Aldehyde content and cross-linking of type III collagen.

Three phosphorylated dinucleosides designated HS1, HS2, and HS3, isolated from the water-mould $\text{Achlya}$, were shown to significantly inhibit ribonucleotide reductase activity from $\text{Achlya}$. All three compounds decreased CDP reduction in fungal extracts by 50% at concentrations of 0.1mM. At the same concentration HS3 also inhibited partially purified CDP reductase from Chinese hamster ovary cells by at least 80% but showed only 10% inhibition with enzyme from $\text{E.}\text{coli}$. ADP reductase activity from $\text{Achlya}$ was inhibited 50% by both HS1 and HS3 at 0.1mM. HS2 however, showed no inhibitory effect on purine reduction. The levels of ribonucleotide reductase during the asexual growth cycle of $\text{Achlya}$ correlated with thymidine uptake into DNA and with the synthesis of HS compounds.     

Conversion of 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one to delta 16-C19 steroids by the reconstituted delta 16-C19-steroid synthetase system

The substrate specificity of the reconstituted delta 16-C19-steroid synthetase system, which catalyzes the formation of 5,16-androstadien-3 beta-ol or 4,16-androstadien-3-one from pregnenolone or progesterone, respectively, was studied. The reconstituted system consisted of a partially purified cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and NADH-cytochrome b5 reductase all from pig testicular microsomes. It was found that 5 alpha-reduced C21 steroids such as 5 alpha-pregnane-3,20-dione, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 beta-hydroxy-5 alpha-pregnan-20-one can be substrates for the enzyme system, resulting in the formation of 5 alpha-androst-16-en-3-one, 5 alpha-androst-16-en-3 alpha-ol and 5 alpha-androst-16-en-3 beta-ol, respectively. The results suggest that 5 alpha-reduced delta 16-C19 steroids might be synthesized from pregnenolone and progesterone via 5 alpha-reduced C21 steroids as intermediates. The pathways would bypass 5,16-androstadien-3 beta-ol and 4,16-androstadien-3-one which have been assumed as obligatory intermediates in the formation of 5 alpha-reduced delta 16-C19 steroids from pregnenolone and progesterone.     

Cytochrome P-450 purified to apparent homogeneity from phenobarbital-induced rabbit liver microsomes: catalytic activity and other properties

Cytochrome P-450 was purified to a content of over 17 nmoles per mg of protein from liver microsomes of phenobarbital-treated rabbits by fractionation with polyethylene glycol 6000, DEAE-cellulose column chromatography, and hydroxylapatite column chromatography in the presence of Renex 690, a nonionic detergent. The purified preparation exhibited a single polypeptide band (molecular weight, 49,000 daltons) when submitted to SDS-polyacrylamide gel electrophoresis. Cytochromes P-420 and $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase were absent. The reconstituted system containing purified cytochrome P-450, reductase, and phosphatidylcholine catalyzed the hydroxylation of benzphetamine, cyclohexane, aniline, and laurate.     

The effect of folate and folate analogs upon dihydrofolate reductase and DNA synthesis in kidneys of normal mice

A single subcutaneous injection of folate, homofolate or MTX resulted in the inhibition of the activity of dihydrofolate reductase in homogenates prepared from the kidneys of normal mice. Stimulation of ³H-thymidine uptake occurred in the kidneys of treated animals approximately 30 hr after administration of either folate or homofolate, and reached a peak 72 hr after administration. The effects of folate and MTX on dihydrofolate reductase activity $\text{in}$$\text{vivo}$ were also determined. One hr after administration of 15 mg/kg methotrexate (MTX) or 300 mg/kg folate, enzyme activity $\text{in}$$\text{vivo}$ was inhibited by 90%.³H-deoxyuridine uptake was neither stimulated nor depressed after treatment with MTX. After administration of folate, uptake of ³H-deoxyuridine was stimulated at approximately 30 hr after drug-treatment and reached a peak at 72 hr after folate administration. Treatment with xanthopterin had no effect on the activity of dihydrofolate reductase $\text{in}$$\text{vitro}$. Xanthopterin stimulated uptake of both deoxyuridine and thymidine in an identical manner.The increased DNA synthesis that occurs in animals after treatment with agents that cause renal damage is distinct from the effect these agents have upon dihydrofolate reductase. Nucleoside incorporation after treatment with folate, homofolate, MTX or xanthopterin cannot be predicted on the basis of enzyme inhibition. Treatment with MTX, folate or homofolate results in enzyme inhibition which is not correlated with the uptake of deoxyuridine into DNA.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

Purification of cytochrome P-450 from liver microsomes of phenobarbital-treated guinea pigs

Cytochrome P-450 was purified from phenobarbital-treated guinea pigs to a specific content of 19.8 nmoles per mg of protein, and was free of cytochrome b₅ and NADPH-cytochrome $\text{c}$ reductase. The purified cytochrome P-450 gave a single protein band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 49,000 was estimated. Benzphetamine N-demethylation activity could be reconstituted by mixing the purified cytochrome, NADPH-cytochrome $\text{c}$ reductase and phosphatidylcholine.     

3-Hydroxy-3-methylglutaryl CoA reductase of Hevea latex: The occurrence of a heat-stable activator in the C-serum

The effect of the C-serum (the cytosol) on the activity of 3-hydroxy-3-methylglutaryl CoA reductase in the latex of $\text{Hevea}\text{brasiliensis}$ was investigated. Depending on the clone from which the latex was obtained, the C-serum was found to depress or activate or have little effect on the enzyme activity. Boiling the C-serum however, resulted in a consistent activation effect in all the clones examined. Optimal activation was obtained with 20 μl boiled C-serum. Dialysis or EDTA (40 mM) treatment of the boiled C-serum did not diminish the activation effect. Although not essential, dithiothreitol complemented the activation effect of the boiled C-serum and the optimal concentration was 10 mM. Trypsin digestion of the boiled C-serum resulted in the complete loss of the activation effect. The activator in the boiled C-serum was salted out by ammonium sulphate at 25 – 100% saturation. Hevein had no effect on reductase activity.     

Presence of peptide hormones that control gonadotropin secretion in extrahypothalamic areas of the brain.

The hypocholesterolemic drug clofibrate (ethyl-α-$\text{p}$-chlorophenoxyisobutyrate) was found to strongly suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) activity in cultured mouse L cells at concentrations of 20 – 50 μg/ml. The half-life ($\text{t1}\text{2}$) of the reductase (approximately 120 min) was strongly reduced when L cells were incubated with cycloheximide plus a maximal inhibitory concentration of clofibrate (50 μg/ml), resulting in a $\text{t1}\text{2}$ value of 10 min. Preliminary kinetic analysis of the inhibition suggested that clofibrate increased the rate of inactivation (or degradation) of the reductase without affecting the rate of enzyme synthesis.     

Ribonucleotide reductase activity in green algae

A ribonucleoside diphosphate reductase is demonstrated in the algae, $\text{Scenedesmus}\text{obliquus}$ and $\text{Chlorella}\text{pyrenoidosa}$. In synchronized cultures an activity maximum at the 12th hour of the cell cycle coincides with maximum DNA production. Induction of reductase activity is prevented by cycloheximide. The enzyme requires dithiols for reduction of CDP $\text{in}\text{vitro}$; it is not significantly stimulated by iron or magnesium ions nor dependent upon deoxyadenosylcobalamin. ATP stimulates the reaction but dATP or dTTP act as inhibitors. The ribonucleotide reductase of green algae differs from the B₁₂-requiring enzyme characterized in $\text{Euglena}\text{gracilis}$.     

An A-nor-3-thia-5beta-pregnane.

The conversion of a 5β-pregnan-3-one into a 3-thia-A-nor-pregnane is described. The single epimer, characterized as the 5β-isomer by nmr spectroscopy, was obtained.A previous report [1] from this laboratory described the synthesis of the epimeric 3-oxa-A-norpregnan-20-ones $\text{1}$ and $\text{2}$ from A-norprogesterone. Recently, the total synthesis of the related A-nor-3-thiaestra-1,-5(10)-diene derivative $\text{3}$ has been described [2]. Continuing interest [3] in A-ring-modified steroids as probes of drug-receptor interactions prompts this report of the preparation of the 3-thia-5β-A-norpregnan-20-one $\text{4}$.     

Regulation of sterol biosynthesis in yeast: induction of 3-hydroxy-3-methylglutaryl-CoA reductase by glucose

Anaerobically cultured yeast cells have a very low HMG-CoA reductase activity and a low sterol content. When these cells are transfered to phosphate buffer containing 1.2 % glucose and held under aerobic conditions, the specific activity of the HMG-CoA reductase increases up to sixfold within 8 hrs. The increase in the reductase activity is paralled by an increase in the sterol content. This induction of HMG-CoA reductase in resting yeast cells is inhibited by cycloheximide indicating that a $\text{de novo}$ synthesis of enzyme protein is mediated by glucose under aerobic conditions. It appears that the regulation of sterol synthesis in yeast is closely connected with the aerobic glucose metabolism.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

The inhibitory effect of ATP on HMGCoA reductase

The inhibitory effect of ATP on HMGCoA reductase activity from rat liver microsomes in the system described by Beg $\text{et al.}$ was examined. The inhibition by magnesium ATP is confirmed but varies widely from zero to complete. A requirement for a cytosolic fraction to enhance the inhibition could not be established. ATP labeled uniformly with ¹⁴C in the adenine portion and ³²P in the terminal phosphate was incubated with the enzyme in a situation where strong inhibition was observed. The enzyme protein was precipitated with trichloroacetic acid, or subjected to column fractionation. No evidence of labeling was found in the protein. Finally, the enzyme protein was specifically isolated by immunoprecipitation with a specific antibody to the HMGCoA reductase. In no instance could labeling of the enzyme protein be detected. These results show that the mechanism of the inhibition does not involve phosphorylation or adenylation of the enzyme protein.     

Evidence for existence and a tentative identification of coenzyme in yeast thiamine pyrophosphokinase.

Thiamine pyrophosphokinase (E.C. 2.7.6.2.) from $\text{Saccharomyces cerevisiae}$ was found to require the presence of a non-protein, non-metal compound for its activity. $\text{myo}$-Inositol was found capable of stimulating the kinase activity in the presumably resolved but otherwise crude sample of the enzyme. The hexytol was also found capable of inducing the enzyme in growing yeast cells. The cultured yeast cells, in which the kinase had been induced, were used as source of the enzyme for its purification. The compound that had been left adsorbed to the final column of DEAE-Sephadex was proved to have a coenzyme activity towards the enzyme and tentatively identified with $\text{myo}$-inositol 1-pyrophosphate. A sample of synthetic $\text{myo}$-inositol 1-pyrophosphate was made and its coenzyme activity was observed.     

Progesterone-induced inactivation of nuclear estrogen receptor in the hamster uterus is mediated by acid phosphatase

Inactivation of hamster uterine estrogen receptor was assayed in nuclear KCl extracts (30 min, 37°C, pH 7.5) after progesterone treatment $\text{in}\text{vivo}$ for 2h. At very low concentrations ($\text{>}$0.05 mM), molybdate and vanadate blocked the progesterone-induced increase in receptor inactivation. In contrast, only high concentrations ($\text{>}$10 mM) of the inhibitors blocked receptor inactivation in extracts from untreated hamsters. Gel electrophoresis and inhibition curves for phosphatases in nuclear extract demonstrated that acid, rather than alkaline, phosphatase activity is most likely responsible for these effects. These data suggest that progesterone antagonizes estrogen action in the hamster uterus by promoting estrogen receptor dephosphorylation leading to inactivation.     

In vitro inhibition of rat uterine contractility induced by 5α and 5β progestins

Ten natural progestins were evaluated for their capacity to inhibit the $\text{in vitro}$ motility of rat's uterus. Progestins with their ring A reduced in the 5β position were significantly more potent than Δ⁴-3 keto and 5α reduced progestins. These last progestins were ineffective to inhibit uterine motility excepting 3α-hydroxy-5α-pregnan-20-one which was slightly less effective than progesterone. The potency of the progestins to inhibit uterine motility was related to their capacity to induce membrane stabilization. The data indicates that 5β, but not 5α reduction of progesterone, may be important for regulating myometrial activity.     

Characterization of a membrane-bound nitrate reductase from Azotobacter chroococcum.

Nitrate reductase from the aerobic bacterium $\text{Azotobacter chroococcum}$ is a soluble enzyme with the characteristic features of Pichinoty's type B nitrate reductase. When cell suspensions of $\text{A. chroococcum}$ are repeatedly subcultured in liquid medium with nitrate as the nitrogen source, most of the nitrate-reducing activity is incorporated into the cytoplasmic membrane. The properties of the particulate nitrate reductase closely resemble Pichinoty's type A enzyme.     

Liver microsomal DT diaphorase: noninvolvement in hydroxylation of benzo(a)pyrene.

A highly purified reconstituted system isolated from the microsomes of 3-methylcholanthrene-treated rats consisting of cytochrome P-448, NADPH-cytochrome $\text{c}$ reductase and synthetic dilauroyl phosphatidylcholine had no DT diaphorase activity, but hydroxylated benzo[a]pyrene at a faster rate than microsomes from 3-methylcholanthrene-treated rats. DT diaphorase purified from liver microsomes of 3-methylcholanthrene-treated rats when added to this reconstituted system did not stimulate or inhibit benzo[a]pyrene hydroxylation, nor could it replace or NADPH-cytochrome $\text{c}$ reductase in supporting the reaction. We therefore conclude that microsomal DT diaphorase is not involved in microsomal hydroxylation of benzo[a]pyrene to its phenolic products despite the observation that both DT diaphorase activity and the hydroxylation of benzo[a]pyrene are induced by 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin     

Influence of progesterone on the initial rate of cholesterol esterification in rat plasma

The effect of progesterone on the initial rate of cholesterol esterification in rat plasma was measured after daily injections of the hormone or following addition of progesterone $\text{in}$$\text{vitro}$. The administration of progesterone did not modify the lecithin:cholesterol acyltransferase (LCAT) activity nor the progress of the enzyme reaction with time. When increasing concentrations of progesterone were added to the medium the percentage of cholesterol esterified per minute decreased progressively. The addition of progesterone also decreased the slope of the time-course reaction. It is suggested that the inhibition of the LCAT activity due to the presence of the hormone would be masked by an increased hepatic production of the enzyme and/or by the alterations that the hormonal treatments produced in the plama lipid levels.     

NADPH-dependent reductase solubilized from microsomes by peroxidation and its activity

Isolated rat liver microsomes were subjected to enzymatic or non-enzymatic lipid peroxidation $\text{in vitro}$. NADPH-dependent cytochrome $\text{c}$ reductase activity was released from the microsomes into the media during peroxidation. This activity could be recovered from the media by DEAE-cellulose chromatography. The recovered enzyme retained high activity for the reduction of cytochrome $\text{c}$ and a lower level of activity for the reduction of cytochrome P-450. The active fractions were capable of enzymatically supporting the peroxidation of isolated mitochondria in the presence of organically complexed Fe⁺³ and NADPH, and in this respect the specific activity was found to be about ten times higher than in microsomes.