The amount of folate compounds rapidly increased during germination of the pea. An especially high increase was observed 2 days after sowing, with a continuously increasing content during germination. Dihydrofolate synthetase [EC 22.214.171.124] activity also increased with the in-crease in the amount of folate compounds, and the maximum activity was observed at 6 days after sowing in the dark and at 8 days after sowing in the light. The dhydrofolate synthetase was localized mostly in the mitochondrial fraction and it was easily extracted from the cell particles by osmotic shock. A similar localization was also observed in spinach leaves. The enzyme which was extracted from the isolated mitochondria was relatively stable in comparison with that extracted from whole cells.
Dihydrofolate synthetase [EC 126.96.36.199] was extracted from the cell particles (mitochondrial fraction) of pea seedlings and purified about 2, 000-fold by ammonium sulfate fraction, DEAE-cellulose column chromatography, Sephadex G-200 gel filtration, and hydroxylapatite column chromatography. The enzyme preparation obtained was con-firmed ultracentrifugally to be in the homogeneous state. The sedimen-tation coefficient of this enzyme was calculated as 3.9 S. The apparent molecular weight of the enzyme was determined to be about 56, 000. Optimum pH for the reaction was 8.8. The enzymatic reaction required dihydropteroate, L-glutamate and ATP as substrates, and divalent (Mg2+ or Mn2+) and univalent (K+, NH4+ or Rb+) cations as cofactors. The enzyme was specific for dihydropteroic acid as the substrate. ATP was not replaceable with any other nucleotides. Km values for dihydropteroate, L-glutamate, ATP, Mg2+, and Mn2+ were 1.0×10-6, 1.5×10-3, 1.0×10-4, 1.1×10-3, and 6.3×10-5M, respectively. The enzymatic reaction was inhibited by the addition of ADP, but not by AMP. This suggests that the product from ATP in the reaction is composed of ADP+Pi. Thus, it is proposed that this enzyme catalyzes the following reaction: Dihydropteroic acid+L-glutamic acid+ATP Mg2+ (or Mat+)→K+ (or NH4+ or Rb+) Dihydrofolic acid+ADP+Pi.
Evidence was presented by paper chromatographic analysis on the occurrence of an enzyme capable of catalyzing a pyrophosphate transfer from ATP to thiamine in green leaves of various plants. The exclusive localization of the enzyme activity in the 105, 000×g supernatant (in a soluble form) was demonstrated by differential centrifugation of a cell homogenate in 0.25M sucrose. The enzyme was purified by column chromatography with DEAF-cellulose and by gel filtration with Sephadex G-150. The partially purified preparation, while contaminated with detectable activity of acid phosphatase, lost the ability of utilizing thiamine monophosphate as the substrate in place of thiamine. These findings lead to the conclusion that thiamine pyrophosphate is formed in green leaves of plants through a direct pyrophosphorylation of thiamine in the presence of ATP and Mg.
During incubation of fertilized hen egg, flavin in egg white migrated into the embryo. The total amount of flavins in the embryo increased with development. On the 7th day of incubation, molar ratio of riboflavin and flavin nucleotides in the embryo was already similar to that of animal tissues. Even though appreciable amounts of FMN and FAD were found in egg white, flavin nucleotides found in the embryo in its early embryonic life cannot be simply ascribed to the migration of these nucleotides originally present in egg white, because egg yolk contains no appreciable amount of flavin nucleotides; the possibility that flavin nucleotides are synthesized from riboflavin in the embryo in its early embryonic life still exists.
1. The highest disaccharidase activity for sucrose, maltose and maltitol was found in the jejunum, followed by the ileum and duodenum. However, the disaccharidase activity for maltitol was extremely low compared with that for sucrose and maltose. 2. For maltitol, the Km value was very large and the Vmax value was very low compared with the values for sucrose and maltose. 3. The initial velocity (v) in the presence of sucrose and maltitol, was equal to the sum of the rates for individual substrates sucrose and maltitol (v1, v2) respectively (v=v1+v2). Thus, no competition between these substrates was observed. In the case of maltose and maltitol, the initial velocity (v) in the presence of both substrates was less than the sum of the individual rates for maltose and maltitol (vl, v2) in the absence of the other substrate (v<vl+v2). This finding demonstrates that there is competition between these two substrates for the same enzyme. Furthermore, the apparent Michaelis constant (Km) and the apparent maximal velocity (Vmax) for pure and mixed substrates, i.e., maltose and maltitol, at various mole fractions of maltose showed dependence on the mole fraction of maltose. The obtained kinetic data provide strong evidence that both maltose and maltitol react at the single active center of maltase.
Studies were conducted to evaluate specific dampening of hyperlipo-genesis (i.e. enhanced lipogenic enzyme activity of the liver in rats refed a high carbohydrate, fat-free diet: F-diet) by exogenous polyunsaturated fatty acids under fixed carbohydrate consumption. In force-feeding of rats with a linoleate-rich diet (F-diet containing 4.5 safflower oil), the lipogenic enzyme activities; fatty acid synthetase (FAS) and malic enzyme (ME), in the liver supernatantt were found to be significantly lower than those in rats force-fed only an F-diet (p<0.02), under conditions of identical consumption of carbohydrate. Among the various methylesters of unsaturated fatty acids administered by gastric intubation at a dose of 0.3g per 100g body weight, arachidonate was most potent in bringing about a significant reduction of hyper-lipogenesis without seriously affecting food intakes. During the same three-day experimental period, fatty acid GLC spectra in both the liver and plasma lipids reflected the exogenous input of PUFA. Plasma total fatty acid concentration (mainly triglyceride) significantly de-creased in the arachidonate group (p<0.01).
Intestinal absorption of cytidine diphosphate choline (CDP-choline), its structural changes in the digestive tract, and hepatic uptake have been investigated in rats using 14C-labeled (14CH3 attached to N of choline) and 3H-labeled (at C5of pyrimidine) compounds. The results indicate that: 1) CDP-choline is relatively stable in the stomach, but is quickly degraded into cytidine and choline in the intestine; 2) The hepatic uptakes of 14C and 3H reach the maximum in two to three hours after oral administration; 3) Whereas the amount of 14C remaining in the gut is inversely related to the hepatic uptake, no similar correlation is seen with 3H-labeled CDP-choline, and 4) Extrahepatic uptake of 14C and 3H is very small. The possibility of phosphorylation in the mucosa of choline and cytidine has been discussed, based on the differences in relative amount of radioactivity in individual broken-down products in the intestinal lumen and mucosa.