Type I collagen in rat skin in an excess of dietary amino acids

Type I collagen in skin of rats who were given redundant amounts of leucin, valine, methionine or phenyl alanine differed essentially from the normal level in the amino acidic composition. Considerable shifts in the value of pI of the proteins under study as compared with the normal level are found during isofocusing. When studying subunits of collagen preparations by the reverse-phase chromatography certain differences typical of each studied state are revealed in the elution profiles in normalcy and under amino acid loadings.     

Importance of amino acid composition to improve skin collagen protein synthesis rates in UV-irradiated mice

Skin collagen metabolism abnormalities induced by ultraviolet (UV) radiation are the major causes of skin photoaging. It has been shown that the one-time exposure of UV irradiation decreases procollagen mRNA expression in dermis and that chronic UV irradiation decreases collagen amounts and induces wrinkle formation. Amino acids are generally known to regulate protein metabolism. Therefore, we investigated the effects of UV irradiation and various orally administered amino acids on skin collagen synthesis rates. Groups of 4-5 male, 8-week-old HR-1 hairless mice were irradiated with UVB (66 mJ/cm2) twice every other day, then fasted for 16 h. The fractional synthesis rate (FSR; %/h) of skin tropocollagen was evaluated by incorporating L-[ring-2H5]-phenylalanine. We confirmed that the FSR of dermal tropocollagen decreased after UVB irradiation. The FSR of dermal tropocollagen was measured 30 min after a single oral administration of amino acids (1 g/kg) to groups of 5-16 UVB-irradiated mice. Branched-chain amino acids (BCAA, 1.34±0.32), arginine (Arg, 1.66±0.39), glutamine (Gln, 1.75±0.60), and proline (Pro, 1.48±0.26) did not increase the FSR of skin tropocollagen compared with distilled water, which was used as a control (1.56±0.30). However, essential amino acids mixtures (BCAA+Arg+Gln, BCAA+Gln, and BCAA+Pro) significantly increased the FSR (2.07±0.58, 2.04±0.54, 2.01±0.50 and 2.07±0.59, respectively). This result suggests that combinations of BCAA and glutamine or proline are important for restoring dermal collagen protein synthesis impaired by UV irradiation.     

Manipulation of amino acid composition in soybean seeds by the combination of deregulated tryptophan biosynthesis and storage protein deficiency

The ability of genetic manipulation to yield greatly increased concentrations of free amino acids (FAAs) in seeds of soybean was evaluated by introduction of a feedback-insensitive mutant enzyme of tryptophan (Trp) biosynthesis into two transformation-competent breeding lines deficient in major seed storage proteins. The storage protein-deficient lines exhibited increased accumulation of certain other seed proteins as well as of FAAs including arginine (Arg) and asparagine in mature seeds. Introduction of the gene for a feedback-insensitive mutant of an α subunit of rice anthranilate synthase (OASA1D) into the two high-FAA breeding lines by particle bombardment resulted in a >10-fold increase in the level of free Trp in mature seeds compared with that in nontransgenic seeds. The amount of free Trp in these transgenic seeds was similar to that in OASA1D transgenic seeds of the wild-type cultivar Jack. The composition of total amino acids in seeds of the high-FAA breeding lines remained largely unaffected by the expression of OASA1D with the exception of an increase in the total Trp content. Our results therefore indicate that the extra nitrogen resource originating from storage protein deficiency was used exclusively for the synthesis of inherent alternative nitrogen reservoirs such as free Arg and not for deregulated Trp biosynthesis conferred by OASA1D. The intrinsic null mutations responsible for storage protein deficiency and the OASA1D transgene affecting Trp content were thus successfully combined and showed additive effects on the amino acid composition of soybean seeds.     

Effect of amino acids on collagen biosynthesis

Summary The effect of various amino acids on collagen biosynthesis was studied in organ cultures of chicken embryo tibiae. Competitive interrelationship between selected amino acids influences independently the uptake of proline and lysine, precursors of hydroxyproline and hydroxylysine, respectively, which are the two amino acids characteristics of collagen. On the basis of these influences, the possibility of biosynthesis of anomalous collagens is stressed. Parallel studies of biosynthesis of hydroxyproline and hydroxylysine are necessary.     

Effect of cycloheximide on protein biosynthesis in rat liver

1. The liver ribosomes of rats given cycloheximide by intraperitoneal injection incorporate less amino acid into protein than ribosomes from control rat liver when they are incubated in vitro with excess of Sephadex-treated cell sap. The effect is rapid, marked and persistent. 2. Cell sap from liver of cycloheximide-treated animals is inhibitory but the inhibition can be relieved almost entirely by treating the cell sap with Sephadex. No damage has been done to the cell-sap factors: it is suggested that the dissolved cycloheximide in the cell sap causes the inhibition. 3. Cycloheximide added in vitro inhibits amino acid incorporation into protein in the presence or absence of polyuridylic acid. The inhibition is lessened by addition of excess of cell sap but is not abolished. 4. The differences between these results and those obtained with mouse liver (Trakatellis, Montjar & Axelrod, 1965) might arise because of species differences in sensitivity to the drug.     

Effects of a methyl-deficient diet on rat liver phosphatidylcholine biosynthesis

To produce a severe choline-methionine deficiency, a synthetic L-amino acid diet, free of choline, methionine, vitamin B12, and folic acid and supplemented with guanidoacetic acid, a methyl group acceptor, was fed to female rats for 2 weeks. The in vitro activity of liver microsomal phosphatidylethanolamine methyltransferase was stimulated twofold when compared with basal diet controls. The activity of choline phosphotransferase was depressed by 86%; thus, the contribution of the methyltransferase in the overall synthesis of phosphatidylcholine apparently increased. However, measurement of the in vivo methylation of phosphatidylethanolamine by incorporation of [1,2-14C]ethanolamine into phosphatidylcholine indicates that the methylation pathway is markedly depressed in methyl deficiency. Hepatic concentrations of the methyltransferase substrate, S-adenosylmethionine, and the inhibitory metabolite, S-adenosylhomocysteine, were significantly altered such that an unfavorable environment for methylation was present in the deficient animal. The ratio of substrate to inhibitor was depressed from 5.2:1 in the controls to 1.7:1 in the livers of methyl-depleted rats. Control of transmethylation in accordance with the availability of substrates, phosphatidylethanolamine, or S-adenosylmethionine, and the level of S-adenosylhomocysteine is discussed.     

Effect of a single dose of dimethylnitrosamine on biosynthesis of nucleic acid and protein in rat liver and kidney

1. Administration of a single dose of dimethylnitrosamine to rats temporarily fed on a protein-deficient diet causes a high incidence of kidney tumours. The effect of such a dose of dimethylnitrosamine (40mg/kg body wt.) on metabolism of nucleic acids and protein in rat liver and kidneys was examined during the week immediately after administration. 2. Incorporation of [(14)C]leucine and [(14)C]orotate into hepatic macromolecules was inhibited within 5h of injection of dimethylnitrosamine, and did not recover for at least 5 days. Interpretation of these results is complicated by the concomitant extensive hepatic necrosis. 3. Renal RNA synthesis was assayed by incorporation of [(14)C]orotate in vivo and measurement of DNA-dependent RNA polymerase activity in vitro. Both systems indicate biphasic inhibition; minimal activity was recorded 9h and 3 days after treatment. Changes in incorporation of [(14)C]leucine into renal protein were similar but less marked. 4. Sucrose-density-gradient analysis of renal cytoplasmic RNA indicated increased synthesis of rRNA 24h after injection of the nitrosamine. The rate of loss of radioactivity from kidney ribosomes pre-labelled with [(14)C]orotate was not modified by dimethylnitrosamine. 5. Dimethylnitrosamine increased incorporation of [(3)H]-thymidine into renal DNA. The three distinct periods of stimulated synthesis observed are discussed, with particular reference to recently published morphological studies of the sequential development of kidney tumours induced by dimethylnitrosamine in protein-depleted rats.     

Natural taxonomy of collagen based on amino acid composition

Hierarchical cluster analysis and principal components have been used to provide a more detailed separation of the collagens into natural taxonomic groupings than previously obtained. These groups strongly reflect the evolutionary development of collagen. The first component separates land- from sea-based animals, primarily based on the hydroxylation of lysine and proline, indicating that control of hydroxylation, a post-translational event, has exerted a dominant influence during evolutionary adaptation. The power of the technique is illustrated by the ability to partially separate the evolutionarily closely related main homothermic species. Furthermore, the genetically different fibrous collagens, Types I and III, are well separated from basement membrane Type IV collagen and the filamentous collagens. The technique could, therefore, in addition to providing a taxonomic grouping, classify any new collagen and provide clues to its evolutionary development.     

Effect of diet and essential amino acids gavage on young rat amino acid metabolism enzymes

• 1.1. The effects of a high-fat, high-energy diet and essential plus semi-essential amino acid gavage on pup rats have been studied (60–65 animals).
• 2.2. The activities of alanine transaminase, adenylate deaminase, glutamine synthetase and serine dehydratase have been tested in liver and muscle.
• 3.3. Plasma was used for the estimation of proteins, urea, amino acids, glucose, lactate, 3-hydroxy-butyrate and acetoacetate.
• 4.4. Liver and muscle glutamine synthetase activities are increased by diet and gavage administered. Hepatic serine dehydratase is inhibited by a cafeteria diet but activated by amino acid gavage. Adenylate deaminase is inhibited by diet and gavage in the liver, but gavage does not affect this enzyme activity in muscle. Liver alanine transaminase is increased by the diet; in the muscle, cafeteria diet and amino acid gavage showed the highest values for this enzyme.
• 5.5. In the plasma, the increase in lactate produced by the diet is inhibited by the amino acids provided. Cafeteria-fed pups showed lower urea levels and higher 3-hydroxybutyrate concentrations in the plasma.
• 6.6. Intracellular glucose is diminished by cafeteria diet. In contrast, the blood cell amino acid concentration increases with diet and gavage supplied.

     

Effects of excess methionine on collagen metabolism: a study on newborn rat skin

We studied the biochemical effects of excess methionine intake on the skin of newborn rats. Group 1 pups were intubated with methionine dissolved in 0.1 ml physiological saline solution in the amount of 0.1 g/100 body wt as a control using a gastric needle. Group 2 pups were given 0.2 g/100 g in the same manner as group 1 as an experimental group. They were intubated every other day from day 3 to 13. On Day 15, [14C]proline was injected intraperitoneally into pups and their skin was removed. 14C total and hydroxyproline uptake was examined in the tissue as well as in the sequential extracts. Although excess methionine intake by the pups did not alter the collagen content of their skin, it caused an increase in the content of type III collagen and a decrease in crosslinked collagen. In addition, newly synthesized collagen in the neutral salt extract increased in the excess methionine group, indicating that crosslinked collagen decreases as excess methionine was intubated. The present study demonstrated that excess methionine in the early lactational period altered the nature of the skin collagen of suckling newborn rat pups.     

Physico-chemical properties and amino acid composition of a highly purified preparation of a specific estrogen-binding protein of the rat liver

The structure and properties of an unusual estrogen-binding protein (UEBP) from male rat liver that was purified by affinity adsorption chromatography was studied. A high degree of purity of UEBP (greater than 99%) associated with appreciable microheterogeneity was demonstrated. The latter seems to be due to partial proteolysis of the protein at the N-end in the course of the isolation procedure. The purified UEBP molecules have the following characteristics: Mr = 31 000 (data from SDS-PAAG electrophoresis), sedimentation coefficient 3.75 S, Stockes'radius 25.6 A, friction coefficient ratio 1.11. The protein absorbance maximum in the UV region lies at 276 nm; extinction coefficient--26, alpha-helix content is 25-30%. The value of equilibrium association constant for estradiol is 5 X 10(7) M-1. Estriol (greater than 100%) and, in a weaker degree, estrone and testosterone (approximately 10%) compete for the binding sites with [3H]estradiol; androsterone has no competitive effect. The amino acid composition of UEBP was determined. The protein was shown to possess a great number of residues carrying hydrophobic side groups (34.4%) and more acidic amino acids over basic ones as well as a low content of cystein, threonine and histidine.     

Crystallization and amino acid composition of cathepsin B from rat liver lysosomes

Cathepsin B (EC 3.4.22.1) from rat liver was crystallized and its amino acid composition was determined. The purified enzyme formed spindle-shaped crystals and its homogeneity was proved by ultracentrifugical analysis. Its S_{20, W} value was 2.5 S and its molecular weight was calculated to be 24,000 from the result of sedimentation equilibrium analysis. Amino acid analysis showed that it contained glucosamine and galactosamine. The activity of the protease was maximal at pH 6.0 with α-N-benzoyl-DL-arginine p-nitroanilide as substrate. The apparent Kms for α-N-benzoyl-DL-arginine p-nitroanilide and α-N-benzoyl-DL-arginine-2-naphthylamide were 1.4 × 10^?2 M and 2.0 × 10^?3 M, respectively     

影响克拉维酸生物合成的氨基酸

发酵液的氨基酸分析显示,谷氨酸,精氨酸,天门冬氨酸,丙氨酸易被棒状链霉菌利用,发酵培养基中添加上述氨基酸后,谷氨酸,精氨酸有利于克拉维酸的生物合成,适时添加谷氨酸,精氨酸可分别提高克拉维酸的产量约25%和12%;而蛋氨酸,半胱氨酸含S氨基酸对克拉维酸生物合成不利,不同来源的黄豆粉作发酵培养基氮源,因其组成中某些氨基酸含量的差异。可使克拉维酸的产量相差百分之十几。