Shigeo Horie

Sensitive assay of cytochrome P450scc activity by high-performance liquid chromatography.

We have developed a simple procedure for analyzing the reaction intermediates and product of the cholesterol side-chain cleavage system by high-performance liquid chromatography with uv absorption monitoring. After the cholesterol side-chain cleavage system had been incubated and the reaction then halted by heat treatment, the product was converted into 3-one-4-en steroid showing intense absorption at 240 nm upon reaction with cholesterol oxidase. The converted steroids were then analyzed by normal-phase HPLC. In consequence, the catalytic activity of the reconstituted adrenocortical cytochrome P450scc system was readily assayed with a sensitivity more than 10-fold higher by this conversion. Also, it was shown that 22R-hydroxy-cholest-4-en-3-one could serve as a good substrate for cytochrome P450scc and that the 20R,22R-dihydroxy derivative could be clearly detected as a reaction intermediate in the reconstituted system.

Purification and properties of cytochrome P-450 (SCC) from pig testis mitochondria

Cytochrome P-450 was purified from pig testis mitochondria to a specific content of 13.1 n mol/mg of protein. The purified preparation was found to contain a single species of P-450, on sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular weight of about 53000 +/- 2000. The cholesterol side chain-cleavage system could be reconstituted by mixing the purified cytochrome P-450, adrenodoxin reductase, adrenodoxin, cholesterol and NADPH. The rate of conversion of cholesterol to pregnenolone was 6.2 n mol/min/n mol of P-450 under the conditions employed. The absorption spectrum of the oxidized cytochrome P-450 had maxima at 416, 530 and 568 nm. The reduced CO-complex of the cytochrome P-450 exhibited an absorption maximum at 448 nm. The purified P-450 was subjected to microsequence analysis and its NH2-terminal amino acid sequence was found to show considerable homology with that of bovine adrenal P-450 (SCC).

Purification of NADPH-cytochrome c reductate from swine testis microsomes by chromatofocusing and characterization of the purified reductase

A purified NADPH-cytochrome c reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was prepared from swine testis microsomes by detergent solubilization followed by a procedure including chromatofocusing. The reductase was eluted at an isoelectric point of 4.8 from the chromatofocusing column. 730-fold purification was achieved with an overall yield of 1.2%. The preparation was found to be homogeneous upon polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate (SDS). Upon SDS-polyacrylamide gel electrophoresis, however, the purified preparation resolved into one major band (Mr 78 000) and two minor bands (Mr 60 000 and 15 000). The enzyme contained about 1 mol each of FMN and FAD, which were both extractable with trichloroacetic acid and also boiling water. The oxidized form of the enzyme showed the absorption spectrum of a typical flavoprotein. Aerobic reduction with NADPH resulted in conversion of the spectrum into one of an air-stable semiquinone form. The activity of the purified preparation was 26 mumol cytochrome c reduced/min per mg protein under the standard assay conditions at 22 degrees C. The enzyme catalyzed the reaction through a ping-pong mechanism.

Mouse strain variations in the magnitude of induction of liver DT-diaphorase and hereditary transmission of the trait

1. Strain variations among mice in terms of cytosolic DT-diaphorase activity were studied in liver, kidney, stomach and heart tissues with or without the administration of 3-tert-butyl-4-hydroxyanisole (BHA). 2. BHA induced DT-diaphorase activity in all strains examined, and the magnitude of induction varied depending on the strain and tissue. Among the 10 inbred strains tested, BALB/c and C57BL mice showed relatively large magnitudes of induction for liver DT-diaphorase, whereas C3H and CBA mice showed relatively small magnitudes. 3. Results of examinations of BALB/c-C3H-F1, -F2 and C57BL-CBA-F1 mice revealed that smaller magnitudes of induction of liver DT-diaphorase were inherited essentially as a dominant trait. The hereditary trait could be adequately explained by postulating two gene loci that regulate the magnitude of induction. 4. The possible significance of DT-diaphorase activity in chemical carcinogenesis was discussed.

Properties of high spin type P-450 preparations from bovine adrenal cortex mitochondria

Abstract A high spin type P-450 preparation (Fraction H) was prepared from bovine adrenocortical mitochondria by extraction with cholate followed by fractionation with ammonium sulfate. Several sub-fractions were further obtained from Fraction H by chromatography and gel filtration. The ratio of heme/protein (nmol/mg) of Fraction H was 4–5 and that of the purest sub-fraction (Fraction A3B) was 12–13. The ratio of A28nm/A393nm of Fraction H was about 3 and that of Fraction A3B was 1–2. All sub-fractions were active for side chain cleavage of cholesterol and (20S)-20-hydroxycholesterol (20α-hydroxycholesterol), but the apparent activity decreased as the purification proceeded in spite of the fact that there was no sign of denaturation of P-450. The activity varied depending on the kind of medium in which steroid substrates were dissolved. The molar ratio of bound cholesterol/heme approached 0.6-0.8 with purification and a small amount of 20α-hydroxycholesterol was also found in the preparations. The bound cholesterol could be metabolized to pregnenolone with a concomitant change of P-450 to a low spin state, and pregnenolone was still bound to P-450 even after re-fractionation. The addition of cholesterol to the low spin form caused a change to a high spin state. On the basis of these observations, the nature of high spin P-450 and the possible requirement of a steroid carrier factor or a structural factor for effective hydroxylation are discussed.

Rat strain variations in liver cytosolic DT-diaphorase activity and possible significance of the trait in carcinogenesis by azo dyes

1. Strain variations among female rats in terms of cytosolic DT-diaphorase activity were studied in liver, heart and glandular stomach tissues with or without administration of 3-tert-butyl-4-hydroxyanisole (BHA). 2. BHA induced liver DT-diaphorase activity in all strains examined, and both the basal and induced activities varied according to strain. Among the five strains tested, Brown Norway (BN) and Sprague-Dawley (SD) rats showed relatively high levels of enzyme activity in the liver, whereas Fischer (F344) rats showed a relatively low level of activity. Results of examination of Fischer-BN-F1 rats indicated that a lower level of liver DT-diaphorase activity was inherited essentially as a dominant trait. 3. Liver DT-diaphorase activity in male rats was significantly lower than in female rats. Small strain variations of the activity, if any, were observed in the heart and stomach cytosolic fractions with or without induction by BHA. The magnitude of induction by BHA was also small, if any, in heart and stomach cytosolic fractions. 4. From these and other observations, we discussed the differences between rats and mice in these strain and tissue variations of DT-diaphorase activity, and also the possible significance of liver DT-diaphorase activity in carcinogenesis by azo dyes.

Cytochrome P-450scc-catalyzed production of progesterone from 22R-hydroxycholest-4-en-3-one by way of 20,22-dihydroxycholest-4-en-3-one

Transient accumulation of a dihydroxylated steroid was found when 22R-hydroxycholest-4-en-3-one was used as the substrate for a reconstituted cholesterol side-chain cleavage system derived from bovine adrenocortical mitochondria. The indications were that the accumulated steroid was an intermediate in the cytochrome P-450scc-catalyzed reaction. The retention time of the accumulated intermediate was identical with that of authentic 20,22-dihydroxycholest-4-en-3-one on HPLC. When 22R-hydroxycholesterol and 22R-hydroxycholest-4-en-3-one were incubated simultaneously, the total amount of reaction products was essentially the same as that observed with 22R-hydroxycholest-4-en-3-one alone. Under the conditions employed, the apparent turnover number of cytochrome P-450scc for 22R-hydroxycholesterol was calculated to be 77 nmol/min/nmol P-450 from the amount of pregnenolone formed, whereas the apparent turnover number for 22R-hydroxycholest-4-en-3-one was 64 nmol/min/nmol P-450 with respect to the intermediate formation and 77 nmol/min/nmol P-450 with respect to the progesterone formation. The apparent turnover number for 20,22-dihydroxycholest-4-en-3-one was about 125 nmol/min/nmol P-450, which was not significantly different from that of 20,22-dihydroxycholesterol. The apparent Km for 22R-hydroxycholesterol was about 20 microM and those for 22R-hydroxycholest-4-en-3-one and 20,22-dihydroxycholest-4-en-3-one were 50 and 40 microM, respectively. Thus, 22R-hydroxycholest-4-en-3-one was efficiently metabolized to progesterone by way of 20,22-dihydroxycholest-4-en-3-one by cytochrome P-450scc.

Mouse and rat strain variations in sensitivity to N-nitroso-diethylamine, hereditary transmission of the trait and the effect of 3-tert-butyl-4-hydroxyanisole on sensitivity

1. Strain variations among male mice were studied in terms of the number of days of survival with chronic administration of N-nitroso-diethylamine (NDEA). Four inbred strains, two F1 progenies and one F2 progeny were tested. 2. BALB/c mice survived for the longest period, whereas C3H mice survived for the shortest time. Results of examinations of BALB/c-C3H-F1, -F2 and C57BL-CBA-F1 mice revealed that the hereditary trait could be adequately explained by postulating two loci of genes or gene clusters that regulate the sensitivity to NDEA. 3. Simultaneous chronic administration of 3-tert-butyl-4-hydroxyanisole (BHA) could prolong the survival period. 4. Preliminary histopathological examinations of the liver tissues revealed that the lesion at the time of death of the mice varied considerably depending on the strain and the length of survival. Evidence for hereditary transmission of the characteristics of histopathological changes, including development of liver hemangiosarcoma, is presented. 5. The strain variations among male and female rats were also studied in terms of the number of days of survival with chronic administration of NDEA. Five strains and one F1 progeny were tested. 6. From these and previous observations, the possible biochemical factors determining sensitivity to NDEA were discussed.

Molecular characteristics of many hemoproteins: A survey of molecular weights, sedimentation coefficients, other molecular parameters and amino acid compositions*

Data on molecular weights, sedimentation coefficients, other molecular parameters and amino acids compositions of many hemoproteins were collected from the literature and studied. The results of the survey gave a general view of the molecular characteristics of hemoproteins and also revealed the presence of various statistical correlations among the molecular parameters and amino acid compositions. Some of the correlations were found to be practically useful for the estimation of number of heme per molecule, molecular weight or partial specific volume. Discussions were made on the possible structural basis of the molecular characteristics of hemoproteins.

PROPERTIES OF HIGH SPIN TYPE P-450 PREPARATIONS FROM BOVINE ADRENAL CORTEX MITOCHONDRIA

A high spin type P-450 preparation (Fraction H) was prepared from bovine adrenocortical mitochondria by extraction with cholate followed by fractionation with ammonium sulfate. Several sub-fractions were further obtained from Fraction H by chromatography and gel filtration. The ratio of heme/protein (nmol/mg) of Fraction H was 4–5 and that of the purest sub-fraction (Fraction A3B) was 12–13. The ratio of A280 nm/A393 nm of Fraction H was about 3 and that of Fraction A3B was 1·2. All sub-fractions were active for side chain cleavage of cholesterol and (20S)-20-hydroxycholesterol (20α-hydroxycholesterol), but the apparent activity decreased as the purification proceeded in spite of the fact that there was no sign of denaturation of P-450. The activity varied depending on the kind of medium in which steroid substrates were dissolved. The molar ratio of bound cholesterol/heme approached 0·6–0·8 with purification and a small amount of 20α-hydroxycholesterol was also found in the preparations. The bound cholesterol could be metabolized to pregnenolone with a concomitant change of P-450 to a low spin state, and pregnenolone was still bound to P-450 even after re-fractionation. The addition of cholesterol to the low spin form caused a change to a high spin state. On the basis of these observations, the nature of high spin P-450 and the possible requirement of a steroid carrier factor or a structural factor for effective hydroxylation are discussed.