Effect of calmodulin on aldosterone synthesis by a cytochrome P-45011 beta-reconstituted system from bovine adrenocortical mitochondria

Bovine adrenocortical calmodulin was purified and its general properties were examined. The latter were similar to those of bovine brain calmodulin. When added to a cytochrome P-450(11)beta-reconstituted system in the presence of dilauroylphosphatidylcholine, calmodulin decreased the rate of aldosterone production from corticosterone from 0.8 to 0.1 nmol/(min X nmol P-450), while it increased the rate of 18-hydroxycorticosterone production from 1.8 to 4.6 nmol/(min X nmol P-450). This effect of calmodulin on steroid production was maximum at a concentration of 1 microM, when 1 microM cytochrome P-450(11)beta was used. The effect was dependent on the presence of Ca2+, and maximal response was observed at less than 1 microM Ca2+. There was essentially no difference in the effect when bovine brain calmodulin was used. Calmodulin induced a change in the activity of cytochrome P-450(11)beta in the presence of a wide concentration range of corticosterone as a substrate. As for 18-hydroxycorticosterone production, calmodulin increased both the maximal activity and the apparent Km for corticosterone, but it decreased the apparent Km for adrenodoxin. Adrenodoxin at a concentration of less than 20 microM did not fully abolish the effect of calmodulin. A small type I difference spectrum appeared when calmodulin was added to cytochrome P-450(11)beta. The difference spectrum increased significantly in the presence of both Ca2+ and adrenodoxin. These results suggest that calmodulin interacts with cytochrome P-450(11)beta in the presence of adrenodoxin and then modulates the activity of aldosterone synthesis catalyzed by cytochrome P-450(11) beta.     

3-Methylhistidine content and pH dependency of ATPase activity of adult and embryonic chicken cardiac ventricular myosins

A distinct difference in the 3-methylhistidine (3-MeHis) content and the pH-dependency curve for calcium-activated adenosine triphosphatase (Ca-ATPase) activity was observed between chicken and mammalian cardiac ventricular myosins. The 3-MeHis content and pH dependency of the Ca-ATPase activity of myosins from adult and embryonic chicken cardiac ventricular muscles and chicken fast white and slow red muscles were almost the same.     

Homeostatic Regulation of Membrane Potential by an Electrogenic Ion Pump against Change in the K+ Concentration of the Extra- and Intra-Organ Perfusion Solutions

The dependence of membrane potentials on changes in the extra-cellularK⁺ concentration [K⁺]_e was investigated in potato tuber sliceswith dripping perfusion, and in growing Vigna hypocotyl segmentswith pressurized intra-organ perfusion methods. Only under anoxiawere the membrane potential of potato tuber slices and the electricpotential difference between the parenchyma symplast and xylem(V_px) of Vigna hypocotyl segments depolarized markedly (46 mVand 42 mV/log[K⁺]_e unit, respectively) with increasing [K⁺]_eabove the critical values. The electric potential differencebetween the parenchyma symplast and organ surface (V_ps of thehypocotyl segments remained nearly unchanged up to 30 mEq [K⁺]_e.Under highly aerobic conditions the membrane potentials wererelatively independent of [K⁺]_e except at very high K⁺ concentrations.V_ps showed even hyperpolarization with the increasing KCl concentrationin the perfusion solution that is not in direct contact withthe surface membrane of the parenchyma symplast. The respiration-dependentelectrogenic components of the membrane potentials regularlyincreased with the increasing [K⁺]_e. A voltage-dependent homeostaticcontrol of membrane potential is discussed. (Received August 13, 1984; Accepted December 21, 1984)     

A streamlined method of subfragment one preparation from myosin

A rapid procedure for isolating subfragment one (SF1) from myosin was found. SF1 can be isolated specifically from proteolytic digests of myosin in the presence of a millimolar concentration of magnesium chloride. Under such ionic conditions all of the rod portion and undigested myosin is selectively precipitated. A nucleotide trapping experiment indicated how important quick preparation of SF1 is for maintaining the active site structure. This method can also be utilized in the preparation of heavy meromyosin.     

Changes of plasma membrane proteins during prespore differentiation in Dictyostelium discoideum

By the use of a shake culture system, we have previously shown (Oyama, M., Okamoto, K., & Takeuchi, I. (1982) J. Cell Sci. 56, 223-232) that both cAMP and cAMP-dependent cell contact are required for prespore differentiation in Dictyostelium discoideum. The present study was undertaken to examine changes of the plasma membrane proteins during prespore differentiation in the shake culture system. Rabbit antibodies prepared against the plasma membrane fraction of the differentiated cells inhibited the reaggregation of the differentiated cells but not that of aggregation-competent cells. This result indicates that new contact sites are formed in the differentiated cells. By the combined use of the antibody-conjugated immuno-adsorbent with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, changes of membrane proteins were analyzed with the cells incubated under various conditions. Three proteins were found to be present specifically in the differentiated cells only in the presence of cAMP, one of which (105K protein) appeared when cells became adhesive, but before prespore specific proteins were detected. Two others (80K and 58K proteins) appeared during prespore differentiation after cells formed agglomerates.     

Partially purified "activated" receptor-glucocorticoid complex from rat liver: regulation of nuclear, chromatin, and DNA-cellulose binding of "activated" complex by pyrophosphate and ATP

The effects of PP1 and ATP on nuclear binding of the "activated" receptor-[3H]-triamcinolone acetonide (TA) complex purified about 3,000-fold from adrenalectomized rat liver were investigated. ATP at up to 5 mM did not affect nuclear binding of the "activated" complex, but PP1 at 2-7 mM greatly enhanced it. However, ATP in the presence of PP1 decreased nuclear binding dose-dependently. Similar results were obtained in the case of chromatin binding, but PP1 alone did not alter DNA-cellulose binding of the "activated" complex, suggesting that the binding sites for chromatin and DNA on the "activated" complex are different. Furthermore, PP1 enhanced ATP-agarose binding of the "activated" complex, indicating that the PP1 binding site(s) on the receptor is different from the ATP binding site(s). The physicochemical properties of the "activated" receptor-glucocorticoid complex bound with ATP and/or PP1 were examined by sucrose density gradient centrifugation and Sephadex G-150 gel filtration. There was no detectable change in the sedimentation coefficient or molecular weight (about 4.2S; Mr approximately equal to 98,000) on binding with ATP and/or PP1. These results suggest that the binding of PP1 and PP1 plus ATP to the "activated" complex caused some allosteric change of the acceptor binding sites of the receptor, resulting in increase or decrease in its binding to nuclei, chromatin, or DNA.     

Selective inhibition of thrombin by (2R,4R)-4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl++ +) sulfonyl]-l-arginyl)]-2-piperidinecarboxylic acid

The potency of thrombin inhibition by 4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-sulfony l]- L-arginyl]-2-piperidinecarboxylic acid (MQPA) depended on the stereoconformation of the 2-piperidinecarboxylic acid moiety. Ki values for bovine alpha-thrombin were 0.019 microM with (2R,4R)-MQPA, 0.24 microM with (2R,4S)-MQPA, 1.9 microM with (2S,4R)-MQPA, and 280 microM with (2S,4S)-MQPA. (2R,4R)-MQPA of the four stereoisomers of MQPA was also the most potent inhibitor for other trypsin-like serine proteases with Ki values of 5.0 microM for trypsin, 210 microM for factor Xa, 800 microM for plasmin, and 1500 microM for plasma kallikrein. Examination of the potency of thrombin inhibition by arginine derivatives related to MQPA in structure suggested the presence of a specific binding site for the carboxamide portion (C-terminal side). The relative inhibitory potency of the four stereoisomers of MQPA for trypsin was nearly identical with that for thrombin, suggesting that the specific binding site for the carboxamide portion is present in both enzymes. Modification of thrombin by phosphopyridoxylation or the presence of heparin did not significantly alter the binding of MQPA.     

Molecular characterization of mutant actin genes which induce heat-shock proteins in Drosophila flight muscles

Heat-shock proteins (hsps) are constitutively induced by the mutant actins in the Drosophila indirect flight muscles (IFM). We compared primary structures of the mutant actin genes (KM75 and HH5) which induce hsps and of the non-inducing alleles (KM129 and KM88). The KM75 actin has lost 20 amino acids at the C-terminus. The HH5 actin has only one amino acid substitution, from Gly-336 to Ser. In KM129, the C-terminal part of actin is replaced by novel amino acids. KM88 is a null allele, with an amber mutation early in the coding region of the mutated actin gene. Although all of the KM75, HH5 and KM129 actins have defects near the C-terminus, only hsp-inducing mutant actins cause enlargement of the IFM nuclei as well as a disruption of myofibrils even in the presence of two copies of the normal genes. We further consider the underlying mechanisms linking these features of the hsp-inducing alleles.     

Detection of carcinogen-induced DNA breaks by nick translation in permeable cells

A nick-translation reaction with $\text{E. coli}$ DNA polymerase I (pol. I) was used to detect $\text{in situ}$ DNA breaks produced by chemical carcinogens. Normal human fibroblasts treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in various doses were permeabilized with lysolecithin, and were nick translated in the presence of [³H]dCTP and pol. I. The radioactivity incorporated increased with MNNG concentration, and was directly proportional to the poly(ADP-ribose) synthetase activity. Other DNA-damaging agents such as bleomycin or 4-nitroquinoline 1-oxide also caused the nick translation rate to increase. When MNNG-treated cells were cultured in fresh medium containing no MNNG, the increase in the rate of nick translation in permeable cells became less and this decrease was abolished by addition of aphidicolin or cytosine arabinoside. The nick translation method described here may be a useful means for estimating intrinsic DNA breaks in cells treated with carcinogens.     

Modulatory Action of Taurine on the Release of GABA in Cerebellar Slices of the Guinea Pig

Abstract: For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization-induced Ca-dependent release of [³H]γ-aminobutyric acid (pH]GABA) and l -[³H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [³H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of l -[³H]glutamate was little affected by taurine. The release of [³H]GABA was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [³H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [³H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca²⁺ into cerebellar tissues was tentatively suggested.