Restriction fragment length polymorphisms detected in N-ras-related sequences of rats and their linkage analyses

Novel restriction fragment length polymorphisms (RFLPs) in inbred rats were revealed with the human N-ras gene as probe. Three fragments hybridizing to the probe were detected by Southern blot hybridization under highly stringent conditions, and one of the fragments showed variation in inbred rat strains. Furthermore, on hybridization under low-stringency conditions, an additional fragment hybridizing to the probe was observed, and this fragment also showed interstrain variation. These two variant fragments showed different distributions in 27 inbred rat strains and segregated in backcross progeny as codominant alleles of independent single autosomal loci. Therefore, the loci for these RFLPs were named Nras-1 and Nras-2, respectively. Analyses of linkages between the RFLPs and 11 other loci revealed that the Nras-2 locus was closely linked to the c locus (3.7 +/- 2.6%), which belongs to rat linkage group I.     

Arachidonic acid stimulates phosphoinositide metabolism and catecholamine release from bovine adrenal chromaffin cells

Arachidonic acid (AA) evoked a dose-dependent increase in the accumulation of inositol phosphates in cultured bovine adrenal chromaffin cells, and this effect was specific for AA. AA also induced a rise in [Ca2+]i, but this rise was markedly reduced by removal of extracellular Ca2+. AA-induced accumulation of inositol phosphates was absolutely dependent on extracellular Ca2+, and nicardipine and nifedine partially reduced it but verapamil had no effect. Moreover, AA dose-dependently stimulated catecholamine release from chromaffin cells in the presence of ouabain, and this effect was specific for AA. AA-induced catecholamine release in the presence of ouabain was also inhibited by nicardipine and nifedipine but not by verapamil. Furthermore, the phospholipase C inhibitor neomycin inhibited the release. These results taken together suggest that AA stimulates catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism in a Ca2(+)-dependent manner.     

Characterization of a cDNA for the unexpressed form of cytochrome P-450g from the (-g) rat and differentiation of its mRNA from that of the (+g) phenotype using specific oligoprobes

Our laboratory recently isolated a cDNA for cytochrome P-450g (IIC13), a male-specific, highly polymorphic P-450 isozyme, from livers of the high phenotype (+g) of Sprague-Dawley rats [McClellan-Green et al. (1989) Biochemistry 28, 5832-5839]. Hybridization studies using a specific oligonucleotide probe for P-450 (+g) indicated that equivalent amounts of P-450g mRNA were present in livers of both the high and low phenotypes (+g and -g) of male Sprague-Dawley, Fischer (inbred -g), or ACI (inbred +g) rats. In the present study, we isolated one full-length and one nearly full-length cDNA clone coding for the unexpressed form of cytochrome P-450g from a cDNA library constructed from mRNA from a (-g) male Sprague-Dawley rat. The longest cDNA had an open reading frame of 1473 nucleotides which coded for a 490 amino acid polypeptide of Mr 55,839. Although the 5'-noncoding leader sequence and the 3'-noncoding region were unchanged, the coding sequence of the (-g) phenotype differed from that of the cDNA isolated from the (+g) phenotype by nine bases changes. These base changes would result in seven amino acid differences between the protein sequences for the two phenotypes. Two specific oligonucleotide probes for (+) P-450g and (-) P-450g containing three base differences between the (+g) and (-g) sequences hybridized differentially to mRNA from the (+g) and (-g) phenotypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of protein kinase C in prostaglandin E2-induced catecholamine release from cultured bovine adrenal chromaffin cells

We recently reported that prostaglandin (PG) E2 stimulated phosphoinositide metabolism in cultured bovine adrenal chromaffin cells and that PGE2 and ouabain induced a gradual secretion of catecholamines from the cells (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). Here we examined the involvement of two signal pathways, Ca2+ mobilization and protein kinase C activation resulting from phosphoinositide metabolism, in the PGE2-induced catecholamine release. Either the Ca2+ ionophore ionomycin or 12-O-tetradecanoylphorbol 13-acetate (TPA) could enhance the release in the presence of ouabain, and ionomycin-induced release was additive to PGE2-induced release, but TPA-induced release was not additive. PGE2 dose-dependently stimulated the formation of diacylglycerol and caused the translocation of 4% of the total protein kinase C activity to become membrane-bound within 5 min. These effects were specific for PGE2 and PGE1 among PGs tested (PGE2 = PGE1 greater than PGF2 alpha greater than PGD2). Furthermore, the phosphoinositide-specific phospholipase C inhibitor neomycin inhibited PGE2-induced accumulation of inositol phosphates, diacylglycerol formation, translocation of protein kinase C, and also stimulation of catecholamine release. Both PGE2- and TPA-induced release were inhibited by the depletion of protein kinase C caused by prolonged exposure to TPA, but ionomycin-induced release was not inhibited. We recently found that the amiloride-sensitive Na+, H+-antiport participates in PGE2-evoked catecholamine release (Tanaka, T., Yokohama, H., Negishi, M., Hayashi, H., Ito, S., and Hayaishi, O. (1990) J. Neurochem. 54, 86-95). In agreement with our recent report, PGE2 and TPA induced a sustained increase in intracellular pH that was abolished by the protein kinase C inhibitor staurosporine but not by the calmodulin inhibitor W-7. Ionomycin also induced a marked increase in intracellular pH, but this increase was abolished by W-7 but not by staurosporine. These results demonstrate that PGE2-induced activation of the Na+, H(+)-antiport and catecholamine release in the presence of ouabain are mediated by activation of protein kinase C, rather than by Ca2+ mobilization, resulting from phosphoinositide metabolism.     

Chemical modification of coenzyme B12-dependent diol dehydrase with pyridoxal 5′-phosphate: Lysyl residue essential for interaction between two components of the enzyme

The apoenzyme of diol dehydrase was inactivated by modification with pyridoxal 5′-phosphate (pyridoxal-P). The inactivation was accompanied by appearance of a new peak at 425 nm which was shifted to 325 nm by reduction with NaBH₄. ?-N-Pyridoxyl lysine was detected by paper chromatography and paper electrophoresis from the hydrolysate of the NaBH₄-reduced enzyme-pyridoxal-P complex. The relationship of inactivation vs pyridoxal-P incorporation as well as kinetic experiments suggests that one lysyl residue per enzyme molecule was essential for catalytic activity, although two to three pyridoxal-P molecules were introduced into the almost completely inactivated enzyme molecule. Both 1,2-propanediol (substrate) and adenosylcobalamin (coenzyme) completely protected the enzyme from inactivation. The result of disc gel electrophoresis showed that the inactivation of diol dehydrase by pyridoxal-P results from irreversible dissociation of the enzyme into subunits upon pyridoxal-P modification. Therefore, it is suggested that this modifiable lysyl residue is essential for subunit interaction to form an active oligomeric enzyme. The inactivated enzyme restored activity by addition of excess component F, but not by S, suggesting that the essential lysyl residue is located in component F of the enzyme. Pyridoxal-P-modified enzyme was no longer able to bind cyanocobalamin (a competitive inhibitor of adenosylcobalamin).     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

Accumulation pattern of arachin and its subunits in maturation of groundnut seeds

The accumulation pattern of arachin and its subunits in growinggroundnuts was investigated. Soluble proteins were extractedfrom the kernels at twelve different stages of maturation (4–16weeks after pegging). Fractionation showed arachin, conarachinII, 5S and 2S protein components with sucrose gradient centrifugation.Ten weeks after pegging, only 35% of the maximum amount of arachinhad accumulated, whereas conarachin II was 85%, the 5S component89%, and the 2S component 76%. Arachin, however, increased rapidlyin the later stage of maturation. No change in the subunit ratioin arachin during seed growth was observed on the patterns ofsodium dodecyl sulfate-gel electrophoresis and gel isoelectricfocusing in the presence of urea. The ratio of the arachin subunitscontained in urea-extractable fraction of the kernels was constantthroughout seed development and was consistent with the subunitratio in arachin. On the other hand, the arachin subunits inthe free forms, if any, accounted for less than 1% of the associatedarachin subunits. Probably, the arachin subunits synthesizedin equimoles are associated into arachin without individualdeposition and are accumulated as arachin associates in growingseeds. (Received July 17, 1980; )     

Synthesis of biologically active cyclic peptides

1. The extent of racemization and the coupling yield in peptide synthesis were studied under high dilution conditions. The azide method yielded the best results. 2. Five linear penta-peptide precursors related to gramicidin S were subjected to cyclization in order to study how the difference in the sequence influences the yield and the ratio of cyclic dimer to monomer. The azide with the sequence of -L -Pro-L -Val-L -Orn(Z)-L -Leu-D -Phe- afforded diZ-gramicidin S in a high yield of 63%. 3. Alternaria mali toxin III, a cyclotetradepsipeptide phytotoxin, was synthesized. The activated linear tetradepsipeptide containing a D -Dap(Z) (N³-Z-D -2,3-diaminopropionic acid) residue at the N-terminus afforded the cyclic precursor (53%). The Dap residue in the precursor was converted into a ΔAla residue by Hofmann degradation to give the desired product.     

Histochemical studies on catecholaminergic cells in the carp retina

Histochemical studies on catecholaminergic cells were conducted with the carp (Cyprinus carpio) retina. Catecholamine (CA)-containing cell bodies appear sparsely distributed among amacrine cells in the innermost cellular row of the inner nuclear layer (INL) and occasionally in the outer half part of the inner plexiform layer (IPL); only exceptionally are they found among ganglion cells. The fluorescent cells interspersed with the amacrine cells and in the IPL send their fiber processes toward both the outer plexiform layer (OPL) and the IPL; the fine fibers form dense networks in the INL and IPL. Pretreatment of the fish with intramuscular injection of reserpine (20 hr prior to enucleation) completely depleted CA from the retina. The fluorescence of catecholaminergic cells was enhanced, and the number of fluorescent cells visible was increased, by intravitreous injection ofl-DOPA, DA, and NA (3 hr prior to enucleation). A combination of pretreatment with intramuscular reserpine and intravitreous NA was particularly effective. These results indicate that catecholamines may play an important role in the modulation of the membrane potential of horizontal cells.