Enzymic mechanism of starch breakdown in germinating rice seeds : 11. Ultrastructural changes in scutellar epithelium

The ultrastructural changes occurring in the scutellar epithelium cells of rice seeds have been studied during germination and early seedling growth. During this time, several prominent structural changes occur, including (a) formation, development, and proliferation of organelles such as mitochondria, rough endoplasmic reticulum, free ribosomes, and Golgi apparatus; (b) folded structural modification of plasmamembranes in later stages; and (c) conspicuous decrease in lipid-storing spherosomes. Glyoxysome-like electron dense particles are detectable but their formation is much less prominent. It is conceivable that all these structural changes are related to the enhancement of the metabolic activities of the epithelial cells including the synthesis of hydrolytic enzymes such as α-amylase and their secretion into the endosperm tissues. Some enzyme activities characteristic of mitochondria and glyoxysomes have been determined using the crude scutellar extracts, and the results dealing with the low activities of the glyoxylate cycle enzymes and palmitoyl-coenzyme A oxidase appear to indicate that fatty acid breakdown is possibly via mitochondrial β-oxidation, although we reserve a definitive conclusion on the glyoxysomes being nonfunctional in fatty acid oxidation in rice seedlings.     

Metabolism of sialic acid in regenerating rat liver.

In regenerating rat liver slices 24 h after partial hepatectomy, the incorporation of [1-14C]glucosamine into 'free sialic acid' (N-acetylneuraminic acid + CMP-N-acetylneuraminic acid) decreased to below 50% of the control values and the incorporation into protein-bound sialic acid decreased to the same extent. The incorporation of [14C]glucosamine into 'free sialic acid' decreased during the period from 6 to 47 h after hepatectomy, showing a minimum at 12 h, and recovered to the control value by 96 h. At 12 h, the activities of UDP-N-acetylglucosamine 2-epimerase (UDP-2-acetoamido-2-deoxy-D-glucose 2-epimerase, EC 5.1.3.14) and N-acyl-D-mannosamine kinase (ATP: 2-acylamino-2-deoxy-D-mannose 6-phosphotransferase, EC 2.7.1.60) in the liver were significantly decreased. The amount of protein-bound sialic acid in the liver was not changed after partial hepatectomy, but the amount in plasma was changed, with a similar pattern to that of the incorporation of [14C]glucosamine into slice 'free sialic acid'. These results indicate that the synthesis of sialic acid in the liver much decreases in the early stage of regeneration and that this may be correlated with the decreased synthesis of plasma sialoglycoproteins.     

Protection by picolinamide,a novel inhibitor of poly (ADP-ribose) synthetase,against both streptozotocin-induced depression of proinsulin synthesis and reduction of NAD content in pancreatic islets

Picolinamide, 2-pyridinecarboxylic acid amide, was found to be a strong inhibitor of poly (ADP-ribose) synthetase of nuclei from rat pancreatic islet cells. Another experiment using isolated pancreatic islets of rats showed that picolinamide protects against streptozotocin-induced depression of proinsulin synthesis as well as against streptozotocin-induced reduction of NAD content. The protection by picolinamide against the NAD depression was considered to be due to the blockage of an increased degradation of NAD mediated by a streptozotocin-induced increase in poly (ADP-ribose) synthetase activity. A possible mechanism of streptozotocin diabetes and its prevention is discussed.     

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 8. Immunohistochemical Localization of beta-Amylase

Rabbit antiserum against β-amylase isolated from germinating seeds of rice was produced, and its specific cross-reactivity with β-amylase was confirmed by means of Ouchterlony double immunodiffusion and immunoelectrophoresis procedures. The cellular localization of β-amylase was studied by indirect fluorescence microscopy of thin sectioned germinating rice seed specimens (1-day stage) which had been fixed and treated with purified rabbit anti-β-amylase immunoglobulin G followed by conjugation with fluorescein isothiocyanate-labeled goat antirabbit immunoglobulin G. It has been demonstrated that β-amylase is uniformly associated with the periphery of starch granules in the starchy endosperm cells. The finding is discussed in relation to the general notion concerning the presence of the latent form of β-amylase bound to protein bodies in cereal seeds.     

A sulfotransferase model: Covalent participation of a macrocyclic oxime in the hydrolysis of 2,4-dinitrophenyl sulfate

The liberation of 2,4-dinitrophenolate ion from 2,4-dinitrophenyl sulfate (DNPS) in aqueous organic solvent with 0.1 N sodium hydroxide was accelerated upon addition of an equimolar amount of Oxime-I (10-hydroxy-11-hydroxyimino[20]-paracyclophane) to the sulfate ester. Oxime-I was found to undergo covalent participation at the oxime group to afford oxime O-sulfonate. The rate acceleration with Oxime-I was larger than that with β-CD (cycloheptaamylose). The catalytic efficiency of Oxime-I has been ascribed primarily to the tighter inclusion of the substrate ester into the more hydrophobic Oxime-I cavity provided by the effective apolar paracyclophane skeleton, as well as to the greater nucleophilicity of the oxime group than of the hydroxyl group in β-CD. Consequently, Oxime-I may be considered as a conventional model for arylsulfatases and sulfotransferases, providing the effective binding process for the substrate.     

Mutagenic activity of amino acid pyrolyzates in Salmonella typhimurium TA 98.

Pyrolyzates of 25 amino acids and 5 indole derivatives were tested for mutagenicity in the histidine-requiring mutant Salmonella typhimurium TA 98. Significant mutagenic activity was detected with pyrolyzates of most of the amino acids. These pyrolyzates required a liver microsomal fraction, as representative of mammalian metabolism, to be detected as mutagens. Among the pyrolyzates tested, the highest mutagenic activity was observed with that of L-tryptophan. As little as 10 microgram of the pyrolyzate of L-tryptophan had detectable mutagenic activity toward TA 98. The optimal pyrolysis temperatures for the formation of mutagenic products were shown to be 500 degrees C for L-tryptophan and 600 degrees C for the other amino acids. The results from pyrolyses of some indole derivatives suggest that an amino group at the alpha-position to the carboxyl group of L-tryptophan plays an important role in the formation of mutagens.     

Effects of tryptic digestion on the enzymatic activites of chicken gizzard myosin.

Tryptic digestion of gizzard myosin resulted in the degradation of the 20K light chain (G1) to its 17K fragment, which could not be phosphorylated. The rapid loss of Ca2+-dependent activation of actomyosin ATPase activity accompanied the degradation of G1. Increase in the Ca2+-ATPase activity and decrease in the EDTA-ATPase activity of myosin accompanied the degradation of myosin heavy chain, but not the cleavage of G1.