Ernst Helmreich

Functional heterogeneity of the intracellular amino acid pool in mammalian cells

Abstract 1. 1. The kinetics of tracer amino acid appearance in the intracellular amino acid pool and in cellular protein was examined under steady-state conditions in the intact rat-diaphragm preparation and in isolated guinea pig and rabbit-lymph node cells. 2. 2. The concentration ratio of tracer amino acid between the extracellular and intracellular amino acid pools approached equilibrium exponentially whereas incorporation into cellular protein proceeded linearly from the earliest time period studied. 3. 3. A theoretical formulation is presented predicting the kinetics of amino acid incorporation into cellular protein if the total intracellular amino acid pool were an obligatory intermediate in protein synthesis. 4. 4. The marked deviation of experimental observations from those theoretically predicted has been interpreted to indicate functional heterogeneity (compartmentation) of the intracellular amino acid pool.

Regulation of glycolysis in muscle

Abstract The question was raised, what causes the sudden increase in the rate of glycolysis when muscle contracts and the equally rapid decrease in rate when the muscle relaxes. For the purpose of this study, isolated frog sartorii were stimulated electrically while immersed in anaerobic Ringers solution without glucose. The rate was varied between 3 and 48 shocks per min and the duration was 30 min. Under these conditions, the conversion of glycogen to lactate is the main energy producing reaction. The results indicated that two reactions are of prime importance in the regulation of glycolysis in the working muscle: the formation of glucose-1-P from glycogen and inorganic P through the action of the phosphorylase system and the removal of fructose-6-P through the phosphofructokinase reaction. From the relation of the concentration of hexosemonophosphates to the flow rate over the glycolytic system it was concluded that during stimulation these two enzyme systems increase their activity synchronously and proportionately. This is in contrast to the action of epinephrine which causes a much greater increase in phosphorylase activity than in phosphofructokinase activity and thereby leads to a large accumulation of hexosemonophosphates. From measurements of tissue concentration and rate of efflux of lactate from muscle immediately after cessation of stimulation it was concluded that lactate formation returned to the resting rate within 5 min. On the basis of measurements of the tissue concentrations of ATP, ADP, AMP, and Pi it was concluded that changes in the concentrations of these compounds in stimulated muscle were too small to permit the increase in enzymatic rates actually observed in vivo. This applies particularly to the phosphorylase reaction for which in vitro data are available which permit predictions of rates at different concentrations of the above reactants. The concentrations of glucose-6-P, fructose-6-P and fructose-1,6-di-P were likewise not well correlated to the rate increase. The only two compounds that increased proportionately with the frequency of stimulation were end products of anaerobic glycolysis in muscle, namely lactate and α- l -glycero-P. A detailed investigation of the phosphorylase b [rlhar2] a interconversion in stimulated muscle suggested that the phosphorylase b kinase underwent an activation-inactivation cycle closely connected with the contraction and relaxation phases of muscle. An analysis of results obtained with a strain of mice incapable of forming phosphorylase a during stimulation indicated that this interconversion, while not essential for lactate formation per se, was of importance for the time of onset and the final speed of lactate formation. A hypothesis based on localization and structural organization of the glycolytic chain in muscle is proposed which could explain the results summarized above. A mechanism is suggested which is similar in nature to the activation and inactivation of actomyosin (and perhaps of phosphorylase b kinase) by Ca++ ions. It contains the idea of access of reactants to the catalyst during contraction and separation from reactants during relaxation.

Purification and functional characterization of the human β2-adrenergic receptor produced in baculovirus-infected insect cells

A human cDNA fragment bearing the complete coding region for the β2‐adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocynnopindolol showed that Sf9 insect cells infected with the recombinant virus expressed ≈ 1 × 104 β2‐adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membraines revealed a molecular weight of ≈ 46000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs‐protein.

Chapter II - Control of Synthesis and Breakdown of Glycogen, Starch and Cellulose

Publisher Summary This chapter discusses the developments related to the control of synthesis and breakdown of glycogen, starch, and cellulose. The main emphasis is laid on the regulation of biosynthetic and degradative pathways. The synthesis of glycogen, in vitro , by the action of a purified enzyme was achieved by C.F. Cori, G. Schmidt and G. T. Cori in 1939. From this time until rather recently the prevalent view among biochemists was that in living systems the same reversible enzymatic reactions are responsible for synthesis and degradation of energy stores. Thus, it was generally accepted that phosphorylase in vivo acted both ways and catalyzed the formation and the breakdown of glycogen. This view was further supported by the fact that the phosphorylase reaction is without a doubt reversible and can form glycogen in vitro . However, thermodynamically, the synthetase reaction favors the formation of α-1,4-glycosidic bonds, which is much more than the phosphorylase reaction and the thermodynamic advantages beat the comparatively low actual activity of the synthetase.

Receptor Modification and Receptor Disposition in Membranes

This report deals with the functional significance of covalent modifications of the s1-adrenoceptor from turkey erythrocytes. Two types of modification are being considered: phosphorylation and glycosylation. The effects of these modifications were tested using a reconstitution assay with pure receptor and GTP-binding proteins incorporated into lipid vesicles.