Kinetics of adrenodoxin reductase oxidation by non-physiologic electron acceptors

The reactions of NADPH oxidation by quinones and inorganic complexes catalyzed by NADPH: adrenodoxin reductase were studied. The catalytic constant for the enzyme at pH 7.0 is 20-25 s-1; the oxidative constants for the quinones vary from 5 X 10(5) to 1.1 X 10(3) M-1 s-1 and show an increase with a rise in the one-electron acceptor reduction potential. The mode of adrenodoxin reductase interaction with oxyquinones differs from that of the enzyme interaction with alkyl-substituted quinones and inorganic complexes. NADPH competitively inhibits electron acceptors, whereas NADP+ is a competitive inhibitor of NADPH and a uncompetitive inhibitor of electron acceptors. (Ki = 25 microM). The depth of FAD incorporation into the enzyme molecule as calculated according to the outer sphere electron transfer theory is 6.1 A.     

Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin

Selective chemical modification of cytochrome P-450SCC has been carried out with lysine-modifying reagents. Modification of cytochrome P-450SCC with succinic anhydride was shown to result in loss of its ability to interact with intermediate electron transfer protein - adrenodoxin. To identify amino acid residues involved in charge-ion pairing with complementary carboxyl groups of adrenodoxin, cytochrome P-450SCC complex with adrenodoxin was modified with succinic anhydride. Adrenodoxin was then removed and cytochrome P-450 was additionally modified with isotopically labelled reagent. Subsequent chymotryptic hydrolysis of [14C]succinylated cytochrome P-450SCC and separation of digest obtained by combining various types of HPLC resulted in seven major radioactive peptides. The amino acid sequence of the peptides was determined by microsequencing. The major amino groups modified with radioactive succinic anhydride were found to be at Lys-73, -109, -110, -126, -145, -148 and -154 in the N-terminal sequence of cytochrome P-450SCC molecule and at Lys-267, -270, -338 and -342 in the C-terminal sequence. The role of electrostatic interactions in fixation of cytochrome P-450SCC complex with adrenodoxin is discussed.     

Inhibition of adrenodoxin reductase by NADP+ and NADPH

Adrenodoxin reductase (EC 1.18.1.2) catalyzes the oxidation of NADPH by 1.4-benzoquinone. The catalytic constant of this reaction at pH 7.0 is equal to 25-28 s-1. NADP+ acts as the mixed-type nonlinear inhibitor of enzyme increasing Km of NADPH and decreasing catalytic constant. NADP+ and NADPH act as mutually exclusive inhibitors relative to reduced adrenodoxin reductase. The patterns of 2',5'-ADP inhibition are analogous to that of NADP+. These data support the conclusion about the existence of second nicotinamide coenzyme binding centre in adrenodoxin reductase.     

Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate]

Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.     

Cross-linking studies of adrenocortical cytochrome P-450scc. Evidence for a covalent complex with adrenodoxin and localization of the adrenodoxin-binding domain

A cleavable cross-linking reagent, dimethyl-3,3'-dithiobispropionimidate, was used to study the molecular organization of adrenocortical cytochrome P-450scc. Extensive cross-linking was found to occur, resulting in the formation of heterologous oligomers up to octamer. The covalently cross-linked complex of adrenocortical cytochrome P-450scc with adrenodoxin has been obtained by using dimethyl-3,3'-dithiobispropionimidate. In the presence of NADPH and adrenodoxin reductase, electron transfer to cytochrome P-450scc occurs in the complex, and, in the presence of cholesterol, the latter effectively oxidizes to pregnenolone. By using covalently immobilized adrenodoxin and heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, the adrenodoxin-binding site was shown to be located in the heme-containing, catalytic domain of cytochrome P-450scc. The data obtained indicate the existence of two different sites on the adrenodoxin molecule that are responsible for the interaction with adrenodoxin reductase and cytochrome P-450scc. This is consistent with the model mechanism of electron transfer in the organized complex.     

Growth-regulator Interactions in the Growth of the Shoot System of Avena sativa Seedlings: I. THE GROWTH OF THE FIRST INTERNODE

1. Segments, 3.5 mm. long, cut from the first internode of Avenasativa seedlings grown in complete darkness respond to bothauxins and gibberellic acid by accelerated extension. 2. The optimum concentration of indole-3-acetic acid (IAA) is10 p.p.m. and of gibberellic acid (GA) is 0.1 p.p.m. 3. The degree of stimulation relative to the growth of controlsegments is affected by the inclusion in the segement of thenode between the internode and coleoptile. Thus the gibberellineffect is greatly increased while the IAA effect is decreased.The optimal concentrations are not affected by inclusion ofthe node. 4. These results can best be explained in terms of the supplyby the node tissue of an endogenous auxin which is necessaryfor the expression of GA action. 5. Numerous factorial experiments demonstrated that there isno detectable interaction between applied IAA and GA in thepromotion of first-internode extension. This implies that thepostulated endogenous auxin which synergized GAA action in (4)is either an active form of IAA produced only in the node tissueor is a completely different auxin. 6. No synergism of growth-promotive action can be detected betweenGA and the two synthetic auxins I-naphthylacetic acid and 2,4-dichlorophenoxyaceticacid. 7. p-chlorophenoxy-iso-butyric acid (PCIB) anc 2,4,6-trichlorophenoxyaceticacid (2,4,6-T) act as weak auxins and thus antagonize competitivelythe promotive action of GA. 8. The anti-auxin -(I-naphythyl-methyl-sulphide)propionic acid(NMSP) antagonizes competitively the promotive action of bothIAA and GA. 9. The facts under (5)–(8) suggest that auxins and GAare acting at the same growth-promotion centres and may competefor them. 10. Growth inhibitions are induced by high concentrations ofPCIB, 2,4,6-T and NMSP. The inhibitions produced by PCIB and2,4,6-T are both synergized by supra-optimal concentrationsof IAA while that of NMSP is synergized by supra-optimal concentrationsof both IAA and GA. This similarity of the effects of IAA andGA suggests that their inhibition actions also are of a closelysimilar nature.     

Cytidine Analogues and Stomatogenic Recovery in Amicronucleate Paramecium tetraurelia and Paramecium jenningsi

Removal of the micronuclei of Paramecium tetraurelia and Paramecium jenningsi by micropipetting generates amicronucleate cell lines. These cell lines go through a period of growth depression for several dozen fissions, but they gradually recover. Amicronucleate cells in the depression period characteristically exhibit abnormal oral development, particularly reduction in the length of the buccal cavity and an abnormal pattern of the oral membranelles. To test the notion that the macronucleus is involved in the recovery of amicronucleate cell lines, DNA demethylation drugs were administered to amicronucleates in the depression period. After at least 4 fissions, the treated amicronucleates were assessed for their progress in recovery by scoring the proportion of cells with normal oral membranelles. Cvtidine analogues which demethylate cytosine specifically at the 5 position, namely 5-azacytidine, 5-aza-2'- deoxycytidine and 5-fluoro-2'-deoxycytidine. promoted recovery of the amicronucleates. Cytidine, 6-azacytidine, 2'-fluoro-2'-deoxy-cytidine and cytosine-β-D-arabinofuranoside did not. These results suggest that (i) 5-methylcytosine is present in the macronucleus of these Paramecium species, probably in small amounts and (ii) recovery of amicronucleates involves demethylation of macronuclear DNA. This implies that in normal cells the micronuclei are involved in maintaining the macronuclear DNA in a methylated state and hence the inactivation of the macronuclear sequences that are to be employed for stomatogenic recovery. A general mechanism for the control of gene expression may therefore be employed for the regulation of specific sequences.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.