Influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase in rat striatal membranes

The influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase activity were investigated in rat striatal membranes. GTP promoted and inhibited the activity at 1 and 100 microM, respectively. The inhibitory effects of GTP were abolished by pretreatment of the membranes with pertussis toxin. GppNHp (guanyl-5'-y1-beta,gamma-imidodiphosphate) exerted only stimulatory effects and pertussis toxin did not affect the effects of GppNHp. GDP at 10 and 100 microM caused significant inhibition which was completely suppressed by pertussis toxin. It is suggested that guanine nucleotide regulates the affinity of as in stimulatory GTP-binding regulatory protein to either beta gamma or catalytic units of adenylate cyclase in a flip-flop manner. Inhibitory GTP-binding regulatory protein seems to play a regulatory role in inhibiting alpha s activity supplying the beta gamma heterodimer.     

Isolation of U1-snRNP(s) from mouse teratocarcinoma cells using immunochemical and biochemical procedures: high proportion of U1a-snRNP to U1b-snRNP

An attempt was made to immunochemically and biochemically purify and characterize the U1-snRNP(s) of mouse embryonal carcinoma cells. The results obtained by RNA analysis of U1-snRNP(s) purified immunochemically from embryoid bodies, F9 cells and PYS-2 cells indicated that the U1-snRNP(s) in these cells consisted of U1a-snRNP and U1b-snRNP. The proportion of U1a-snRNP to U1b-snRNP was also found to be high in the embryoid bodies and F9 cells. The U1a-snRNP predominance in U1-snRNP population was also detected in PYS-2 cells. The immunochemically purified U1-snRNP population from liver nuclei of 129 syngeneic male mouse (129/sv), a host mouse for transplantable tetratocarcinoma OTT6050, and ICR male mouse, contained approximately equal levels of the two U1-snRNP species (U1a- and U1b-snRNP). Partially purified U1-snRNP from embryoid bodies was also obtained by elution from a DEAE-Sepharose column at around 0.18 M NH4Cl or by fractionation by 5-20% linear sucrose gradient centrifugation. The electrophoretic RNA profiles of the partially purified U1-snRNP of embryoid bodies were similar to those obtained immunochemically.     

Aldose reductase inhibitors: flavonoids, alkaloids, acetophenones, benzophenones, and spirohydantoins of chroman

The inhibitory activity of various compounds, including 12 flavonoids, 10 alkaloids, 15 benzophenones, 5 acetophenones, and 7 spirohydantoins of chroman, was tested on rabbit lens aldose reductase, an enzyme involved in complications of diabetes. Almost all compounds tested were found to inhibit the enzyme at low concentrations (10(-5) M). The most potent inhibitor was 2R,4S-6-chloro-2-methylspiro(chroman-4,4'-imidazo-lidine+ ++)-2',5'-dione with an I50 value of 4.7 x 10(-8) M; other spirohydantoins showed similar potency. Polyhydroxybenzophenones were also potent inhibitors with an I50 value of about 10(-7) M. The possible structure-inhibitory activity relationships of the compounds tested are discussed.     

Isolation of porcine follicular fluid inhibin of 32K daltons

Purification of ovarian inhibin from porcine follicular fluid was performed by using an bioassay based upon the suppression of spontaneous FSH release from cultured cells of rat anterior pituitary. The presence in the follicular fluid of four molecular forms of inhibin activity corresponding to Mr 100K, 80K, 55K and 32K was revealed by SDS-gel electrophoresis under non-reducing conditions. The smallest inhibin amongst them, named 32K inhibin, eliciting about 70% of the total activity in the follicular fluid, was separated by gel filtration in the presence of 8 M urea. By subsequent ion-exchange chromatography, followed by RP-HPLC, 32K inhibin was purified to homogeneity with a 8,000 fold purification factor in a yield of 12%. The purified 32K inhibin was found to comprise two polypeptide subunits (Mr 20K and 13K), linked by disulfide bridges and to specifically suppress the secretion of FSH, but not of LH from the pituitary cells.     

Promotion of Rooting in Azukia Cuttings by Possible Glycoproteins Extracted from Lentinus edodes Culture

Macromolecules purified from Lentinus edodes mycelia cultureenhanced adventitious root formation in Azukia epicotyl cuttings.Partial purification by sequential column chromatographies gavematerial composed of 71% polysaccharides and 29% proteins. Thesugar moiety consisted of mainly xylose, glucose and arabinose,the sum of their contents being more than 76% of the total carbohydrates.The protein moiety consisted of mainly glycine and serine, whichaccounted for more than 40% of the total amino acid residues. (Received June 9, 1984; Accepted November 12, 1984)     

Purification and Properties of Sweet Potato NADPH-Cytochrome c (P-450) Reductase

NADPH-cytochrome c reductase, strictly NADPH-cytochrome P-450reductase, was purified by chromatography through DEAE-cellulose,2',5'-ADP-Sepharose, and Sephadex G-100 columns after solubilizationfrom microsomes from Ceratocystis fimbriata-infected sweet potatoroot tissue with Emulgen 913. The enzyme existed in three formsafter solubilization which migrated to positions correspondingto molecular weights of 81,000, 75,000 and 72,000 on an SDS-polyacrylamidegel. Trypsin treatment of the enzyme species with the largestpolypeptide yielded the species with the smallest one. Aftersucrose density gradient centrifugation of the pellet fractionobtained by centrifugation at 100,000?g of the crude extract,the enzyme species with the largest polypeptide was presentin the particulate fractions, whereas that with the smallestone was only found at the top of the gradient. We conclude thatthe enzyme species with the largest polypeptide is in an intact,amphipathic form, whereas that with the smallest one, and probablyalso the other species, is its hydrophilic domain produced byan endogenous protease(s). The K_m values of the enzyme in theintact form for NADPH and cytochrome c were 7.7 and 2.3 µM,respectively. ¹ Present address: Laboratory of Food Hygienics, Faculty ofAgriculture, Kagawa University, Miki-cho, Kida-gun, Kagawa 761-07,Japan. (Received September 6, 1984; Accepted December 27, 1984)     

A Comparison of Tomato Fruit Chloroplast and Chromoplast DNAs as Analyzed with Restriction Endonucleases

Chromoplast DNA was isolated from ripe-red tomato fruits, andits structure compared with that of chloroplast DNAs from maturegreen fruits and leaves. There was a good correspondence betweenthe structures analyzed by BamHI or EcoRI digestion and by hybridizationwith a probe for the gene of the ribulose 1,5-bisphosphate carboxylase/oxygenaselarge subunit. ¹ Present address: Koryo International College, Nisshin, Aichi470-01, Japan. (Received November 5, 1984; Accepted February 6, 1985)     

Direct-acting mutagenicity of N4-aminocytidine derivatives bearing alkyl groups at the hydrazino nitrogens.

To investigate the mechanism of N4-aminocytidine-induced mutagenesis, N'-alkyl-N4-aminocytidines and N4-alkyl-N4-aminocytidines were prepared and their mutagenicity on bacteria were assayed. N'-Methyl-N4-aminocytidine, N'-(2-hydroxyethyl)-N4-aminocytidine and N',N'-dimethyl-N4-aminocytidine showed direct-acting mutagenicity on S. typhimurium TA100 and E. coli WP2 uvrA, tester strains that are sensitive to base-pair substitutions. In contrast, N4-methyl-N4-aminocytidine, N4-(2-hydroxyethyl)-N4-aminocytidine and N4,N'-dimethyl-N4-aminocytidine were not mutagenic on these bacteria. Since N'-methyl-N4-aminocytidine does not form hydrazones, the possibility that N4-aminocytidine causes mutation due to its reactivity with carbonyl compounds has been excluded. Furthermore, the fact that only those alkyl N4-aminocytidines having a hydrogen on the nitrogen at position 4 are mutagenic is consistent with the previously proposed mechanism in which the tautomerization between the amino and the imino forms of N4-aminocytosine allowing an ambiguous base pairing is the cause of the mutagenesis.     

Light-induced linear dichroism in photoreversibly photochromic sensor pigments. – VI. Relation between the two pigments of the mycochrome system in Alternaria cichorii

Conidiation in Alternaria cichorii Nattras is reversibly stimulated by near ultraviolet radiation (NUV, ca 313 nm) and inhibited by blue light (ca 450 nm) and seems to be a mycochrome-mediated process. After induction with plane-polarized NUV, blue light polarized perpendicularly to the NUV was more effective in counteracting the induction than was blue light polarized parallel to the NUV. From this the conclusions are drawn that (a) both the blue-absorbing component (presumably a flavo-protein) and the P_NUV of the mycochrome system are membrane-bound and that (b) the transition moment associated with blue light absorption in the presumed flavoprotein forms an angle of at least 53° with the transition moment associated with NUV absorption in P_NUV.