The two components of lipovitellin and the three major components of yolk granules, phosvitin, α- and β-lipovitellin, have been separated by gradient elution chromatography on TEAE-cellulose. A 0.2 M phosphate buffer (pH 6.8) had the necessary ionic strength to dissolve these proteins and when applied in this solvent all components except β-lipovitellin were retained by the column. A linear gradient of ionic strength (limit buffer 0.2 M phosphate plus 0.5 M NaCl) was used to remove the other components. Recovery was essentially complete and the composition and properties of the individual components were similar to those obtained by previous chromatographic methods that gave only partial recovery. An additional component eluted after α-lipovitellin and before phosvitin, previously observed in Dowex-1 separations, was also observed by the present method. The composition, sedimentation behavior, and absorption spectra of this component indicate that it is a soluble complex of phosvitin and lipovitellin. When granules are dissolved in alkaline solvents (pH 9.4) of low ionic strength (0.05), phosvitin is not evident as a separate component during ultracentrifugation, but appears as the ionic strength is increased.
The metabolism of propionic acid by maturing wheat plants was investigated by use of the radioactive tracers propionate-1-C 14 , -2-C 14 , and -3-C 14 . Carbon 2 of propionate was most extensively incorporated into kernel components and yielded kernel protein of high specific activity, glutamic acid being particularly radioactive. Carbon 3 was also preferentially incorporated into glutamic acid but was not as efficient in this regard as was carbon 2. Carbon 1 of propionate was extensively respired as carbon dioxide. It did not label glutamic acid extensively. Partial degradation of glutamic acid from kernel protein hydrolyzates showed that carbon 1 of propionate labelled carbon 1 of glutamate more than it did other glutamate carbons. Carbon 2 of propionate preferentially labelled carbon 4 of glutamate and carbon 3 preferentially labelled carbon 5 of glutamate. Similar data were obtained by examining the carbon-14 distribution in free glutamic acid obtained from wheat seedlings labelled with radioactive propionate-1-C 14 , -2-C 14 , and -3-C 14 .The results are interpreted as evidence that propionate is degraded by conversion of carbon 1 to carbon dioxide and by utilization of carbons 2 and 3 as acetate, with carbon 3 behaving as the carboxyl carbon of acetate. They accord with views on the mode of propionate metabolism derived from studies with plant tissue slices.
A method of double isotope derivative assay of aldosterone, corticosterone, and cortisol is described. It involves acetylation with acetic- 3 H anhydride of high specific activity followed by the purification of the steroids through four paper chromatographies and the formation of a derivative by chromium trioxide oxidation. The method is particularly suited to the quantitative measurement of these steroids in the incubation media of either rat or beef adrenal slices.
It has been observed that after long standing a reduction occurs in the activity of glucuronidase preparations of mammalian origin (e.g. Ketodase) on sodium pregnanediol glucuronidate (NaPG), either as a pure solution or in urine. This deterioration is characterized by increases in the optimum pH and in the amount of enzyme required to yield a complete hydrolysis of the NaPG. The fact that complete enzyme activity on pure NaPG solutions is restored by the addition of propylene glycol suggests that this deterioration might involve some glucuronyl transfer mechanism. Propylene glycol increases the activity of enzyme on pure NaPG solution by a factor of 6–15, reducing the amount of enzyme required to give a 95% hydrolysis in 4 hours from about 150 units/ml to 10 units/ml or by reducing the time required for 10 units/ml of enzyme to effect this hydrolysis from 24 to 4 hours. The presence of inhibitors in urine reduces this factor to 2, thus it requires half as much enzyme to effect a 95% or complete hydrolysis of the NaPG of urine in the presence of propylene glycol as in its absence. About two-thirds of the inhibitors toward the Ketodase hydrolysis of the NaPG in urine can be removed by simple (NH4)2SO4precipitation and extraction techniques. Thus 25 units/ml urine equivalent are capable of giving a complete hydrolysis of the NaPG in such extracts in the presence of propylene glycol, compared to 150 units/ml for the original urine under similar circumstances or 300–600 units/ml in the absence of propylene glycol.
A method for the separation, determination, and characterization of 2-amino-2-deoxy-D-glucose (D-glucosamine) and 2-amino-2-deoxy-D-galactose (D-galactosamine) is presented. Treatment of 2-acetamido-2-deoxy-α-D-glucose and 2-acetamido-2-deoxy-α-D-galactose in pyridine solution with trimethylchlorosilane and hexamethyldisilazane results in a rapid conversion of the glycoses to their respective trimethylsilyl 3,4,6-tri-O-trimethylsilyl-2-acetamido-2-deoxy-α-D-glycosides which are sufficiently stable and volatile to allow their separation and quantitative analysis to be made by gas–liquid partition chromatography. The two trimethylsilyl derivatives, collected by preparative gas–liquid partition chromatography, were crystalline compounds which had sharp melting points and characteristic infrared spectra and specific optical rotations. Quantitative analyses of mixtures of 2-amino-2-deoxy-D-glucose hydrochloride and 2-amino-2-deoxy-D-galactose hydrochloride were made by gas chromatographic analysis of their trimethylsilyl derivatives formed after prior conversion to their N-acetyl derivatives.The analytical procedure was applied to the characterization of 2-amino-2-deoxy-D-glucose in hyaluronic acid and 2-amino-2-deoxy-D-galactose in chondroitin sulphate. The quantitative procedure was also successfully applied to the analysis of mixtures of hyaluronic acid and chrondroitin sulphate by the gas–liquid partition chromatographic determination of the 2-amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-galactose in the hydrolyzates prepared from synthetic mixtures of the two mucopolysaccharides.
Attempts were made to detect antigenic fragments in the sera or spleens of rabbits given a single intravenous injection of bovine serum albumin, human gamma-globulin, or human serum albumin. The results obtained, using a number of different methods for the detection of antibody–antigen interaction (double diffusion in gel, immunoelectrophoresis, inhibition of hemagglutination), failed to provide any evidence in favor of the presence of antigenic fragments either in the circulation or in the spleen of the experimental animal.
Biological tests were carried out to study the effect of INCAP (Institute of Nutrition of Central America and Panama) Vegetable Mixture 9, casein, and skim milk on the carcass, liver, blood, and bone composition of rats. At low levels of protein intake body and liver fat levels were higher in the animals fed the vegetable mixture. Higher values for total serum protein, albumin, globulin, and urea nitrogen concentration were obtained with skim milk, but the albumin/globulin ratio and the red and white blood cell count were similar for both protein foods. The fresh weights of the femur and tibia were found to be significantly lower for the rats fed Vegetable Mixture 9, while bone moisture and fat were significantly higher. Likewise, the percentages of ash, calcium, and phosphorus were also significantly lower in the animals fed the vegetable mixture.At higher levels of protein intake, carcass and liver fat were again higher for the animals fed the vegetable mixture. The blood analyses revealed no differences between the two proteins tested, except for a higher urea content in the animals fed skim milk.The differences in carcass, liver, and bone composition between animals fed the vegetable mixture and the animal proteins were probably due to minor essential amino acid deficiencies in the mixture, since supplementation with lysine, threonine, and methionine resulted in similar chemical composition values. Higher protein levels of intake also decreased the differences in carcass, liver, and bone composition of rats fed INCAP Vegetable Mixture 9, casein, or skim milk.
An attempt was made to produce 'forced methylation' and subsequent reduction of lecithin content of livers of rats fed a semipurified diet. The addition of guanidoacetic acid to the diet of the rats did not alter either the total phospholipid extracted from their livers or the liver lecithin content significantly. This constant pattern was observed whether choline was included in the diet or not. However, in animals fed a diet deficient in choline, the ratio of lecithin and cephalin extracted from their livers was altered, although the total phospholipid content remained constant.
Aldolase activity and nitrogen content of the muscle were determined in hereditary muscular dystrophic mice and their normal litter mates at various ages. Aldolase activity was found to decrease in dystrophic muscle when expressed per mg of wet tissue but showed an increase at later stages of the disease when expressed per mg of total nitrogen in muscle. Total nitrogen content of dystrophic muscle decreased considerably during the evolution of the disease. In normal mice, the muscle aldolase activity increases with age.
16-Epiestriol has been isolated in crystalline form from hen urine and characterized on the basis of the melting points of the isolated product and of two of its derivatives (acetonide, triacetate) and on the chromatographic behavior of the isolated product and five of its derivatives in each of three different solvent systems.
Studies of the inhibition of yeast adenylosuccinase by the products of the reaction it catalyzes, as well as studies of the ability of the products to protect the enzyme from inhibitors, indicate that fumarate leaves the enzyme before adenylic acid. Evidence to be presented indicates that the binding of adenylic acid to the enzyme is by the phosphate group and either the amino group or the purine ring. Evidence is also cited for the existence of a sulphydryl group at the active center of the enzyme, apparently close to the C–N bond that is cleaved by the enzyme.
In the isolated zona glomerulosa of beef adrenal cortex, progesterone, 11β-hydroxyprogesterone, 11-desoxycorticosterone, and corticosterone are precursors of aldosterone. 18-Hydroxylation is probably a rate-limiting step in the biosynthesis of aldosterone. At low concentration Metopirone selectively inhibits 18-hydroxylase activity. The sequence of hydroxylation of the steroid molecule proceeds effectively in the order 11, 17, 21. The main action of ACTH is to increase cortisol production by the fasciculate–reticularis. In the present in vitro system, the production of aldosterone by the glomerulosa is not affected by either ACTH or angiotensin.
Methanol-C14was rapidly metabolized by carrot tissue slices, pea cotyledons, soybean cotyledons, castor bean endosperm, beet storage tissues, and mature beet leaves. With the exception of beet storage tissues, carbon dioxide was a chief product of methanol metabolism. In all tissues, methanol carbon was also incorporated in the organic acids, sugars, amino acids, and the insoluble residue. Serine, methionine, methionine sulfone, and methionine sulfoxide were important labelled components present in the amino acid fractions separated. Degradation of the serine-C14that was produced by carrot tissues metabolizing methanol-C14showed that the bulk of the label was present in the 3-position. The results are interpreted as indicating that methanol can act as a precursor of the carbon-1 units that are to be utilized in transmethylation reactions leading to serine and methionine biosynthesis. In addition, methanol can be oxidized to carbon dioxide by these tissues, and this reaction possibly involves dehydrogenase systems.
The effects of hypoglycin A on the metabolism of L-leucine-C14, L-alanine-C14, and L-glutamic-acid-C14by rat liver slices have been investigated. Hypoglycin exerted markedly inhibitory effects on the conversion of leucine-C14to fatty acid, cholesterol, and CO2. Conversion of alanine-C14and glutamic acid-C14to fatty acids was also inhibited by hypoglycin. No effects of hypoglycin on the conversion of C14-amino acids into protein or glycogen were demonstrated.
Ten antigens have been distinguished in the sera of cocks and non-laying hens by immunoelectrophoretic analysis (IEA) against homologous rabbit antisera. An additional antigen has been found in the sera of laying hens by IEA against anti-laying-hen serum.Four of the serum antigens obtained by IEA have been correlated electrophoretically with the filter paper electrophoretic fractions serum albumin, serum α2-globulin, serum β-globulin, and serum γ-globulin.Serum albumin, serum α1-globulin, serum β-globulin, and serum γ-globulin have been identified immunologically with alpha-livetin, a livetin antigen (livetin 2), gamma1-livetin and gamma2-livetin respectively. Another antigen of cock serum and hen serum has been identified by IEA with a livetin antigen detected by IEA of livetin against anti-cock, anti-hen, and anti-yolk sera.
Yeast cells grown anaerobically on glucose supplemented with yeast extract, hydrolyzed casein, and oleic acid developed a respiratory capacity on exposure to air. The time course of development of respiration was characteristic with an abrupt onset of oxygen consumption. The magnitude of the uptake of oxygen and the kinetics of its onset were determined by the concentration of glucose to which the yeast was exposed during growth and by the relative amounts of glucose and yeast present during the time of adaptation. A source of amino acids was necessary during the time of adaptation for the consistent development of respiration. Under the growth conditions used, adaptive development of respiration occurred most efficiently when the cells were harvested immediately prior to the onset of the stationary phase. Cytochrome oxidase was virtually absent from the anaerobically grown cells. The kinetics of its adaptive formation were not the same as those for the development of respiration. Cytochrome oxidase appeared before respiration became appreciable. When these anaerobically grown yeast cells were added to the adaptation medium there was a burst of gas output. The identity of the gas is unknown but it is likely to be CO2, H2S, or H2. The role of glucose and other factors in the adaptive development of respiration in yeast is discussed.
The effects of x irradiation on spermntozoal hyaluronidase activity and release were investigated to test the enzyme release theory and to give a measure of fertility other than loss of mobility. Rats and gninea pigs were given whole- body x-ray doses up to 1000 rads with higher doses to the gonads. Cell counts were then made of epididymnl spermatozoa and hyaluronidase activity was estimated turbidimetrically. Hyaluronidase activity and release was not significartly affected by doses of x-irradiation, and the permeability of the acrosomal membrane was apparently undamaged. Therefore, it was concluded that the temporary loss of fertility in irradiated males is due to sperm depletion as a result of the destruction of stem cells such as spermatogonia. (H.M.G.)
The acute hypoxia, caused by severe blood loss, gives rise to the rapid breakdown of glycogen in the liver and concurrent increase in the concentration of ATP in the mitochondria. The increase in adenosine triphosphate (ATP) continues until the onset of the reversible phase of shock. The glycogen reserve approaches depletion during the late reversible phase. Simultaneously, the generation of ATP in the mitochondria ceases and the concentration begins to fall. It would appear that at this time the adenylate kinase mechanism in the mitochondrial membrane comes into play to convert the adenosine diphosphate (ADP) into ATP and adenosine monophosphate (AMP). As the condition becomes irreversible the residual ATP and phosphorylated intermediates of the Embden–Meyerhof system undergo rapid hydrolysis with liberation of AMP and inorganic phosphate in the cytoplasm.The concentration of the pyridine nucleotides undergoes no change in any of the liver cell components until the onset of the irreversible phase of failure. Thereafter, these nucleotides undergo a progressive conversion to the reduced form.
An α-galactosidase from watermelon seeds has been separated from invertase and β-galactosidase by ammonium sulphate precipitation followed by elution from a DEAE-cellulose column with phosphate buffers. This enzyme is capable of hydrolyzing galactose residues from melibiose, raffinose, and stachyose. The pH optimum with the three substrates is close to 4.2. The enzyme is inhibited by the products of the reaction, and, in the case of melibiose, by the substrate itself.
