Purification and properties of reductases for aromatic aldehydes and ketones from guinea pig liver.

NADPH-dependent enzymatic reduction of aromatic aldehydes and ketones observed in the cytosol of guinea pig liver was mediated by at least three distinct reductases (AR 1, AR 2, and AR 3), which were separated by DEAE-cellulose chromatography. By several procedures AR 2 and AR 3 were purified to homogeneity, but AR 1 could be purified only 30-fold because of the small amount. These enzymes were found to have similar molecular weights of 34,000 to 36,000 and similar Stokes radii of about 2.5 nm. AR 3 was identical to aldehyde reductase [EC 1.1.1.2] in substrate specificity for aromatic aldehydes and D-glucuronate and specific inhibition by barbiturates. AR 1 and AR 2 acted on aromatic ketones and cyclohexanone as well as aromatic aldehydes at optimal pHs of 5.4 and 6.0, respectively, and were immunochemically distinguished from AR 3. AR 1 was the most sensitive to sulfhydryl reagents, and AR 2 was more stable at 50 degrees C than the other enzymes. Similar heterogeneity was observed in the kidney enzymes, but other tissues had little aldehyde reductase activity and contained only AR 3. In addition, lung contained a high molecular weight aromatic ketone reductase different from the above reductases.     

Inheritance of extra triradii of palmar interdigital areas II and IV.

Data obtained from 423 individuals were analyzed for mode of inheritance of extra triradii on palmar interdigital area II and IV separately, and in combination. It indicates that a single dominant allele A for extra triradius in the area II, and an allele D in the area IV, may be responsible for the presence of the patterns in the respective area, with 75 to 80 per cent penetrance. There are numbers of individuals in which extra triradii are present in both interdigital areas II and IV. The mode of inheritance has been estimated from the data obtained and was found to have co-dominant alleles A and D for the presence in their respective areas. Further analysis revealed the existence of three alleles. A and D for presence, and 0 for absence. The alleles A and D are co-dominant, if combined in the genotype AD, but either allele is dominant in heterozygous combination with 0.     

Biochemical basis for the resistance of guinea pigs to carcinogenesis by 2-acetylaminofluorene

Studies were made on why guinea pigs are resistant to carcinogenesis by 2-acetylaminofluorene. Cytochrome P-448 and arylhydrocarbon hydroxylase were not induced in either the microsomes and nuclei of guinea pigs by 3-methylcholanthrene treatment. 3-Methylcholanthrene treatment caused only 2-fold increase in the binding of 2-acetylaminofluorene to DNA in nuclei isolated from guinea pigs, while it caused 17-fold increase in the binding in rat nuclei. Microsomes from 3-methylcholanthrene treated rats had 5 times more effect than Microsomes from 3-methylcholanthrene treated guinea pigs on the binding of 2-acetylaminofluorene to DNA of nuclei from untreated guinea pigs. N-Hydroxy-2-acetylaminofluorene combined equally well with the DNA of rats and guinea pigs. In guinea pigs, there was a good correlation between the low inducibility of cytochrome P-448 and the low binding of 2-acetylaminofluorene to DNA. Our results clearly showed that guinea pigs are resistant to tumor induction by 2-acetylaminofluorene through inability to carry out the first step of activation of 2-acetylaminofluorene.     

Temperature dependent ionic structure of phospholipid monolayers

Radiotracer studies of calcium adsorption to dipalmitoylphosphatidyl-alkanolamine monolayers measured at various temperatures showed that the binding constant of calcium increased with temperature up to around 30°C but then decreased on exceeding this critical temperature.The temperature dependent ionic structure of ampholytic phospholipid monolayers are discussed.     

Pumpkin (Cucurbita sp.) seed globulin I. Purification, characterization, and subunit structure

A heat stable globulin present in the cotyledons of pumpkinseeds was prepared as crystals which were soluble in a dilutesaline solution below pH 4.5 or in a solution with a high ionicstrength at neutral pHs. The protein was nearly homogeneousby ultracentrifuge analysis, and had a molecular weight of about112,000 daltons. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisseparated the globulin into two subunits, and ß,corresponding to molecular weights of about 63,000 and 56,000daltons, respectively. By reduction of disulfide bonds, thetwo subunits were each separated into two polypeptide chainswith molecular weights of around 36,000 and 22,000 daltons,judged by gel electrophoresis. The amino acid composition ofwhole globulin indicated high contents of arginine, glutamicacid and aspartic acid. The total number of half-cystine residuewas nine and only one residue was shown to be free. The subunitstructure of the globulin is discussed. The protein has beenshown to have oxaloacetate decarboxylase activity, and thisfact was confirmed. However, the activity decreased markedlyat pH 4.5 in a fairly short period. It did not require Mn⁺⁺,and the K_m for oxaloacetate was determined to be 4.1 mM. (Received April 9, 1976; )     

Degradation of rod outer segment proteins by cathepsin D.

The degradation of proteins of the rod outer segment (ROS) fraction by partially purified cathepsin D [EC 3.4.23.5] from the retinal pigment epithelium was studied. The ROS fraction, prepared from bovine eyes by sucrose density gradient centrifugation, had little cathepsin D activity. Partially purified cathepsin D, obtained from crude extract of bovine retinal pigment epithelium using bovine serum albumin as a substrate, hydrolyzed the porteine of the ROS fraction. The rate of degradation of ROS proteins was proportional to both the enzyme concentration and the incubation time. With ROS proteins as substrate, the optimal pH of cathepsin D was about 3.5. The degradation of ROS proteins was inhibited by pepstatin.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF HUMAN CEREBROSPINAL FLUID

Abstract— Cyclic 3',5'-AMP (cAMP) and cyclic 3',5'–GMP (cGMP) phosphodiesterase activities were found in human cerebrospinal fluid (CSF) using low substrate concentration (0.4μM). More rapid hydrolysis of cGMP than that of cAMP was observed in human CSF. However, cGMP hydrolytic activity of CSF was very much lower (0.3 pmol/min/ml CSF) than that of human cerebral cortex (33.7 nmol/min/g wet cortex). The pH optimum was found to be 8.0 (cGMP phosphodiesterase) and 7.5 (cAMP phosphodiesterase). The maximum stimulation of both cAMP and cGMP phosphodiesterase was achieved at 4 mM-MgCl₂. Cyclic AMP had relatively little effect on the hydrolysis of cGMP in CSF and the cortex, while cGMP inhibited hydrolysis of cAMP in both tissues. Snake venom was found to stimulate cAMP and cGMP phosphodiesterase activity of CSF, by 60% and 110% respectively. This stimulation by snake venom was also observed in the cortex phosphodiesterase, but was not observed in human plasma or thyroid phosphodiesterase. When CSF was applied to Sepharose 6B column, cGMP phosphodiesterase was separated into three different molecular forms. A plot of activity against substrate concentration using peak I (largest molecular size) revealed a high affinity ( K _m= 2.6μM) and a low affinity ( K _m= 100μM) for cAMP suggesting the existence of at least two molecular forms of the enzyme. On the other hand, using a cGMP as substrate the only one K _m value (1.90 μm) was obtained. These K _m values of CSF enzymes described above were close to those obtained from human cerebral cortex preparations. The enzyme under peak I corresponded to the cortex enzyme when judged from its molecular size and stimulation by snake venom. It seems likely from our results that at least a part of CSF phosphodiesterase originates from the central nervous system.     

A dynamic model of the receptive field of L-cells in the carp retina

A dynamic model of the receptive field of L2-cells in the carp retina is developed by using our experimental results on the basis of physiological and morphological evidences. Linear spatial summation is assumed in the model for the interactions among L2-cells. Linear forward and feedback loops are also assumed for the interactions between L2-cells and cones. The model has dynamic properties similar to the ones of the receptive field of L2-cells: L2-cells respond faster as the size of a light spot is enlarged and the L2-cells nearer to the center of the light spot respond faster. It is suggested that the faster responding properties of L2-cells are due to the feedback action.     

Relationship between changes in properties and contents of riboflavin derivatives of NADPH-cytochrome P-450 reductase in the liver microsomes of riboflavin-deficient rats

Weanling male rats were fed a riboflavin-deficient diet for 5-8 weeks, and the decrease in NADPH-cytochrome P-450 reductase (FpT) activity in the liver microsomes was compared with the contents of riboflavin derivatives. The decrease of FpT activity for the reduction of cytochrome c was greater than that for the reduction of ferricyanide. The FpT's of riboflavin-deficient and control rats were indistinguishable in the Ouchterlony immunodiffusion test against anti-FpT, and were shown to have the same molecular weight of 78,000 by SDS-polyacrylamide slab gel electrophoresis. However, the purified FpT of the riboflavin-deficient rats contained 14.2, 4.9, and 1.9 nmol of FAD, FMN, and riboflavin per mg of protein, respectively, while that of the control rats contained 10.6 and 9.5 nmol of FAD and FMN per mg of protein, respectively. After riboflavin injection into the riboflavin-deficient rats, NADPH-cytochrome c reductase activity and FMN content of the FpT were restored to the control levels in 36 h, NADPH-ferricyanide reductase activity recovered in 18 h, and riboflavin content diminished in 18 h. On incubation of the purified FpT of the riboflavin-deficient rats with FMN, NADPH-cytochrome c reductase activity and FMN content were restored to those of control rats. These results indicated that a part of FMN in the FpT of the riboflavin-deficient rats was replaced with FAD and riboflavin.