Search PubMed⌕ Search

Biomedical subjects

D B Johnson

Publications and source records attributed to D B Johnson.

At least 73 records · Page 4Linked to original sources
PubMed1979

Glucuronidation of lipophilic substrates: preparation of 3-benzo[a]pyrenyl-beta-D-glucopyranosiduronic acid in multimilligram quantities by microsomal UDP-glucuronyl transferase.

A convenient method for the enzymic conversion of multimilligram quantities of 3-hydroxybenzo[a]pyrene to 3-benzo[a]pyrenyl-beta-D-glucopyranosiduronic acid in 90% yield is described. Commercially available freeze-dried rabbit liver microsomes were incubated in the presence of UDPGA, 3-hydroxybenzo[a]pyrene, and Triton X-100 detergent (Figure 1). The course of the biosynthetic reaction was followed by fluorimetry. The glucuronide product was extracted from the acidified incubation supernate with ethyl acetate and the acid function of the glucuronide was utilized in an acid-base extraction procedure to purify the glucuronide from biological and unreacted starting material. The glucuronide precipitated from ethyl acetate and was collected by centrifugation. High pressure liquid chromatography and spectroscopic techniques were used to verify the structure and purity of 3-benzo[a]pyrenyl-beta-D-glucopyranosiduronic acid.

Animals↗
PubMed1978

Studies on the stability of immobilized xanthine oxidase and urate oxidase.

The stability of immobilized preparations of xanthine oxidase and urate oxidase was studied, and optimized, because of the potential joint use of both enzymes in clinical analysis. Xanthine oxidase was immobilized on cellulose, Sepharose, hornblende, Enzacryl-TIO, and porous glass. Thehalf-lives of these preparations at 30 degree C ranged from 40 min to 5.0 hr. In this respect immobilized enzyme resembled soluble enzyme in dilute solution (0.11 mg/ml), when the half-live was about 3.5 hr. More concentrated enzyme solution (1 mg/ml) had a half-life of 64 hr, and was, therefore, considerably more stable than the untreated immobilized xanthine oxidase preparations. Inclusion of albumen in storage and assay buffer increased the half-life of bound xanthine oxidase. So also did treatment with glutaraldehyde: in the case of xanthine oxidase bound to Enzarcyl-TIO such treatment increased the half-life at 30 degree C from 3 hr to about 100 hr. Immobilized xanthine dehydrogenase was more stable than immobilized xanthine oxidase: the dehydrogenase lost no activity during continuous assay for 5 hr at 30 degree C. The stability of immobilized urate oxidase depended on the quantity of enzyme used and on the time of stirring during immobilization: thus a preparation was made (by stirring urate oxidase (48 mg/g support) with Enzacryl-TIO for 24 hr) which lost no activity during 350 hr at 30 degree C.

Enzymes, Immobilized↗
PubMed1978

Preparation, isolation, analysis, and characterization of 3-benzo[a]pyrenyl-beta-D-glucopyranosiduronic acid: a metabolite of 3-hydroxybenzo[a]pyrene with potentially high carcinogenic activity.

The aglycone, 3-hydroxybenzo[a]pyrene, was metabolized to 3-benzo[a]pyrenyl-beta-D-glucopyranosiduronic acid in the presence of uridine 5'-diphosphoglucuronic acid and rabbit liver microsomes. The course of the biosynthetic reaction was followed by fluorimetry and reverse-phase, paired-ion high pressure liquid chromatography (HPLC). Also, the HPLC system was used to analyze for glucuronide and 3-hydroxybenzo[a]pyrene during the isolation procedure. The existence of a glucuronide of 3-hydroxybenzo[a]pyrene was determined by radiotracer and enzymic techniques, utilizing the HPLC system. Field desorption and direct inlet mass spectral techniques were used to characterize the 3-hydroxybenzo[a]pyrene glucuronide.

Animals↗
PubMed1976

A sterospecific synthesis of 7alpha-hydroxycholesterol.

The five step synthesis of 7alpha-hydroxycholesterol utilizes the solvolysis of 7alpha-bromocholesterol benzoate with potassium acetate in acetic acid as the key step in controlling the stereospecificity of the reaction sequence. This reaction yields 7alpha-acetoxycholesterol benzoate with retention of configuration at position seven. The diester is readily reduced with lithium aluminum to 7alpha-hydroxycholesterol.

Hydroxycholesterols↗
PubMed1976

The use of [7alpha-3H]- and [7alpha, 7beta-3H]cholesterol in the enzymic assay of cholesterol 7alpha-hydroxylase.

A tritium release method is described for following the enzymic conversion of cholesterol to 7alpha-hydroxycholesterol. Incubations of rat liver subcellular preparations (containing microsomes) with [7alpha-3H]cholesterol or [7alpha,7beta-3H]cholesterol release the labeled hydrogen in the 7alpha position as 3H2O which, after counting, allows for the determination of the fraction of exogenous cholesterol converted to 7alpha-hydroxycholesterol. These findings document those recently reported by Van Cantfort, Renson, and Gielen (1975. Eur J. Biochem. 55:23). Analysis of incubation mixtures containing both [4-14C]cholesterol and either [7alpha-3H] or [7alpha,7beta-3H]cholesterol demonstrate that one atom of hydrogen (from the 7alpha position) is incorporated into H2O for every molecule of exogenous cholesterol that is converted to 7alpha-hydroxycholesterol. In the case of [7alpha-3H]cholesterol no label is retained by the product. With [7alpha,7beta-3H]cholesterol, one atom is released as 3H2O and one is retained by the product in the 7beta position. Microsomal incubations with [7alpha,7beta-3H]cholesterol were performed, followed by the acetylation of the steroid fractions with [14C]acetic anhydride. If intermixing of exogenous with endogenous cholesterol were complete during the enzymic reaction, one would expect the 3H: 14C ratio of the isolated cholesterol acetate to be four times that observed in the 7alpha-acetoxycholesterol acetate. Average values of 4.23 in one series and 4.03 in a second series indicate that intermixing was sufficiently complete to use the tritium release method as an indicator of mass conversion.

Animals↗