The interaction of rat liver sterol carrier p rotein (SCP) and 7-dehydrocholesterol or vitamin D3 was analyzed by the method of Scatchard plots and gel filtration on Sephadex G-100. Scatchard plots of binding data revealed that the binding behavior for 7-dehydrocholesterol was monophasic, while that for vitamin D3 was biphasic. In 7-dehydrocholesterol, the apparent number of binding sites and the apparent association constant K were 0.72 nmoles/mg protein and 8.75×10-7M-1, respectively. On the other hand, vitamin D3 showed two types of binding sites differing in affinity. The apparent number of binding sites and the K for high affinity binding were 0.65 nmoles/mg protein and 7.08×10-7M-1, those for low affinity binding were 1.51 nmoles/mg protein and 0.36×10-7M-1, respectively. Gel filtration of SCP on Sephadex G-100 column gave three protein peaks (peaks I, II and III protein according to the elution orders; Ve/Vo=1.0, 1.56 and 2.29, respectively). 7-Dehydrocholesterol was able to bind with peak III protein, while vitamin D3 was bound peaks II and III protein, respectively. The molecular weight of peak II protein was estimated to be 44, 000 and that of peak III protein was 16, 000. From these results, it was clearly demonstrated that SCP was involved in the transformation of 7-dehydrocholesterol to cholesterol and could also bind vitamin D3.
This investigation was undertaken to decide if tocopherol is actually depleted in the fetus and newborn infant by measuring liver tocopherol level. First, a reliable assay method was established as follows. The colorimetric values obtained by Emmerie-Engel's principle from liver extracts which were eluted through a floridin column were corrected by using the ratios of the tocopherol band area to the total scanning values of TLC analysis by densitometry carried out simultaneously. The recovery of tocopherol added to the liver homogenate was always greater than 90%, as measured in this procedure. Thus, tocopherol levels were determined in the human fetal, infant and adult liver with this method. The levels were very low in the fetal liver even though they gradually increased throughout fetal life. After birth, a slight elevation in tocopherol levels was observed during infancy when compared with those in fetal life, but they were still one-third or less of the adult level.
When adenine was added to the non-growing cell medium of Eremothecium ashbyii, riboflavin production of the mold was increasingly inhibited with increasing concentration of adenine. Under these condi-tions, a cationic compound was accumulated in the mycelia. The com-pound was isolated from the mycelia and highly purified. The purified compound was proved to be S-adenosylhomocysteine through IR analysis. In the control experiment, or in the addition of other purines which stimu-lated riboflavin production of the mold, another cationic compound was accumulated in the non-growing cells. The compound was largely accumulated in the presence of both adenine and methionine, isolated from the mycelia and purified. The purified compound was concluded to be S-adenosylmethionine through IR and NMR analyses. The signifi-cance of these compounds on the riboflavin biosynthetic pathway was argued under “DISCUSSION.”
A mutant strain of Escherichia coil 64-21, requiring either high concentrations of hydroxymethylpyrimidine or low concentrations of thiamine, has been produced from mutant 26-43 which requires the thiazole moiety of thiamine. The concentration of hydroxymethyl-pyrimidine required for a half maximal growth of mutant 64-21 was approximately 10, 000 times higher than that of thiamine. Mutant 26-43 cells were able to take up hydroxymethylpyrimidine and then utilized it as a precursor for thiamine biosynthesis, whereas mutant 64-21 cells could not take up hydroxymethylpyrimidine in a significant amount when it was supplied at 0.01mM. Increasing concentrations of external hydroxy-methylpyrimidine resulted in increasing the amount of intracellular hydroxymethylpyrimidine compounds but the amounts proved to be always far less than the amounts taken up by mutant 26-43 cells. In addition, phosphomethylpyrimidine kinase activity was deficient in this organism and the reaction did not proceed unless a high concentration of hydroxymethylpyrimidine monophosphate was added. Another mutant, 70-23, requiring thiamine in an intact form, lacked phospho-methylpyrimidine kinase activity and also proved to be lacking in the ability to take up hydroxymethylpyrimidine. These results lead to the conclusion that phosphomethylpyrimidine kinase might participate in the passage of hydroxymethylpyrimidine through the cell membrane and the enzyme in mutant 64-21 could not catalyze the reaction from hydroxy-methylpyrimidine monophosphate to the pyrophosphate unless a high concentration of hydroxymethylpyrimidine, hence, hydroxymethylpyri-midine monophosphate, was available intracellularly.
Dihydrofolate synthetase activity is widely distributed in various microorganisms and mushrooms. Animals and microorganisms i.e., rat and chicken (in the liver), L. casei and P. cerevisiae, which require essentially pteroylglutamic acid as a nutrient for growth showed no detectable dihydrofolate synthetase activity. S. faecalis R, which can replace pteroylglutamic acid with pteroic acid as a nutrient for growth, had little dihydropteroate synthetase activity but showed normal dihydro-folate synthetase activity. This suggests that the nutritional requirements for folate compounds shown in vivo is in good agreement with the results obtained with dihydropteroate and dihydrofolate synthetase activities in vitro, and that the pathway through dihydropteroic acid as an intermediate is the main route in folate biosynthesis in nature.
Male guinea pigs fed a cholesterol diet for 1-8 weeks showed a marked increase in platelet aggregation induced by ADP. Analyses of lipids indicated that dietary cholesterol resulted in highly signi-ficant increases in plasma cholesterol, β-lipoprotein and phospholipid levels, and platelet cholesterol contents. Plasma triglyceride levels in cholesterol-fed animals increased significantly only at the 8th week of feeding. Platelet phospholipid levels in cholesterol-fed animals increased significantly at the 2nd and 4th week of feeding. There were statistically insignificant increases in plasma and platelet free fatty acid. Fibrinogen levels in cholesterol-fed animals showed an increase at the 2nd and 4th week of feeding, but clotting tests, such as prothrombin time, recalcifi-cation time and Stypven time, were not changed. When platelets from animals fed on a control diet were exposed to plasma from cholesterol-fed animals, there was a notable increase in platelet aggregation in comparison with plasma from animals fed on a control diet. An increase of platelet aggregation was also observed, when platelets from cholesterol-fed animals were mixed with the plasma from not only cholesterol-fed animals but also animals fed on a control diet. As the cholesterol in plasma and platelets was assumed to be a possible factor in the increase of platelet aggregation, β-lipoproteins (cholesterol-rich proteins) were isolated from plasma of cholesterol-fed animals and animals fed on a control diet, and β-lipoprotein fractions were found to cause a marked increase in platelet aggregation induced by ADP. The results of this study indicate that cholesterol feeding causes an increase of platelet aggregation, and that the causative factor of the increase is assumed to be β-lipo-proteins.
The effect of supplementation of limiting amino acids on rats fed a 10 or 20% wheat gluten diet containing 5% tyrosine has been investigated. Growth-limiting amino acids in a 20% wheat gluten diet were lysine and threonine. When rats were fed a 20% wheat gluten diet containing excess tyrosine, the addition of 1.0% lysine⋅HCl or 1.0% lysine⋅HCl and 0.4% threonine completely prevented the development of tyrosine toxicity, and was accompanied by a lowering of free tyrosine concentrations in plasma, liver and muscle. In rats receiving a 10% wheat gluten diet, lysine, threonine and methionine were limiting for growth. The addition of 1.0% lysine⋅HCl to the 10% wheat gluten plus 5% tyrosine diet failed to alleviate the tyrosine toxicity, but the supplement of 1.0% lysine⋅HCl and 0.4% threonine could partially alleviate the toxicity. The combined addition of 1.0% lysine⋅HCl, 0.4% threonine and 0.5% methionine was most effective. The free tyrosine concen-trations in plasma, liver and muscle were lowered greatly by the supple-mentation of three limiting amino acids, but amounts of free tyrosine and phenolic metabolites excreted in the urine did not decrease with ad-dition of these limiting amino acids.
The nutritional quality of rice protein was compared with that of whole egg protein by slope ratio assay. Diets for each food at four levels of protein, 4, 6, 10 and 15% and a protein-free diet were given to male weanling rats of the Sprague-Dawley strain for 21 days. The slopes of the regression lines of the whole egg and rice groups calculated from the changes of body weight (Y in g/21 days) with nitrogen intake (X in g/21 days), including and (excluding) zero protein group were, respectively, Y=27.39 X-12.26 (Y=24.41 X-1.86) and Y=13.86 X -8.06 (Y=12.54 X +0.50). Assuming a potency of 100 for the egg protein, the relative potency of rice estimated from body weight gain with nitrogen intake was 51 (51). The values for rice calculated from body water gain and nitrogen retention with nitrogen intake were, respectively, 51 (47) and 46 (44). These values were compared with RNV of several varieties of conventional rice and high-protein rice.
The trypsin inhibitor in eggplant, Solanum melongena L., was isolated and purified by the improved method with the techniques of dialysis using acetylated cellulose tube and ion-exchange chromato-graphy on DEAE-Sephadex. The final preparation was found to be homogeneous by disc and SDS-polyacrylamide gel electrophoreses. This inhibitor had the molecular weight of about 6, 200, the pI value of 4.7, and furthermore characteristic amino acid composition lacking in trypto-phan, histidine, valine and methionine. The trypsin inhibition data indicated that the purified inhibitor combined with bovine trypsin [EC 188.8.131.52] in the molar ratio of 1:1. These properties of this inhibitor were in agreement with those of the dialyzable eggplant trypsin inhibitor pre-viously purified, indicating that the dialyzable and non-dialyzable inhi-bitors in eggplant are identical.
In order to identify the functional groups which really con-tribute to the carbon dioxide gas adsorption by proteins, ε-amino groups of lysine residues of egg albumin were chemically modified with trinitro-benzene sulfonic acid to various degrees. About 60% of the total amount of carbon dioxide gas adsorbed by solid egg albumin diminished by complete modification. The amount of carbon dioxide gas adsorbed by lysozyme, its hydro-lyzates and gelatin hydrolyzates depended upon the lysine content, arginine content and average molecular weight. The good correlation was obtained between the amount of carbon dioxide gas adsorbed and the total of lysine and arginine content of them. The ability of carbon dioxide gas adsorption by α-amino group of amino acids and oligopeptides was found to be developed by the elongation of the peptide chain of glycine and other amino acid, by the removal of α-carboxyl group of histidine and tyrosine to corresponding amines and by the esterification of α-carboxyl group of leucine with p-nitrophenol. These results clearly indicate that CO2 binding sites in protein in the gas-solid phase system are ε-amino, α-amino and guanidinium groups.
The effects of various additives on the reaction of rennin with κ-casein were investigated by using carboxymethylcellulose. Both urea and sodium 1-anilino-8-naphthalenesulfonate effectively inhibited rennin action at concentrations larger than 2M and 2mM, respectively. These reagents, however, activated the enzyme action at the lower concentrations. Both αs- and β-caseins had some ranges of concen-trations in which the rennin reaction was activated. Calcium chloride had an inhibitory effect on the rennin action. Neither mercaptoethanol nor KCl had any appreciable effect on the enzymatic hydrolysis of β-casein. These results are analyzed in terms of the association and dissociation of β-casein due to the presence of these additives in the reaction solutions.