Hydrolysis of storage proteins in barley endosperms : analysis of soluble products

Soluble products, released by the hydrolysis of hordeins into the media of barley (Hordeum vulgare cv. Perth) half-seeds were analyzed. Large polypeptide fragments (methanol-insoluble) were identified using the Western immunoblot technique with the antibodies prepared against B and C polypeptides of hordein. A number of hordein IgG-reacting bands were noted in the samples from dry kernels. In samples incubated in the absence of gibberellic acid, polypeptide fragments in the size range of 25 to 30 kilodaltons appeared within 24 hours, and those in the size range of 40 kilodaltons became more prominent. In samples incubated in the presence of gibberellic acid, polypeptide fragments in the size range of 45 to 67 kilodaltons were less apparent and those in the size range of less than 15 kilodaltons were more pronounced. The hordein-related polypeptide fragments were present in low amounts after 72 hours in the presence of gibberellic acid. Methanol-soluble peptides were fractionated, on the basis of size, into two broad peaks. In the absence of gibberellic acid, there was no significant change in their profile over a 72 hour incubation period. In the presence of this growth substance, however, there was a decrease in the proportion of large size peptides (50-70 amino acid residues in length), and an increase in the levels of small peptides (15-35 amino acid residues in length) and amino acids. Our interpretation of the results is that the release of the initial large polypeptide fragments from hordein proteins is mediated by a protease(s) whose appearance is not dependent on the exogenously added gibberellic acid. Further hydrolysis is, however, mediated by proteases induced in the presence of this growth substance.     

Alkaloids of Litsaea laeta and L. salicifolia

The structure for laetine (2-hydroxy-1-methyl 10,11-methylenedioxy noraporphine), a new alkaloid from the bark of Litsaea laeta has been established. N,O-Dimethylharnovine and glaucine were also isolated from the same source. C-2 hydroxy aporphines are characteristic of L. laeta. Two known alkaloids, dicentrinone and nordicentrine, have also been isolated from the leaves of L. salicifolia.     

Structure of asclepin and some observations on the NMR spectra of Calotropis glycosides

Asclepin has been shown to be 3′-O-acetylcalotropin using various chemical and physical methods. The NMR data of all the Calotropis glycosides have been analysed in conjunction with that of asclepin, certain anomalies have been pointed out and revised structures have been proposed for the acetylated glycosides.     

Steroids and related products. 38. 11-Aza steroids. 3. The synthesis of 11-aza 9 -steroids. I

The synthesis of 3,20-dioxygenated 11-azapregnanes, with a “normal” (a) configuration.in position 9, is described. The saturation of the 8,9-double bond of previously described unsaturated precursors can be effected not only by reduction of 9,11-iminium salts, but also catalytically, under pressure, in the presence of perchloric acid, It is shown that the 7-position of 8,9-unsaturated 11-aza steroids can be functionalized with the chromic acid-pyridine complex, the double bond of the resulting 7-ketone being prone to Birch reduction. Other, direct, pathways from saturated 11-nor 9,12-seco steroids to saturated 11-aza 9α-steroids have been explored. The crucial difficulty inherent in this approach stems from the fact that in displacement reactions involving an axial 9α-substituent, elimination competes successfully with substitution.     

Polyamine Metabolism in Ripening Tomato Fruit : II. Polyamine Metabolism and Synthesis in Relation to Enhanced Putrescine Content and Storage Life of a/c Tomato Fruit

The fruit of the Alcobaca landrace of tomato (Lycopersicon esculentum Mill.) have prolonged keeping qualities (determined by the allele a/c) and contain three times as much putrescine as the standard Rutgers variety (A/c) at the ripe stage (ARG Dibble, PJ Davies, MA Mutschler [1988] Plant Physiol 86: 338-340). Polyamine metabolism and biosynthesis were compared in fruit from Rutgers and Rutgers-a/c—a near isogenic line possessing the allele a/c, at four different stages of ripening. The levels of soluble polyamine conjugates as well as wall bound polyamines in the pericarp tissue and jelly were very low or nondetectable in both genotypes. The increase in putrescine content in a/c pericarp is not related to normal ripening as it occurred with time and whether or not the fruit ripened. Pericarp discs of both normal and a/c fruit showed a decrease in the metabolism of [1,4-¹⁴C]putrescine and [terminal labeled-³H]spermidine with ripening, but there were no significant differences between the two genotypes. The activity of ornithine decarboxylase was similar in the fruit pericarp of the two lines. Arginine decarboxylase activity decreased during ripening in Rutgers but decreased and rose again in Rutgers-a/c fruit, and as a result it was significantly higher in a/c fruit than in the normal fruit at the ripe stage. The elevated putrescine levels in a/c fruit appear, therefore, to be due to an increase in the activity of arginine decarboxylase.     

Comparisons of Peptide hydrolase activities in cereals

Carboxypeptidase activity (hydrolysis of N-carbobenzoxy-l-phenylalanyl-l-alanine) is high in a number of temperate zone cereals, originating in Asia Minor (wheat, barley, oats, wild oats, rye, triticale) compared to other cereals originating in central America or Asia (maize, sorghum, rice). However, endopeptidase activity (hydrolysis of azocasein or hemoglobin) is relatively much higher in the latter group. Comparison of trichloroacetic acid (TCA)-soluble products derived from the hydrolysis of hemoglobin showed that carboxyterminal amino acids (histidine, arginine, and tyrosine), are released when extracts from wheat and barley endosperms are used. With extracts from corn endosperms, much more TCA-soluble ultraviolet- absorbing material is released, but very little is released as free amino acids within the first 2 hours and the expected C-terminal amino acids of hemoglobin are not detected in significant amounts. These results suggest that the method of hydrolysis of the storage proteins may be significantly different in these two classes of cereals.     

Moonlighting protein in Starkeyomyces koorchalomoides: Characterization of dihydrolipoamide dehydrogenase as a protein acetyltransferase utilizing acetoxycoumarin as the acetyl group donor

In this report we have identified for the first time a transacetylase (TAase) in a mesophilic fungi Starkeyomyces koorchalomoides catalyzing the transfer of acetyl group from polyphenolic acetate (PA) to a receptor protein glutathione S-transferase (GST). An elegant assay procedure was established for TAase based on its ability to mediate inhibition of GST by 7,8-diacetoxy-4-methylcoumarin (DAMC), a model PA. Utilizing this assay procedure, S. koorchalomoides TAase was purified to homogeneity. TAase was found to have MW of 50 kDa. The purified enzyme exhibited maximum activity at 45 °C at pH 6.8. The N-terminal sequence of purified fungal TAase (ANDASTVED) showed identity with corresponding N-terminal sequence of dihydrolipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme and an E3 component of pyruvate dehydrogenase complex (PDHC). TAase was found to have all the properties of LADH and avidly interacted with the anti-LADH antibody. TAase catalyzed acetylation of GST by DAMC was identified by LC–MS/MS and a single lysine residue (Lys-113) was found to be acetylated. Further, recombinant LADH from Streptococcus pneumoniae lacking lipoyl domain was found to exhibit little TAase activity, suggesting the role of lipoyl domain in the TAase activity of LADH. These observations bear evidence for the protein acetyltransferase activity of LADH. Such an activity of LADH can be attributed as a moonlighting function of the enzyme.     

Detection of reactive oxygen species (ROS) by the oxidant-sensing probe 2′,7′-dichlorodihydrofluorescein diacetate in the cyanobacterium Anabaena variabilis PCC 7937

The generation of reactive oxygen species (ROS) under simulated solar radiation (UV-B: 0.30 Wm⁻², UV-A: 25.70 Wm⁻² and PAR: 118.06 Wm⁻²) was studied in the cyanobacterium Anabaena variabilis PCC 7937 using the oxidant-sensing fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA is a nonpolar dye, converted into the polar derivative DCFH by cellular esterases that are nonfluorescent but switched to highly fluorescent DCF when oxidized by intracellular ROS and other peroxides. The images obtained from the fluorescence microscope after 12 h of irradiation showed green fluorescence from cells covered with 295, 320 or 395 nm cut-off filters, indicating the generation of ROS in all treatments. However, the green/red fluorescence ratio obtained from fluorescence microscopic analysis showed the highest generation of ROS after UV-B radiation in comparison to PAR or UV-A radiation. Production of ROS was also measured by a spectrofluorophotometer and results obtained supported the results of fluorescence microscopy. Low levels of ROS were detected at the start (0 h) of the experiment showing that they are generated even during normal metabolism. This study also showed that UV-B radiation causes the fragmentation of the cyanobacterial filaments which could be due to the observed oxidative stress. This is the first report for the detection of intracellular ROS in a cyanobacterium by fluorescence microscopy using DCFH-DA and thereby suggesting the applicability of this method in the study of in vivo generation of ROS.