Incorportion of oxygen-18 into the 25-position of cholecalciferol by hepatic cholecalciferol 25-hydroxylase.

The oxygen enzymically inserted as a hydroxy function by rat liver post-mitochondrial fraction into the 25-position of cholecalciferol to giver 25-hydroxycholecaliferol is derived exclusively from molecular O2. Therefore like the other two cholecalciferol hydroxylases, i.e. 25-hydroxycholecalciferol 1alpha-hydroxylase and 25-hydroxycholecalciferol 24-hydroxylase, the cholecalciferol 25-hydroxylase is also a mono-oxygenase ('mixed-function oxidase').     

Effect of oestrogen and 1,25-dihydroxycholecalciferol on 25-hydroxycholecalciferol metabolism in primary chick kidney-cell cultures.

Primary cultures of chick kidney cells convert 25-hydroxycholecalciferol into more-polar metabolites. Cells from vitamin D-deficient chicks have high 25-hydroxycholecalciferol 1 alpha-hydroxylase (1 alpha-hydroxylase) activity, but no 25-hydroxycholecalciferol 24-hydroxylase (24-hydroxylase) activity. Physiological concentrations of 1,25-dihydroxycholeclaciferol suppress 1 alpha-hydroxylase and induce 24-hydroxylase activity. The inhibition of 1 alpha-hydroxylase preceded the induction of 24-hydroxylase. In contrast, oestradiol-17 beta had no effect on the activity of either hydroxylase under a variety of experimental conditions. These results clearly demonstrate that 1,25-dihydroxycholecalciferol, but not oestrogen, acts directly on the kidney cells to regulate the metabolism of 25-hydroxycholecalciferol.     

Separation of the primary dehydrogenase from the cytochromes of the nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium

A selective extraction procedure was developed for sequentially extracting a fraction containing the primary dehydrogenase and a fraction containing the cytochromes of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase of Bacillus megaterium KM membranes. The primary dehydrogenase (NADH-2,6-dichlorophenolindophenol oxidoreductase) activity was extracted from sonically treated membranes with 0.4% sodium deoxycholate for 30 min at 4 C. The insoluble residue was extracted with 0.4% sodium deoxycholate in 1 m KCl for 30 min at 25 C. A combination of the two extracts and dilution in Mg(2+) gave good recovery of the original membrane NADH oxidase activity. The primary dehydrogenase fraction contained 41% of the membrane protein, no cytochromes, flavine adenine dinucleotide as the sole acid-extractable flavine, and most of the membrane ribonucleic acid (RNA). The cytochrome-containing fraction had 16% of the membrane protein, 61% of the membrane cytochrome with the same relative amounts of cytochromes a and b as the original membrane, no acid-extractable flavine, little RNA, and no oxidoreductase activity. The oxidoreductase fraction remained soluble after removal of deoxycholate whereas the cytochrome fraction became insoluble after removal of deoxycholate-KCl, but the precipitated fraction could be redissolved in 0.4% sodium deoxycholate. Treatment of both fractions with ribonuclease to destroy all of the RNA present did not affect the ability of the fractions to recombine into a functional oxidase unit. Treatment of either fraction with phospholipase A prevented restoration of a functional oxidase when the oxidoreductase and cytochrome fractions were treated in solution, but no affect on restoration of oxidase was observed when the phospholipase A treatment was carried out with the soluble oxidoreductase fraction and the insoluble cytochrome fraction.     

Preparation of chick brain synaptosomes and synaptosomal membranes.

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome c: oxygen oxidoreductase (EC 1.9.3.)), monoamine: oxygen oxidoreductase (deaminating) EC 1.4.3.4), rotenone-insensitive NADH: cytochrome c oxidoreductase (EC 1.6.99.3), NADPH: cytochrome c oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, (Na+ -K+)-activated ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

Purification and resolution of NADH diaphorase activity from NADPH diaphorase-linked: O2 oxidoreductase activity of human neutrophils

Intrinsic NADPH diaphorase activity is a component of the membrane-bound NAD(P)H:O2 oxidoreductase of human neutrophils. NADH-specific diaphorase activity is also present in membrane fractions rich in oxidoreductase activity. Studies were undertaken to determine whether the NADH diaphorase might also be intrinsic to the oxidoreductase. The latter diaphorase was freed from the membrane by detergent extraction and partially purified approximately 80-fold. Its apparent molecular weight following solubilization in deoxycholate and Tween-20 was 204 000 +/- 10 000. The specific activity of the partially purified diaphorase with ferricyanide as electron acceptor was 7.6 X 10(3) mU/mg protein, its pH optimum was 7.0, and its Km for NADH was 13 microM. It is completely devoid of NADPH diaphorase activity, lacks the capacity to reduce molecular oxygen, yet readily reduces ferricyanide, 2,6-dichlorophenolindophenol and ferricytochrome c. Whereas the NADH diaphorase was freed from the particulate fraction of cell lysates by extraction in 10 mM Tris-HCl buffer (pH 8.6) made up in 15% glycerol and 0.5% Tween-20, NADPH-dependent diaphorase and superoxide-generating activities also present in the membrane were not. These observations make it unlikely that the principal membrane-bound NADH diaphorase found in human neutrophils is a component of the NAD(P)H:O2 oxidoreductase, despite its common association in the same particulate fraction of cell lysates.     

Characteristics of the rat liver microsomal enzyme system converting cholecalciferol into 25-hydroxycholecalciferol. Evidence for the participation of cytochrome p-450.

Properties of the rat hepatic cholecalciferol 25-hydroxylase have been studied. An assay system has been developed in which 25-hydroxycholecalciferol production is linear for at least 2h in both homogenates and microsomal fraction. Furthermore, the initial reaction velocity is linearly related to the amount of liver tissue or microsomal fraction. This enzyme system also metabolizes an analogue of cholecalciferol, namely dihydrotachysterol 3, into 25-hydroxydihydrotachysterol 3. The 25-hydroxylase is in the microsomal fraction and not in mitochondria. It has a Km of 44 nM for cholecalciferol and 360 nM for dihydrotachysterol 3. Its activity is not altered by dietary concentrations of calcium and phosphorus. Vitamin D-deficient rats have higher activities of the hepatic 25-hydroxylase than those receiving 25 ng of cholecalciferol daily. The 25-hydroxylase is inhibited by metyrapone. An atmosphere of CO/O2 (9:1, v/v) inhibits the reaction by 87%. This inhibition is partially reversed by white light. Additionally, cholecalciferol and 25-hydroxycholecalciferol competitively inhibit aminopyrine demethylase. These results support the idea that the cholecalciferol 25-hydroxylase is a cytochrome P-450-dependent mono-oxygenase.     

Oxygen toxicity in Astasia

1. Exposure of Astasia longa to oxygen+carbon dioxide (95:5) at atmospheric pressure leads to an inhibition of growth rate and of respiration. Growth resumes at the normal rate as soon as the oxygenation is discontinued, but respiration recovers more slowly. 2. Mitochondria prepared from cells exposed to oxygen+carbon dioxide (95:5) during growth have considerably decreased activities of succinate-cytochrome c oxidoreductase, NADH-cytochrome c oxidoreductase, succinate dehydrogenase and succinate oxidase activities as compared with mitochondria obtained from cells exposed to air+carbon dioxide (95:5). Cytochrome oxidase activity is not appreciably inhibited by exposure of the cells to 95% oxygen. 3. The mitochondrial fraction of Astasia contains rhodoquinone. The rhodoquinone concentration increases in cells exposed to 95% oxygen. The content of ergosterol-containing compounds also increases in the mitochondria of cells exposed to 95% oxygen. There is little change in the ubiquinone content of the mitochondrial fraction. The ubiquinone of Astasia appears to be ubiquinone-45.     

Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. II. Kinetics of reoxidation of the reduced enzyme

The pre-steady-state kinetics of reoxidation of NADH:Q oxidoreductase present in submitochondrial particles has been studied by the freeze-quench method. It was found that at pH 8 only 50% of the Fe-S clusters 2 and 4 and 75% of the clusters 3 were rapidly reoxidised after transient and complete reduction by a pulse of NADH in the presence of excess NADPH. Thus, NADPH keeps 50% of the clusters 2 and 4 and 25% of the clusters 3 permanently reduced at this pH. Since NADH oxidation is nearly optimal at this pH, whereas NADPH oxidation is virtually absent, it was concluded that these permanently reduced clusters were not involved in the NADH oxidation activity. Incomplete reoxidation of the clusters 2, 3 and 4 after a pulse of NADH was also found in the absence of NADPH, both at pH 6.5 and at pH 8. A pulse of NADPH given at pH 6.5, where NADPH oxidation by oxygen is nearly optimal, caused a slow reduction of 50% of clusters 2 and 4 and 30% of the clusters 3, which persisted for a period of at least 15 s. It was concluded that these clusters were not involved in the oxidation of NADPH by oxygen, as catalysed by the particles. As a working hypothesis a dimeric model for NAD(P)H:Q oxidoreductase is proposed, consisting of two different protomers. One of the protomers, containing FMN and the Fe-S clusters 1-4 in stoichiometric amounts, only reacts with NADH, and its oxidation by ubiquinone is rapid at pH but slow at pH 6.5. The other protomer, containing FMN and the clusters 2, 3 and 4, reacts with both NADH and NADPH and has a pH optimum at 6-6.5 for the reaction with ubiquinone.     

Evidence for two independent pathways of electron transfer in mitochondrial NADH:Q oxidoreductase. II. Kinetics of reoxidation of the reduced enzyme

The pre-steady-state kinetics of reoxidation of NADH:Q oxidoreductase present in submitochondrial particles has been studied by the freeze-quench method. It was found that at pH 8 only 50% of the Fe-S clusters 2 and 4 and 75% of the clusters 3 were rapidly reoxidised after transient and complete reduction by a pulse of NADH in the presence of excess NADPH. Thus, NADPH keeps 50% of the clusters 2 and 4 and 25% of the clusters 3 permanently reduced at this pH. Since NADH oxidation is nearly optimal at this pH, whereas NADPH oxidation is virtually absent, it was concluded that these permanently reduced clusters were not involved in the NADH oxidation activity. Incomplete reoxidation of the clusters 2, 3 and 4 after a pulse of NADH was also found in the absence of NADPH, both at pH 6.5 and at pH 8. A pulse of NADPH given at pH 6.5, where NADPH oxidation by oxygen is nearly optimal, caused a slow reduction of 50% of clusters 2 and 4 and 30% of the clusters 3, which persisted for a period of at least 15 s. It was concluded that these clusters were not involved in the oxidation of NADPH by oxygen, as catalysed by the particles. As a working hypothesis a dimeric model for NAD(P)H:Q oxidoreductase is proposed, consisting of two different protomers. One of the protomers, containing FMN and the Fe-S clusters 1–4 in stoichiometric amounts, only reacts with NADH, and its oxidation by ubiquinone is rapid at pH but slow at pH 6.5. The other protomer, containing FMN and the clusters 2, 3 and 4, reacts with both NADH and NADPH and has a pH optimum at 6–6.5 for the reaction with ubiquinone.     

Preparation of chick brain synaptosomes and synaptosomal membranes

A method is described for the preparation of synaptosomes and synaptosomal membranes from chicken brain. Procedures for isolating rat synaptosomal membranes could not be used directly; several modifications of existing procedures are reported. Purity of the subcellular and subsynaptosomal fractions was monitored by electron microscopy and measurements of ferrocytochrome $\text{c}$: oxygen oxidoreductase (EC 1.9.3.1.), monoamine: oxygen oxidoreductase (deaminating) (EC 1.4.3.4), rotenoneinsensitive NADH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.3), NADPH: cytochrome $\text{c}$ oxidoreductase (EC 1.6.99.1), orthophosphoric monoester phosphohydrolase (EC 3.1.3.2), ATP phosphohydrolase (EC 3.6.1.4), and levels of RNA. Microsomes are the main contaminant of the synaptosomal membrane fraction. Mitochondrial and lysosomal enzymes occur in lesser amounts. No myelin contamination was observed. Marker enzymes for contaminants suggest that these synaptosomal membranes are as pure as membranes described by others, and the specific activity of a neuronal membrane marker, $\text{(}\text{Na}{}^{\mathrm{+}}\text{?}\text{K}{}^{\mathrm{+}}\text{)-}\text{activated}$ ATPase, is as high as other preparations. Levels of this enzyme in the membrane fraction are enriched 13-fold over homogenate ATPase levels.     

Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7alpha-hydroxylase in the rat

Specific activities of the hepatic microsomal enzymes 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase and cholesterol 7alpha-hydroxylase were studied in rats fed sterols and bile acids. The administration of bile acids (taurocholate, taurodeoxycholate, taurochenodeoxycholate) at a level of 1% of the diet for 1 wk reduced the activity of HMG CoA reductase. Taurocholate and taurodeoxycholate, but not taurochenodeoxycholate, inhibited cholesterol 7alpha-hydroxylase. Dietary sitosterol produced increases in the specific activity of HMG CoA reductase (3.6-fold) and cholesterol 7alpha-hydroxylase (1.4-fold), and biliary cholesterol concentrations in this group more than doubled. Compared with controls fed the stock diet, the simultaneous administration of sitosterol and taurochenodeoxycholate resulted in a 60% decrease of HMG CoA reductase activity and no change in cholesterol 7alpha-hydroxylase activity or biliary cholesterol concentration. Rats fed sitosterol plus taurocholate had nearly normal HMG CoA reductase activity, but cholesterol 7alpha-hydroxylase was inhibited and biliary cholesterol remained high. Bile acid secretion rates and biliary bile acid composition were similar in controls and sterol-fed animals. In all groups receiving bile acids, biliary secretion of bile acids was nearly doubled and bile acid composition was shifted in the direction of the administered bile acid. It is concluded that the composition of the bile acid pool influences the hepatic concentrations of the rate-controlling enzymes of bile acid synthesis.     

The reductive metabolism of diaziquone (AZQ) in the S9 fraction of MCF-7 cells: Free radical formation and NAD(P)H: Quinone-acceptor oxidoreductase (DT-diaphorase) activity

The S9 fraction of MCF-7 human breast carcinoma cells has NAD(P)H (quinone-acceptor) oxidoreductase activity as measured by the reduction of dichlorophenol-indophenol (DCPIP). This reduction is dependent on the activators Tween-20 and bovine serum albumin and it is inhibitable by dicumarol. The S9 fraction also has cytochrome c reductase activity which is approximately 29 times less than the two-electron reduction activity of NAD(P)H (quinone-acceptor) oxidoreductase. Diaziquone (AZQ) is a substrate for this NAD(P)H oxidoreductase active S9 fraction as judged by its enzymatic reduction detected spectrophotometrically and by electron spin resonance spectroscopy. Two-electron mediated enzymatic reduction of AZQ was evidenced by the formation of the colorless dihydroquinone (AZQH2) which could be followed at 340 nm. The production of the dihydroquinone was inhibitable by dicumarol implicating NAD(P)H oxidoreductase in its formation. Under aerobic conditions, electron spin resonance spectroscopy showed evidence for the production of AZQ semiquinone (AZQH) and oxygen radicals. Under anaerobic conditions no oxygen radicals were observed, but the semiquinone was stable for hours. These results are also inhibitable by dicumarol and suggest a two-step one-electron oxidation process of the dihydroquinone. The production of semiquinone and oxygen radicals as detected by electron spin resonance spectroscopy was more sensitive to dicumarol when NADPH was used as cofactor (68% inhibition of OH and 65% inhibition of AZQH) than when NADH was used (28% inhibition of OH and 5% inhibition of AZQH). This suggests that NADH flavin reductases play a more important role in the one-electron reduction pathway of AZQ in MCF-7 S9 fraction than NADPH reductases. The reduction of AZQ by NAD(P)H (quinone-acceptor) oxidoreductase may play an important role in the bioreductive alkylating properties of AZQ.     

Subcellular Distribution of Enzymes in Ochromonas malhamensis*

SYNOPSIS. The activity and distribution of 7 enzymes in Ochromonas malhamensis were studied. Subcellular organelles were separated by centrifugation at 648,000 g min to precipitate the larger particles; the resulting supernatant was centrifuged at 5,560,000 g min to separate the microsomal fraction from the supernatant. Sixty-four percent of the cytochrome oxidase (1.9.3.1 ferrocytochrome c:oxygen oxidoreductase, 81% of the catalase (1.11.1.6 hydrogen-peroxide: hydrogen-peroxide oxidoreductase) and 70% of the urate oxidase (1.7.3.3 urate:oxygen oxidoreductase) activity was associated with the larger particles, altho only 20% of the total protein was found in this fraction. Three acid hydrolases, cathepsin (3.4.4.9 cathepsin C, acid phosphatase (3.1.3.2 orthophosphoric monoesterphosphohydrolase) and acid ribonuclease (2.7.7.17 ribonucleate nucleotido-2′-transferase) were found mostly in the supernate (50-60%, yet their latency and their similar subcellular distribution indicated the presence of lysosomes. After 2.5 hr centrifugation in a sucrose density gradient (ρ= 1.08–1.25, the acid hydrolases showed a broad distribution which differed greatly from cytochrome oxidase associated with mitochondria. Catalase, which could not be separated from cytochrome oxidase by centrifuging on this gradient, had a different distribution after centrifugation on a kinetic gradient. Urate oxidase had a similar distribution to catalase and both these enzymes were latent, indicating the presence of peroxisomes.     

Enzymatic and energetic properties of the aerobic respiratory chain-linked NADH oxidase system in the marine bacterium Pseudomonas nautica

Membranes of Pseudomonas nautica, grown aerobically on a complex medium, oxidized both NADH and deamino-NADH as substrates. The activity of membrane-bound NADH oxidase was activated by monovalent cations including Na+, Li+, and K+. The activation by Na+ was higher than that by Li+ and K+. The maximum activity of NADH oxidase was obtained at about pH 9.0 in the presence of 0.08 M NaCl. The NADH oxidase activity was completely inhibited by 60 microM 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO), while the NADH:quinone oxidoreductase activity was about 37% inhibited by 60 microM HQNO. The activities of NADH oxidase and NADH:quinone oxidoreductase were about 40% inhibited by 60 microM rotenone. The fluorescence quenching technique revealed that electron transfer from NADH to ubiquinone-1 (Q-1) or oxygen generated a membrane potential (deltapsi) which was larger and more stable in the presence of Na+ than in the absence of Na+. However, the All was highly sensitive to a protonophore, carbonyl-cyanide m-chlorophenylhydrazone (CCCP) even at alkaline pH.     

Comparison of the redox activities in plasma membranes from roots and shoots of etiolated bean seedlings

Summary Plasma membrane (PM) vesicles were purified in parallel from the roots and shoots of 6-day-old etiolated bean (Phaseolus vulgaris L.) seedlings, grown in water culture at 25 °C, by aqueous polymer two-phase partitioning. The purity of PM fractions was determined by measuring the activity of known marker enzymes (vanadate-sensitive Mg-ATPase, 1,3--glycan synthase, latent ID-Pase, cytochromec oxidase, and antimycin-A-insensitive cytochromec reductase). NADH-(acceptor) oxidoreductase activities were determined with the following electron acceptors: ferricyanide, cytochromec, duroquinone, monodehydroascorbate, Fe³⁺-EDTA, and oxygen. Cytochromeb content was also determined. In general, results show that redox activities are higher in the root PM than in the shoot PM which follows the glycan synthase II (PM marker) pattern. The relative activities of the distinct redox enzymes measured (activity profile) are remarkably similar in the root and shoot PM preparations. The cytochromeb content and level of ascorbate reduction were also similar in both plant organs. However, the ratio of NADH-(acceptor) oxidoreductase activity to cytochrome content was signifcantly higher in roots when compared to the shoots.Abbreviations CCO cytochromec oxidase - CCR cytochromec reductase - GSII 1,3--glycan synthase - MF microsomal fraction - N-CC-OR NADH-cytochromec oxidoreductase - N-DQ-OR NADH-duroquinone oxidoreductase - N-FC-OR NADH-ferricyanide oxidoreductase - N-FE-OR NADH-Fe³⁺-EDTA oxidoreductase - N-MDA-OR NADH-monodehydroascorbate oxidoreductase - PM plasma membrane     

Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from human term decidua

Kinetics and regulation of 25-hydroxycholecalciferol 1 alpha-hydroxylase from cells isolated from term human decidua were studied. The production of 1 alpha,25-dihydroxycholecalciferol (calcitriol) was linear with time for up to 6 h and was directly proportional to the number of cells up to 20 X 10(6)/dish at a substrate concentration of 100 nM. Under these conditions the apparent Km was 88 nM and the Vmax 3.0 pmol/10(6) cells. The production of [3H]calcitriol was inhibited by 0.1 nM (P less than 0.01) and 1 nM (P less than 0.005) unlabeled calcitriol. Unlike the kidney enzyme and for reasons that remain unclear, neither inorganic phosphate salts nor parathyroid hormone had any acute effect on the calcitriol production. Further studies are required to delineate the regulatory mechanism of this enzyme.     

Enzymatic properties of mouse 25-hydroxyvitamin D3 1 alpha-hydroxylase expressed in Escherichia coli.

Renal 25-hydroxyvitamin D3 1 alpha-hydroxylase cDNA cloned from the kidneys of mice lacking the vitamin D receptor was expressed in Escherichia coli JM109. As expected, the bacterially-expressed enzyme catalyzes the 1 alpha-hydroxylation of 25-hydroxyvitamin D3 with a Michaelis constant, K(m), value of 2.7 microM. Unexpectedly, the enzyme also hydroxylates the 1 alpha-position of 24,25-dihydroxyvitamin D3 with a K(m) of 1.3 microM, and a fourfold higher Vmax/K(m) compared with the 25-hydroxyvitamin D3 hydroxylase activity, suggesting that 24,25-dihydroxyvitamin D3 is a better substrate than 25-hydroxyvitamin D3 for 1 alpha-hydroxylase. In addition, the enzyme showed 1 alpha-hydroxylase activity toward 24-oxo-25-hydroxyvitamin D3. However, it showed only slight activity towards 23,25-dihydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, and no detectable activity towards vitamin D3 and 24,25,26,27-tetranor-23-hydroxyvitamin D3. These results suggest that the 25-hydroxyl group of vitamin D3 is essential for the 1 alpha-hydroxylase activity and the 24-hydroxyl group enhances the activity, but the 23-hydroxyl group greatly reduced the activity. Another remarkable finding is that living recombinant E. coli cells can convert the substrates into the 1 alpha-hydroxylated products, suggesting the presence of a redox partner of 1 alpha-hydroxylase in E. coli cells.     

Enzymatic properties of human 25-hydroxyvitamin D3 1alpha-hydroxylase coexpression with adrenodoxin and NADPH-adrenodoxin reductase in Escherichia coli.

We have cloned human 25-hydroxyvitamin D3 1alpha-hydroxylase cDNAs from normal subjects and patients with pseudovitamin D-deficient rickets (PDDR), and expressed the cDNAs in Escherichia coli JM109 cells. Kinetic analysis of normal 1alpha-hydroxylase in the reconstituted system revealed that Km values for 25(OH)D3 and (24R), 25(OH)2D3 were 2.7 and 1.1 microM, respectively. The lower Km value and higher Vmax/Km value for (24R),25(OH)2D3 indicated that it is a better substrate than 25(OH)D3 for 1alpha-hydroxylase. These results are quite similar to those of mouse 1alpha-hydroxylase. To establish a highly sensitive in vivo system, 1alpha-hydroxylase, adrenodoxin and NADPH-adrenodoxin reductase were coexpressed in E. coli cells. The recombinant E. coli cells showed remarkably high 1alpha-hydroxylase activity, suggesting that the electrons were efficiently transferred from NADPH-adrenodoxin reductase through adrenodoxin to 1alpha-hydroxylase in E. coli cells. Using this system, the activities of four mutants of 1alpha-hydroxylase, R107H, G125E, R335P and P382S, derived from patients with PDDR were examined. Although no significant reduction in expression of these mutants was observed, none showed detectable activity. These results strongly suggest that the mutations found in the patients with PDDR completely abolished 1alpha-hydroxylase activity by replacement of one amino acid residue.     

A possible restriction of ferro- and ferricyanide oxidoreductase activities of rat liver mitochondria by the outer membrane

In this work, various ferro-ferricyanide oxidoreductase activities of rat liver mitochondria were studied to find conditions under which the outer membrane might restrict the flux of these highly charged non-biological anions. When the isotonic low ionic strength medium was supplemented with 25mM KCl, a several-fold increase in the succinate-ferricyanide reductase activity of mitochondria and in the rate of external NADH oxidation in the presence of ferrocyanide was observed. Mitochondrial respiration with 5mM ferrocyanide was almost completely inhibited after consumption of 3.8-18.5% of the dissolved oxygen, depending on the medium and the presence of 2,4-dinitrophenol. These and other experimental data together with mathematical modeling of the redox-state equilibrium suggest that the measured activities might be restricted by two factors: first, the permeability of the outer mitochondrial membrane and second, a strong influence of the ionic strength of incubation media on the intermembrane space redox reactions.     

Biosynthesis of streptomycin enzymic oxidation of dihydrostreptomycin (6-phosphate) to streptomycin (6-phosphate) with a particulate fraction of Streptomyces griseus

Resting cells and to a greater extent permeabilized cells of Streptomyces griseus can oxidize dihydrostreptomycin to streptomycin. The dihydrostreptomycin oxidoreductase activity was localized in the 100 000 × g particulate fraction. Sucrose density gradient centrifugation of the particulate suspension gave a band at a density of 1.09 which consisted mainly of membrane vesicles. This fraction had high dihydrostreptomycin oxidoreductase activity. S. griseus protoplasts also contain high oxidoreductase activity. These data are consistent with localization of the enzyme in the cell membrane. Dihydrostreptomycin and dihydrostreptomycin 6-phosphate can both serve as substrates for the oxidoreductase, but the phosphate was the better substrate in the cell free system. Addition of cofactors was not required for the bound dihydrostreptomycin oxidoreductase. The electron acceptor for the oxidation is unknown. Oxidation of dihydrostreptomycin 6-phosphate to streptomycin 6-phosphate very probably represents the penultimate step in the biosynthesis of streptomycin.