and inhibited by high concentrations of F-. Mn2+, Co2+ and Sr2+ appear to be in vitro Ca2+ substitutes for both enzyme systems. On this basis, Triton X-100 treatment results in about a three-fold increase in specific activity of the Ca2+ dependent cyclase component but a complete abolition of the NaF stimulated component.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Convenient non-chromatographic assays for the microbial deconjugation and 7alpha-OH bioconversion of taurocholate.

We described two convenient assay methods to estimate bile acid deconjugation and bile acid bioconversion at the 7alpha-OH position by individual microorganisms grown in media containing taurocholic acid. The methods are based on (i) a selective chemical assay for taurine conjugates previously described and (ii) the use of a cell-free preparation of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli to directly quantify 7alpha-OH groups. These non-chromatographic approaches have been applied to the study of three model strains of intestinal organisms, E. coli, Bacteroides fragilis, and Clostridium perfringens, grown in standard media in the presence of purified tritiated taurocholate. Assay results were confirmed by thin-layer chromatography solvent systems designed to separate conjugated from unconjugated bile acid and unmodified cholic acid nucleus from 7alpha-OH bioconversion product(s) (primarily 3alpha, 12alpha dihydroxy, 7-keto-cholanoic acid). In addition, 7alpha-hydroxysteroid dehydrogenase activity was demonstrated in cell-free extracts of all three organisms. Of the three organisms, only C. perfringens was demonstrated to (i) deconjugate taurocholic acid, (ii) contain 3alpha-hydroxysteroid dehydrogenase activity, (iii) convert cholic acid into at least five labeled metabolites visible on thin-layer chromatography, and (iv) catalyze significant tritium exchange with water in the medium.

Bacteroides fragilis↗