K.L. Manchester

Isolation of labelled aminoacyl transfer RNA from muscle Studies of the entry of labelled amino acids into acyl transfer RNA linkage in situ and its control by insulin

Abstract Methods have been investigated for measurement of the specific activity of labelled amino acids contained in aminoacyl-tRNA of muscle after incubation of diaphragm or perfusion of heart with labelled amino acids. Extraction of tissue homogenates with hot trichloroacetic acid, alkali or hot NaCl solution were all found to solubilise sufficient protein to make measurement of radioactivity specifically derived from aminoacyl-tRNA impossible. Treatment of the pH 5 precipitate or 100 000×g supernatant with phenol and/or bentonite resulted in reasonable purification of tRNA as judged by density gradient profiles. Two extractions with bentonite at pH6 without phenol was found to be a satisfactory compromise in terms of yield, purity of product and ease of manipulation. By such means aminoacyl-tRNA labelled with tyrosine and leucine was satisfactorily isolated, but the correlation of radioactivity with absorbance during gradient analysis was poor when glycine, proline and methionine were the source of label. The specific activity calculated for the amino acid of the aminoacyl-tRNA appeared in most cases to reach values close to that of the free amino acid added to the system. Turnover of the aminoacyl group was rapid. Labelling of the leucyl- and tyrosyl-tRNA was raised in the presence of insulin.

Comparison of ability of Mg and Mn to activate the key enzymes of glycolysis

Mg and Mn are activating ions for all enzymes employing adenine nucleotides and for many others. Ca by contrast is often an inhibitor. Interaction between the ions has been suggested as a possible mechanism involved in the control of metabolism including the pathways of glycolysis and gluconeogenesis. We have already published some kinetic parameters for the interaction of these ions with the enzymes of hepatic gluconeogenesis [ 11. The present study is concerned with the effects of bivalent ions on the activities of key glycolytic enzymes of liver.

Diminution in starvation of capacity of cell sap to support protein synthesis in cell‐free systems

A diminished capacity of sap from tissues of diabetic animals to support incorporation of amino acids into protein in cell-free systems has several times been noted [l-3] . Conversely there is evidence that long-term protein malnutrition enhances the activity of the hepatic amino acid activating enzymes [4, 51. There are surprisingly no reports of the effect of fasting on the capacity of sap to support protein synthesis by isolated ribosomes, though after 6 days without food the hepatic content of amino acid activating enzymes is said to be unchanged [4] _ We find that sap from liver and muscle of fasted rats has a lower capacity to support protein synthesis and that this decrease is not attributable to change in the size of the pool of free amino acids.

Evaluation of kinetic parameters for uptake of amino acids by cells: Effect of insulin on the accumulation of aminoisobutyrate and cycloleucine by isolated rat diaphragm muscle and chick embryo hearts

An attempt has been made to evaluate some of the kinetic parameters governing the uptake of amino acids by muscle by measuring the steady-state concentration ratios established on incubation of the tissue in vitro. The data are analysed in terms of the steady state resulting from a balance between saturable uptake and efflux processes in conjuction with diffusion. For aminoisobutyrate and cycloleucine the data were compatible with efflux being solely by diffusion, but it was also possible to fit the data to conditions such that efflux by a saturable process and by diffusion were both occurring. Measurement of initial entry rates after preloading hearts with aminoisobutyrate gave results consistent with the first possibility only. Such measurements also suggested that the parameters governing a saturable uptake process changed during the course of uptake, but that the rate constant for diffusion remained the same. The rate of approach to equilibrium was found to be faster the higher the external concentration of amino acid. Both these observations would be consistent with accumulated amino acid progressively interfering with further operation of a carrier. For aminoisobutyrate the enhancement of accumulation in the presence of insulin appeared to result from an increase in the maximal velocity of the transport rate rather than from any change in apparent Km. For cycloleucine there was some diminution in the Km with insulin but again a rise in the transport rate.

Effects of denervation hypertrophy in rat diaphragm muscle on the activity of ribosomes and sap fractions in protein synthesis

Abstract Rat hemidiaphragm undergoes a pronounced transient hypertrophy in the first few days following unilateral nerve section. Ribosomes have been prepared from the hypertrophying tissue 1, 3 and 10 days after denervation. Enhanced capacity for incorporation of [ 14 C]leucine into protein was found after 3 days, though not after as short an interval as 1 day or as long as 10. Activity of the ribosomes in poly(U) directed incorporation of [ 14 C]phenylalanine was unchanged. The possibility of an increased level of mRNA or of growing peptide chains in the polysomes of the tissue denervated three days is considered. The capacity of sap extracts prepared from diaphragm to support incorporation of [ 14 C]leucine by leg muscle polysomes was also found to be enhanced by the third day after nerve section. No effect was seen after 1 day, but there was possibly still increased activity after 10 days. Hypertrophy of the denervated rat diaphragm thus shows a co-ordinated enhancement of polysome activity and of capacity of sap to support incorporation, which correlates with an increased capacity for amino acid accumulation and maintenance of tissue amino acid levels.

The actions of insulin and glucagon on glucose metabolism and on related enzyme activities in the isolated perfused rat liver

Abstract 1. 1. The metabolic response of perfused livers to glucagon or insulin stimulation has been followed, measuring glucose, amino acid, urea, lactate, pyruvate, lipid and β-hydroxybutyrate levels in the perfusate. 2. 2. Analysis of these perfused livers shows changes in the activities of some of the key glycolytic, gluconeogenic and lipogenic enzymes. Lactate perfusion increases pyruvate carboxylase and decreases isocitrate dehydrogenase; these changes are prevented by insulin and glucagon respectively. Under these conditions, both hormones decrease phosphofructokinase, and insulin also increases fructose-1, 6-diphosphatase. At endogeneous substrate levels, insulin looses its effect on the latter enzyme. In both types of perfusion insulin produces a 50% drop in phosphoenolpyruvate carboxykinase and an increase in pyruvate kinase. 3. 4. Neither hormone has any effect on glucokinase, hexokinase, glucose-6-phosphatase, acetyl-CoA carboxylase, ATP-citrate lyase or malic enzyme.

Influence of denervation of the free amino acids of the rat diaphragm

Abstract The concentrations of free amino acids in diaphragm muscle have been measured at intervals following unilateral nerve action. Marked increases of alanine, glycine, glutamine and the serine/asparagine peakd were seen between 3 and 7 days, but little change occurred for most other amino acids. Comparison is made with the data on changes of amino acid concentration in muscle in response to insulin, in liver during regeneration and with metabolic changes taking place in the denervated tissue.

Effects of manganese on the activity of glycolytic and gluconeogenic enzymes in the perfused rat liver

We have shown previously [l] that pyruvate carboxylase activity declines when liver from the fasted rat is perfused with glucose containing medium. Conversely addition of lactate to the perfusate enhances the activity of both pyruvate carboxylase and phosphoenolpyruvate carboxykinase [2]. It appears that the hepatic activity of these two enzymes may fluctuate depending on the concentration of the final product and on the supply of precursors for transformation. Administration of manganese results in a rapid increase in serum glucose [3] and is reported to enhance glucose production by the perfused liver [4]. It is a potent activator of several glycolytic and gluconeogenic enzymes including specifically pyruvate carboxylase and phosphoenolpyruvate carboxykinase [5,6]. We here report that addition of manganese to the perfused rat liver prevents the increase in activity of the two enzymes otherwise induced by lactate. This seemingly contradictory event is interpreted to mean that the extra functional capacity of pyruvate carboxylase and phosphoenolpyruvate carboxykinase resulting from manganese activation minimises the influences which would otherwise induce extra enzyme synthesis.

Suppression of pyruvate carboxylase by glucose in the perfused rat liver.

The hepatic levels of three of the key enzymes of gluconeogenesis, pyruvate carboxylase (E.C. 6.4.1. l), phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) and glucose-6-phosphatase (E.C. 3.1.3.9) rise in fasting and conversely decrease on refeeding [l-5] , the fourth enzyme, fructose-l ,6-diphosphatase (E.C. 3.1.3.1 l), decreasing during an overnight fast [6-71 . The levels of these enzymes are also elevated in diabetes and lowered on treatment with insulin [8-121. It is thus likely that insulin rather than food per se is the suppressor of the enzyme levels in the fedstate, the glucose intake stimulating insulin secretion. If this is so, it is not to be expected that addition of glucose would influence the levels of these enzymes in the perfused liver. This appears to be the case for three of the four enzymes, but we find that the activity of pyruvate carboxylase does decline under such conditions.