scholarly journals Inhibitory effect of pH5 enzyme from non-lactating bovine mammary gland on various stages of protein synthesis in the rat liver amino acid-incorporating system

1969 ◽  
Vol 115 (4) ◽  
pp. 671-678 ◽  
Author(s):  
M. D. Herrington ◽  
A. O. Hawtrey
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Amino Acid ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Inhibitory Effect ◽  
Bovine Mammary Gland ◽  
Free System ◽  
Enzyme Fraction

1. pH5 enzyme from non-lactating bovine mammary gland was found to contain potent inhibitors of protein synthesis in the rat liver cell-free system. These inhibitors affect (a) formation of aminoacyl-tRNA where tRNA represents transfer RNA, (b) transfer of labelled amino acids from rat liver amino[14C]acyl-tRNA to protein in rat liver polyribosomes, and (c) incorporation of 14C-labelled amino acids into peptide by rat liver polyribosomes supplemented with rat liver pH5 enzyme. 2. Increasing amounts of pH5 enzyme from bovine mammary gland progressively inhibited the incorporation of labelled amino acids into protein by a complete incorporating system from rat liver. Approx. 80% inhibition was observed at a concentration of 2mg. of protein of pH5 enzyme from bovine mammary gland. The inhibitory effect of the bovine pH5 enzyme fraction could not be overcome by the addition of increasing amounts of rat liver pH5 enzyme. 3. Fractionation of bovine pH5 enzyme with ammonium sulphate into four fractions showed that all the fractions inhibited the incorporation of 14C-labelled amino acids in the rat liver system, but to varying extents. The highest inhibition observed (90%) was exhibited by the 60%-saturated-ammonium sulphate fraction. 4. Heat treatment of bovine pH5 enzyme at various temperatures caused only a partial loss of its inhibitory effect on labelled amino acid incorporation by the rat liver system. Treatment at 105° for 5min. resulted in the bovine pH5 enzyme fraction losing 30% of its inhibitory activity. 5. pH5 enzyme from bovine mammary gland strongly inhibited the charging of rat liver tRNA in the presence of its own pH5 enzymes. 6. The transfer of labelled amino acids from rat liver amino[14C]acyl-tRNA to protein in a system containing rat liver polyribosomes and pH5 enzyme was almost completely inhibited by bovine pH5 enzyme at a concentration of 2mg. of protein of the enzyme fraction. 7. One of the inhibitors of various stages of protein synthesis in rat liver present in bovine pH5 enzyme was identified as an active ribonuclease, and the second inhibitor present was shown to be tRNA.

scholarly journals Evidence for the absence of the terminal adenine nucleotide at the amino acid-acceptor end of transfer ribonucleic acid in non-lactating bovine mammary gland and its inhibitory effect on the aminoacylation of rat liver transfer ribonucleic acid

1970 ◽  
Vol 116 (3) ◽  
pp. 405-414 ◽  
Author(s):  
M. D. Herrington ◽  
A. O. Hawtrey
Keyword(s):  
Amino Acids ◽  
Mammary Gland ◽  
Amino Acid ◽  
Rat Liver ◽  
Ribonucleic Acid ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Inhibitory Effect ◽  
Bovine Mammary Gland ◽  
Amino Acid Acceptor

1. tRNA isolated from non-lactating bovine mammary gland competitively inhibits the formation of aminoacyl-tRNA in the rat liver system. 2. Non-lactating bovine mammary gland tRNA and twice-pyrophosphorolysed rat liver tRNA are unable to accept amino acids in a reaction catalysed by aminoacyl-tRNA synthetases from either rat liver or bovine mammary gland. Deacylated rat liver tRNA can however be aminoacylated in the presence of either enzyme. 3. Bovine mammary gland tRNA lacks the terminal adenine nucleotide at the 3′-terminus amino acid acceptor end, which can be replaced by incubation in the presence of rat liver nucleotide-incorporating enzyme, ATP and CTP. 4. The enzymically modified bovine tRNA (tRNApCpCpA) can bind labelled amino acids to form aminoacyl-tRNA, which can then transfer its labelled amino acids to growing polypeptide chains on ribosomes. 5. Molecules of rat liver tRNA or bovine mammary gland tRNA that lack the terminal adenine nucleotide or the terminal cytosine and adenine nucleotides inhibit the aminoacylation of normal rat liver tRNA to varying degrees. tRNA molecules lacking the terminal -pCpCpA nucleotide sequence exhibit the major inhibitory effect. 6. The enzyme fraction from bovine mammary gland corresponding to that containing the nucleotide-incorporating enzyme in rat liver is unable to catalyse the incorporation of cytosine and adenine nucleotides in pyrophosphorolysed rat liver tRNA and deacylated bovine tRNA. This fraction also markedly inhibits the action of the rat liver nucleotide-incorporating enzyme.

Isolation and partial characterization of amphibian tyrosine oxidase polysomes

Development ◽  
1970 ◽  
Vol 24 (1) ◽  
pp. 109-118
Author(s):  
E. L. Triplett ◽  
R. Herzog ◽  
L. P. Russell
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Enzymic Activity ◽  
Antigenic Determinants ◽  
Free System ◽  
A Cell ◽  
Generating System ◽  
Soluble Components

A population of polysomes isolated from frogskinis capable of supporting protein synthesis in a cell-free system containing an energy generating system, ‘soluble components’, and amino acids. These polysomes catalyse the oxidation of DOPA after gentle trypsinization, and they also have antigenic determinants attributable to tyrosine oxidase. Skin polysomes sedimented in 10–30 % sucrose gradients contain tyrosine oxidase peaks of enzymic activity at the bottom and top of the tube and in the 250 S regions. A peak of tyrosine oxidase antigenic acitvity is found in the 250–350S region of the gradient. Polysomes resolved on the gradient retain the ability to support protein synthesis in a cellfree system. All 250–350S particles capable of supporting the incorporation of [14C]amino acid into tyrosine oxidase are precipitable with tyrosine oxidase antibodies. It is probable that 250–350S tyrosine oxidase antibody precipitates contain only polysomes for this protein.

Sulfur amino acid metabolism in the whole body and mammary gland of the lactating Saanen goat

1999 ◽  
Vol 50 (3) ◽  
pp. 413 ◽  
Author(s):  
J. Lee ◽  
R. J. Knutson ◽  
S. R. Davis ◽  
K. Louie ◽  
D. D. S. Mackenzie ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Amino Acid ◽  
Significant Proportion ◽  
Oxidation Products ◽  
Whole Body ◽  
Acid Uptake ◽  
Saanen Goat ◽  
Arterial Blood

Five multiparous Saanen goats in late lactation were infused with 35S-cysteine into the mammary gland via the external pudic artery. A further 2 goats were infused with 35S-methionine via the same artery and later with 35S-methionine into the jugular vein. Total uptake of cysteine from the arterial blood supply by the mammary gland was approximately 6% of the 35S-cysteine flux past the gland, whereas uptake of methionine was 30–40%. Total mammary uptake of cysteine was also lower than that of methionine when expressed as a percentage of whole body utilisation (6.5 and 14%, respectively). The uptake from the blood did not account for output in the milk for either cysteine or methionine. Both amino acids were highly conserved by the gland as shown by little release of any degraded constitutive protein amino acids and no evidence of oxidation products of either cysteine or methionine being released into the blood. Comparison of 35S activity in the milk from the infused and non-infused sides of the gland showed up to 10% trans-sulfuration of methionine to cysteine within the gland, none of which was exported in the venous drainage. Total ATP production by one side of the gland was 12.1 mol/day or 13 mmol/min.kg mammary tissue, of which 15% was required for gland protein synthesis. The experimental measurements from both the cysteine and methionine infusions were used to solve a model of gland amino acid uptake and partitioning. Modelling radioactivity of both amino acids in the blood, intracellular free pool, and milk protein suggested that a single intracellular pool cannot be the only source of amino acid for protein synthesis. The model also provides support for the hypothesis that a significant proportion of the uptake of at least some amino acids by the mammary gland is from intracellular hydrolysis of extracellularly derived peptides.

scholarly journals Amino Acid-Mediated Intracellular Ca2+ Rise Modulates mTORC1 by Regulating the TSC2-Rheb Axis through Ca2+/Calmodulin

2021 ◽  
Vol 22 (13) ◽  
pp. 6897
Author(s):  
Yuna Amemiya ◽  
Nao Nakamura ◽  
Nao Ikeda ◽  
Risa Sugiyama ◽  
Chiaki Ishii ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Growth Regulator ◽  
Inhibitory Effect ◽  
Environmental Cues ◽  
Gtpase Activating Protein ◽  
Mechanistic Target Of Rapamycin ◽  
Rag Gtpases ◽  
Mtorc1 Activation

Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca2+/calmodulin (CaM). We showed that the amino acid-mediated increase of intracellular Ca2+ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca2+/CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca2+/CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.

scholarly journals Effects of a High-Grain Diet With a Buffering Agent on Milk Protein Synthesis in Lactating Goats

2021 ◽  
Vol 8 ◽  
Author(s):  
Meilin He ◽  
Xintian Nie ◽  
Huanhuan Wang ◽  
Shuping Yan ◽  
Yuanshu Zhang
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Amino Acid ◽  
Milk Production ◽  
Milk Protein ◽  
Time Of Flight ◽  
Lactating Goats ◽  
Rumen Acidosis ◽  
Buffering Agent

Chinese dairy industries have developed rapidly, providing consumers with high-quality sources of nutrition. However, many problems have also appeared during the development process, especially the low quality of milk. To improve milk quality, a large amount of concentrated feed is usually added to the diet within a certain period of time, which increases the milk production to a certain extent. However, long-term feeding with high-concentration feed can lead to subacute rumen acidosis. Therefore, the present study aimed to determine the effect of adding a buffer on subacute rumen acidosis, and the improvement of milk production and milk quality. We also aimed to study the mechanism of promoting mammary gland lactation. A total of 12 healthy mid-lactating goats were randomly divided into two groups, they were high-grain diet group (Control) and buffering agent group. To understand the effects of high-grain diets with buffers on amino acids in jugular blood and the effects of amino acids on milk protein synthesis, Milk-Testing™ Milkoscan 4000, commercial kits, and high-performance liquid chromatography (HPLC) measurements were integrated with the milk protein rate, the amino acid concentration in jugular venous blood samples, quantitative real-time PCR, comparative proteomics, and western blotting to study differentially expressed proteins and amino acids in mammary gland tissues of goats fed high-grain diets. Feeding lactating goats with buffering agent increased the percentage of milk protein in milk, significantly increased the amino acid content of jugular blood (p < 0.05), and increase the amino acid transporter levels in the mammary gland. Compared with the high-grain group, 2-dimensional electrophoresis technology, matrix-assisted laser desorption/ionization-time of flight/time of flight proteomics analyzer, and western blot analysis further verified that the expression levels of beta casein (CSN2) and lactoferrin (LF) proteins in the mammary glands of lactating goats were higher when fed a high-grain diets and buffers. The mechanism of increased milk protein synthesis was demonstrated to be related to the activation of mammalian target of rapamycin (mTOR) pathway signals.

Effects of increased supply of essential amino acids on the metabolism of the isolated perfused guinea-pig mammary gland

1979 ◽  
Vol 46 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Andrew R. Peters ◽  
Stephen Alexandrov ◽  
T. Ben Mepham
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Uptake ◽  
Essential Amino Acids ◽  
Acid Uptake ◽  
Isoleucine Leucine ◽  
Group 1

SUMMARYThe effects of high rates of infusion of essential amino acids on amino acid uptake by the isolated perfused guinea-pig mammary gland were studied. Infusion of methionine, tyrosine, phenylalanine, histidine and tryptophan (designated group 1) resulted in significant increases in the uptakes of tyrosine, phenylalanine and histidine. Methionine, tryptophan and other essential amino acids were not significantly affected. Infusion of threonine, valine, isoleucine, leucine, lysine and arginine (designated group 2) resulted in significant increases in uptake of all these amino acids. Group 1 amino acid uptake was not significantly affected. Infusion of all the essential amino acids (i.e. groups 1 and 2 together) resulted in significant increases in all their uptakes. Using as index ‘the predicted rate of protein synthesis’, infusion of group 1 and 2 together led to an apparent 27% increase in protein synthesis. The above results are discussed in relation to the control of milk protein synthesis by limiting essential amino acids.

Steps in amino-acid incorporation into mammary tissue

1958 ◽  
Vol 149 (936) ◽  
pp. 392-400 ◽  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Amino Acid ◽  
De Novo ◽  
Protein Biosynthesis ◽  
Mammary Tissue ◽  
Mammary Cells ◽  
Intact Cells ◽  
Ideal System

The mammary gland in full lactation had for long been recognized as an ideal system for the study of the biosynthesis of protein. The discoveries during the last 5 years of the incorporation of labelled amino acids into the microsomes of cell homogenates and of other reactions of amino acids which might be on the pathway to protein synthesis, encouraged us to study the fate of amino acids in various systems prepared from mammary cells. De novo protein synthesis had not yet been proved in any system which contained no intact cells. So far no net increase in any defined protein fraction during incubations has been found or indeed looked for in our experiments. Naturally one hopes that such studies of the fate of labelled amino acids in cell-free preparations will reveal the detail of enzymic reactions which will prove to be part of the mechanisms of protein biosynthesis. Three types of reactions of amino acids in cell-free preparations from homogenates of many tissues have been studied most extensively. (1) The acyl activation of amino acids to form amino acid-acid adenylates in the presence of ATP and ‘activating enzymes’. (2) The formation of compounds of cell sap-ribonucleic acid ( SRN A ) with amino acids in the presence of ATP and ‘activating enzymes’. (3) The incorporation of amino acids into intracellular particles either from free amino acid or by transfer from amino-acid- SRN A compounds in the presence of ATP , guanosine triphosphate ( GTP ) and ‘activating enzymes’. In this paper we are giving a survey of the results of studies on these three types of reactions in systems prepared from mammary tissue and we are relating these to results obtained with other systems elsewhere. Some comparative studies of the incorporation of labelled amino acids into protein fractions of intact mammary cells (minced tissue) are also presented. All the original results given here were obtained from experiments with guinea-pig mammary gland preparations from animals 2 to 6 days after parturition. Experimental detail will be reported elsewhere.

scholarly journals Effects of High-Grain Diet with Buffering Agent on the Milk Protein Synthesis in Lactating Goats

2020 ◽  
Author(s):  
Meilin He ◽  
Xintian Nie ◽  
Huanhuan Wang ◽  
Shuping Yan ◽  
Yuanshu Zhang
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Amino Acid ◽  
Western Blot ◽  
Milk Protein ◽  
Mtor Pathway ◽  
Production Performance ◽  
Dairy Goats ◽  
Buffering Agent

AbstractFeeding of straw as main roughage with numerous high-grain diets improves the performance of ruminants but it can easily lead to subacute ruminal acidosis. In recent years, buffering agent is applied to prevent the acid poisoning of ruminants and improve the production performance of ruminants in animal husbandry. it is necessary to understand feeding high-grain diet with buffering agent which transport carriers amino acids mainly take amino acids into the mammary gland and the signal mechanism of amino acids in the mammary gland synthesize milk proteins. To gain insight on the effects of a high-grain diet with buffering agent on the amino acids in the jugular blood, and the effects of amino acids on the synthesis of milk protein, commercial kit and high performance liquid chromatography (HPLC) were applied to determine the concentration of amino acids of jugular blood samples, quantitative real-time PCR, comparative proteomic approach and western blot were employed to investigate proteins differentially expressed in mammary tissues and the mechanism of amino acids on the synthesis of milk protein in mammary gland of lactating dairy goats fed high-grain diet with buffering agent or only high-grain diet.Results showed that feeding high-grain diet with buffering agent to lactating dairy goats could outstanding increase amino acid content of jugular blood (p<0.05), and mRNA transcriptional level of amino acid transporters in the mammary gland were also increased; the CSN2 and LF protein expression level were significant higher by 2-DE technique, MALDI-TOF/TOF proteomics analyzer and western blot analysis further validated in mammary of lactating dairy goats compared with high-grain group; the research on the mechanism of milk protein synthesis increasing suggested that it was related to the activation of mTOR pathway signaling.Feeding of high-grain diet with buffering agent promoted the jugular vein blood of amino acids concentration, and more amino acids flowed into the mammary. In addition, milk protein synthesis was increased and the increase of milk protein synthesis was related to the activation of mTOR pathway signalling.

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