Cytochrome P-450 and 18-oxygenase system from beef adrenocortical mitochondria - inhibitory effect of puromycin.

$\text{Trans}$-3-dehydro-D, L-ornithine and $\text{trans}$-1, 4-diamino-2-butene have been synthesized and shown to be potent competitive inhibitors of ornithine decarboxylase. The K_I′S for $\text{trans}$-3-dehydro-L-ornithine and $\text{trans}$-1, 4-diamino-2-butene are 2.2 and 2.0 μM respectively. Both analogs bind much more tightly to the enzyme than either ornithine or putrescine. Studies of chick embryo muscle cells in culture show results consistent with reversible inhibition of division and/or fusion by addition of $\text{trans}$-3-dehydro-D, L-ornithine to the culture medium.     

Danazol inhibits human adrenal 21-and 11β-hydroxylation invitro

The effects of danazol on steroidogenesis $\text{in}$$\text{vitro}$ in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant K_I = 0.8 μM) and the mitochondrial 11β-hydroxylase (K_I = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal $\text{in}$$\text{vitro}$.     

Paradoxical activation of Leptomonas NAD-linked alpha-glycerophosphate dehydrogenase by ethidium and antrycide.

Antrycide and ethidium bromide — 2 cationic trypanocides — inhibited NAD-linked α-glycerophosphate dehydrogenase from $\text{Leptomonas}$ sp. The kinetics of enzyme inhibition was determined by Lineweaver-Burk, Dixon, or direct linear plots. Inhibition by Antrycide was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. With dihydroxyacetone phosphate at saturation, Antrycide inhibition was also noncompetitive with respect to Mg²⁺ (K_i = 115 μM) and spermidine (K_i = 85 μM). Inhibition by ethidium in the presence of saturating dihydroxyacetone phosphate, was noncompetitive for Mg²⁺ (K_i = 400 μM) but mixed for spermidine (K_i = 495 μM); inhibition was noncompetitive for dihydroxyacetone phosphate in the presence of saturating Mg²⁺ or spermidine. Rabbit-muscle α-glycerophosphate dehydrogenase was inhibited at all concentrations of Antrycide and ethidium tested, but the $\text{Leptomonas}$ enzyme was stimulated up to 3.5-fold by low concentrations of inhibitors in the absence of polyamine. New chemotherapeutic possibilities may thus be opened and an evolutionary distinction between trypanosomatid and mammalian enzyme.     

Evidence for the regulation of pyridoxal 5′-phosphate formation in liver by pyridoxamine (pyridoxine) 5′-phosphate oxidase

Pyridoxamine (pyridoxine) 5′-phosphate oxidase (EC 1.4.3.5) purified from rabbit liver is competitively inhibited by the reaction product, pyridoxal 5′-phosphate. The K_i, 3 μM, is considerably lower than the K_m for either natural substrate (18 and 24 μM for pyridoxamine 5′-phosphate and 25 and 16 μM for pyridoxine 5′-phosphate in 0.2 M potassium phosphate at pH 8 and 7, respectively). The K_i determined using a 10% rabbit liver homogenate is the same as that for the pure enzyme; hence, product inhibition $\text{in}\text{vivo}$ is probably not diminished significantly by other cellular components. Similar determinations for a 10% rat liver homogenate also show strong inhibition by pyridoxal 5′-phosphate. Since the reported liver content of free or loosely bound pyridoxal 5′-phosphate is greater than K_i, the oxidase in liver is probably associated with pyridoxal 5′-phosphate. These results also suggest that product inhibition of pyridoxamine-P oxidase may regulate the $\text{in}\text{vivo}$ rate of pyridoxal 5′-phosphate formation.     

Detection of aryl hydrocarbon hydroxylase activity in normal and neoplastic human breast epithelium

Studies were conducted to determine whether normal and/or neo-plastic (MCF-7) human breast epithelial cells contain the microsomal aryl hydrocarbon hydroxylase (AHH) which catalyses the conversion of polycyclic aromatic hydrocarbons (PAH) to carcinogenic intermediates. Low constitutive levels of AHH activity were found in homogenates of both normal human breast epithelial and MCF-7 cells. The addition of 7,12-dimethylbenz(a)anthracene (DMBA) to the culture medium of either cell type significantly increased AHH activity. Peak induction of hydroxylase activity occurred following the $\text{in}$$\text{vitro}$ addition of 10 μM DMBA. A time course of DMBA-induced AHH activity in both normal human breast epithelium and MCF-7 cells revealed maximal induction 16 hr after 10 μM DMBA was added to the culture medium. Benzo(a)pyrene (BP), 3-methylcholanthrene (MCA) and benz(a)anthracene (BA) also induced AHH activity in normal and MCF-7 cells. For example, the addition of 10 μM BP to the culture medium of either normal human breast epithelial or MCF-7 cells for 16 hr increased AHH activity 13.8 and 65.3-fold, respectively. For all PAH, the magnitude of AHH induction was substantially greater in MCF-7 than normal breast epithelial cells. Finally, α-naphthoflavone inhibited BA-induced AHH activity in MCF-7 cells. The study demonstrates the presence of a PAH-inducible AHH enzyme(s) in normal human breast epithelial cells grown in primary culture and in the human breast tumor cell line, MCF-7.     

Nδ-(phosphonacetyl)-L-ornithine,a potent transition state analogue inhibitor of ornithine carbamoyltransferase

$\text{N}$^δ-(Phosphonacetyl)-L-ornithine, a transition state analogue for the reaction catalyzed by ornithine carbamoyltransferase (EC 2.1.3.3), was synthesized. It strongly inhibited bovine liver ornithine carbamoyltransferase. The inhibition was competitive with respect to carbamoyl-phosphate; the apparent $\text{K}$_m values for carbamoyl-phosphate were 15 μM in 0.05 M $\text{N}$-2-hydroxyethylpiperazine-$\text{N′}$-2-ethanesulfonate (pH 7.2) and 33 μM in 0.1 M Tris-HCl (pH 8.5), and the inhibition constants at pH 7.2 and 8.5 were 7.1 and 4.7 nM, respectively. The inhibition was non-competitive with L-ornithine, the other substrate of the enzyme. This analogue may provide an effective reagent for the elucidation of carbamoyl-phosphate metabolism and its regulation in the liver of ureotelic animals.     

m-Octopamine as a substrate for monoamine oxidase.

$\text{m}$-Octopamine was characterized as substrate for monoamine oxidase (MAO) in rat brain and liver mitochondria. The $\text{K}$m and $\text{V}$max values of the brain enzyme were 735 μM and 32.5 nmoles/mg protein/30 min, and those of the liver enzyme 351 μM and 125 nmoles/mg protein/30 min, respectively. The inhibition experiments with clorgyline and deprenyl showed that $\text{m}$-octopamine was a common substrate for type A and type B MAO, though a major part of the activity was due to type A enzyme.     

Mutational alteration of Bacillus subtilis DNA polymerase 3 to hydroxyphenylazopyrimidine resistance: polymerase 3 is necessary for DNA replication

A spontaneous mutant of $\text{Bacillus}\text{subtilis}$ resistant to killing by two hydroxyphenylazopyrimidines has been isolated. The DNA polymerase III of this mutant is resistant to inhibition by these drugs. The K_i for 6-(p-hydroxyphenylazo)-uracil (HPUra) is 20 μM, about 40 times higher than the K_i of the wild-type enzyme. The mutant and wild-type polymerases behave similarly during purification, are sensitive to N-ethylmaleimide and to 0.1 M KCl, and have the same K_m for dGTP (0.5 μM). The HPUra inhibition of both enzymes is attenuated competitively by dGTP. We conclude that polymerase III is the target for hydroxyphenylazopyrimidines $\text{in}\text{vivo}$, and since the drugs specifically inhibit replicative DNA synthesis, polymerase III is necessary for DNA replication.     

Tryptophan uptake by Mycobacterium tuberculosis H37Rv--inhibition by isoniazid.

The effect of various sub-inhibitory concentrations of isoniazid on tryptophan uptake by $\text{Mycobacterium tuberculosis}$ H₃₇Rv grown $\text{in vitro}$ and $\text{in vivo}$ was studied. Uptake, measured after 3 minutes of drug exposure was inhibited mildly by 0.1 μg/ml and 0.2 μg/ml concentration and completely by 0.3 μg/ml. However, with the minimal inhibitory concentration (MIC)⁷ of 0.5 μg/ml, not only inhibition but also a strong efflux of the preformed tryptophan pool were observed. The results are discussed in the light of the theory that isoniazid interferes with the cell wall mycolate synthesis.     

The enzyme "aldehyde oxidase" is an iminium oxidase. Reaction with nicotine delta 1'(5') iminium ion.

The second of the two reaction steps involved in the metabolic transformation of (?)-nicotine to (?)-cotinine $\text{(}\text{3}\text{) (}\text{i.}\text{e.}$, the oxidation of the intermediate $\text{2}\text{)}$ is mediated mainly, if not solely, by the enzyme aldehyde oxidase (EC 1.2.3.1). Of the molecular species that constitute $\text{2}$, nicotine Δ^1′(5′) iminium ion $\text{(}\text{2a}\text{)}$ appears to serve as the substrate. The enzyme has a strong affinity for $\text{2a}$, as shown in a study on the inhibition of the oxidation of 3-(aminocarbonyl)-1-methylpyridinium chloride. This study gave a value of K_i = 6 μM; K_m = 2 μM (pH 7.4). Mainly in view of this finding, “iminium oxidase” seems to be a more adequate name than “aldehyde oxidase” for this enzyme.     

3β-Hydroxysteroid dehydrogenaseΔ5−4isomerase activity in the rhesus monkey placenta and fetal adrenal,testis and ovary during late gestation

3β-Hydroxysteroid $\text{dehydrogenase}\text{Δ}{}^{\mathrm{5?4}}\text{isomerase}$ (3β-HSDH) was measured in the rhesus monkey ($\text{Macaca mulatta}$) placenta, fetal adrenal (whole organ minus medulla), testis and ovary during late gestation (Days 145–162). Activities were evaluated from the conversion of [³H]-pregnenolone to [³H]progesterone. The maximum enzyme velocity (V_m) in adrenal microsomes (100,000 g pellet) was significantly higher (146 nmoles progesterone/h x mg^?1protein) than in microsomes from the other tissues. Testicular V_m was greater than either ovarian or placental V_m which were not different from one another (11.5 versus 1.9, 1.2 nmoles progesterone/h x mg^?1protein, respectively). Apparent Michaelis-Menten constants in the adrenal, placenta, testis and ovary averaged 1.8,2.5,0.27 and 0.16 μM, respectively. In some cases, substrate inhibition was noted. Estimated dissociation constants for pregnenolone were 2.3 μM (adrenal), 2.1 μM (placenta), 0.74 μM (testis) and 0.13 μM (ovary). 3β-HSDH was less active in a crude mitochondrial preparation from the fetal adrenal (10,000 g pellet) than in microsomes, whereas activity in the placenta and testis appeared to be equally distributed between mitochrondria and microsomes.Rate measurements were consistent with the apparent potentials of these organs to synthesize their characteristic hormones. Thus, 3β-HSDH activity may be an important rate determining step in hormone synthesis. The importance of substrate inhibition in progesterone formation remains to be assessed.     

Inhibition of nucleic acid synthesis in leukemia 1210 cells by antimetabolites of coenzyme Q10

Thirteen diversified antimetabolites of coenzyme Q₁₀ which have antitumor activity $\text{in vivo}$ were tested for inhibition of uptake of tritiated thymidine and uridine into DNA and RNA, respectively, of L1210 cells grown in tissue culture. Eight of these antimetabolites have inhibitory activities of the same order of magnitude as the used anticancer drugs, rubidazone and ellipticine. 5-ω-Phenylpropylmercapto-2,3-dimethoxy-1,4-benzoquinone was particularly potent to inhibit nucleic acid synthesis; ED₅₀ for DNA = 2.1 μM and ED₅₀ for RNA = 4.0 μM.     

Succinylacetone pyrrole,a powerful inhibitor of vitamin B12 biosynthesis: Effect on δ-aminolevulinic acid dehydratase

Succinylacetone, a competitive inhibitor (K_I = 400 μM) of δ-aminolevulinic acid dehydratase of $\text{Clostridium}\text{tetanomorphum}$, is converted non-enzymatically upon incubation with δ-aminolevulinic acid to succinylacetone pyrrole, a much stronger competitive inhibitor (K_I = 5 μM) of the enzyme. A similar effect is seen in vivo: when present in the growth medium at concentrations of about 1 μM, the pyrrole decreases the level of corrinoids produced by this organism by half, while succinylacetone at 200 μM causes only 19 per cent inhibition of corrinoid formation. Levulinic acid is a much weaker inhibitor in vitro and in vivo. The inhibition by succinylacetone pyrrole is considered to be due to its structural resemblance to δ-aminolevulinic acid rather than to porphobilinogen, the reaction product of δ-aminolevulinic acid dehydratase: succinylacetone, succinylacetone pyrrole, and levulinic acid all contain a succinyl group.     

Regulation of prostaglandin metabolism: inhibition of 15-hydroxyprostaglandin dehydrogenase by thyroid hormones

15-Hydroxyprostaglandin dehydrogenase has been purified from swine kidney to a specific activity of near 100 miliunits per mg of protein. The purified enzyme was found to be inhibited by thyroid hormone analogues of which triiodothyroacetic acid was the most potent inhibitor. The concentration required for 50% inhibition was 5 μM for triiodothyroacetic acid. The inhibition by thyroid hormones was uncompetitive and non-competitive with regard to NAD⁺ and prostaglandin E₁, respectively. The sensitivity of this enzyme to thyroid hormones suggests that these hormones may regulate the metabolism of prostaglandins $\text{in vivo}$.     

Oxygen exchange associated with electron transport and photophosphorylation in spinach thylakoids

O₂ uptake in spinach thylakoids was composed of ferredoxin-dependent and -independent components. The ferredoxin-independent component was largely 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) insensitive (60%). Light-dependent O₂ uptake was stimulated 7-fold by 70 μM ferredoxin and both uptake and evolution (with O₂ as the only electron acceptor) responded almost linearly to ferredoxin up to 40 μM. NADP⁺ reduction, however, was saturated by less than 20 μM ferredoxin. The affinity of O₂ uptake for for O₂ was highly dependent on ferredoxin concentration, with $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(O}{}_2\text{)}$ of less than 20 μM at 2 μM ferredoxin but greater than 60 μM O₂ with 25 μM ferredoxin. O₂ uptake could be suppressed up to 80% with saturating NADP⁺ and it approximated a competitive inhibitor of O₂ uptake with a K_i of 8–15 μM. Electron transport in these thylakoids supported high rates of photophosphorylation with NADP⁺ (600 μmol ATP/mg Chl per h) or O₂ (280 μmol/mg Chl per h) as electron acceptors, with $\text{ATP}\text{2}\text{e}$ ratios of 1.15–1.55. Variation in $\text{ATP}\text{2}\text{e}$ ratios with ferredoxin concentration and effects of antimycin A indicate that cyclic electron flow may also be occurring in this thylakoid system. Results are discussed with regard to photoreduction of O₂ as a potential source of ATP in vivo.     

Inhibition of bovine adrenocortical cytochrome P-450scc by 3,3'-dimethoxybenzidine

The effect of 3,3'-dimethoxybenzidine ($\text{o}$-dianisidine) on the conversion of cholesterol to pregnenolone was investigated in a reconstituted side chain cleavage system using enzymes purified from bovine adrenal cortex; d-$\text{p}$-aminoglutethimide was also assayed under similar conditions for comparison. 3,3'-Dimethoxybenzidine was found to be a potent inhibitor of pregnenolone formation, causing 50% inhibition at a concentration of 1.5 μM when using 70 μM cholesterol — this dose is approximately one fourth that required of 3-methoxybenzidine and one twentieth that required of benzidine for equal inhibition. In the same system, d-$\text{p}$-aminoglutethimide exhibited an I₅₀ value of about 55 μM. No effects of 3,3'-dimetoxybenzidine on adrenodoxin reductase or adrenodoxin activities could be detected, and inhibition of side chain cleavage could be relieved by dilution suggesting that the inhibitor acts by reversibly binding to cytochrome P-450scc.     

Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I₁ (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both $\text{in}$$\text{vitro}$ and $\text{in vivo}$. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB₄ synthesis in human peripheral neutrophils with an ID₅₀ of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.     

1,4-Methylhistamine is a specific substrate for type B monoamine oxidase

1,4-Methylhistamine was characterized as substrate for monoamine oxidase (MAO) in rat liver mitochondria. The $\text{K}$_m and $\text{V}$_max values were 38.8 μM and 6.33 nmoles/mg protein/60 min, respectively. The inhibition experiments with clorgyline and deprenyl, the selective inhibitors for type A and type B MAO, showed that 1,4-methylhistamine was specific for type B MAO.     

Induction by retinoic acid of NAD+-glycohydrolase activity of myelomonocytic cell lines HL-60, THP-1 and U-937, and fresh human acute promyelocytic leukemia cells in primary culture

HL-60, a human promyelocytic leukemia cell line, is induced to differentiate by retinoic acid to mature granulocytes. We have now found that after the addition of 1 μM retinoic acid to HL-60 cultures an increase in NAD⁺-glycohydrolase (NADase) activity is detected by 6 hr and after a 33-fold increase in activity reaches a plateau by 24 hr. Cycloheximide inhibits completely the retinoic acid-induced increase in NADase activity indicating that enzyme induction requires protein synthesis $\text{de}\text{,}\text{novo}$. An increase of NADase activity was found not only in HL-60 cells but also in two human monoblast cell lines (U-937 and THP-1) and fresh cells in primary culture from two patients with acute promyelocytic leukemia. An increase in synthesis $\text{de}\text{,}\text{novo}$ of NADase does not appear to be obligatory for differentiation of HL-60 because there was no increase of NADase activity in HL-60 cells induced to differentiate with either dimethylsulfoxide, hypoxanthine, butyrate, or 1, 25-dihydroxycholecalciferol and there were marked increases in NADase activity at concentrations of retinoic acid having little or no effect on differentiation.