Leonard S. Jefferson

The hormonal control of glycogen metabolism: dephosphorylation of protein phosphatase inhibitor-1 in vivo in response to insulin.

Oyer the past few years evidence has accumulated that protein phosphatase-I is of central importance in the regulation of glycogen metabolism in mammalian skeletal muscle. This enzyme catalyses the dephosphorylation of glycogen phosphorylase, phosphorylase kinase and glycogen synthase, and therefore carries out the reactions which inhibit glycogenolysls or activate glycogen synthesis (reviewed [ 1,2]). Protein phosphatase-I has been shown to be inhibited in vitro by two heat-stable proteins termed inhibitor-l and in~~ibitor-2, one of which (inhibitor-l) is only an inhibitor after it has been phosphorylated by cyclic AMP-dependent protein kinase [3,4]. Inhibitor-l and inhibitor-2 have been purified to homogeneity in this laboratory [5,6] and characterized’ extensively [ 1,7-93. Inhibitor-l is phospho~lated by cyclic AMP-dependent protein kinase in vitro at a similar rate to phosphorylase kinase and glycogen synthase 171. It inhibits the phosphorylase phosphatase, phosphorylase kinase phosphatase and glycogen synthase phosphatase activities of protein phosphatase-I in a reversible non-competitive manner,and halfmaximal inhibition is observed at 3-4 nM [S]. The concentration of ~nhibitor”l in rabbit skeletal muscle is 1.8 PM 161, which is higher than the concentration of protein phosphatase-1 [ 11. The properties of inhibitor-l determined in vitro are consistent with the idea that this protein is important in the regulation of protein phosphatase-1 in vivo. It has been demonstrated that inhibitor-l is phosphorylated in rabbit skeletal muscle in vivo, and that its degree of phosphorylation increases markedly in response to adrenaline [IO]. Here we demonstrate

Regulation of guanine nucleotide exchange through phosphorylation of eukaryotic initiation factor eIF2alpha. Role of the alpha- and delta-subunits of eIF2B.

The guanine nucleotide exchange activity of eIF2B plays a key regulatory role in the translation initiation phase of protein synthesis. The activity is markedly inhibited when the substrate, i.e. eIF2, is phosphorylated on Ser51 of its α-subunit. Genetic studies in yeast implicate the α-, β-, and δ-subunits of eIF2B in mediating the inhibition by substrate phosphorylation. However, the mechanism involved in the inhibition has not been defined biochemically. In the present study, we have coexpressed the five subunits of rat eIF2B in Sf9 cells using the baculovirus system and have purified the recombinant holoprotein to >90% homogeneity. We have also expressed and purified a four-subunit eIF2B complex lacking the α-subunit. Both the five- and four-subunit forms of eIF2B exhibit similar rates of guanine nucleotide exchange activity using unphosphorylated eIF2 as substrate. The five-subunit form is inhibited by preincubation with phosphorylated eIF2 (eIF2(αP)) and exhibits little exchange activity when eIF2(αP) is used as substrate. In contrast, eIF2B lacking the α-subunit is insensitive to inhibition by eIF2(αP) and is able to exchange guanine nucleotide using eIF2(αP) as substrate at a faster rate compared with five-subunit eIF2B. Finally, a double point mutation in the δ-subunit of eIF2B has been identified that results in insensitivity to inhibition by eIF2(αP) and exhibits little exchange activity when eIF2(αP) is used as substrate. The results provide the first direct biochemical evidence that the α- and δ-subunits of eIF2B are involved in mediating the effect of substrate phosphorylation.

Binding of radioactive α-difluoromethylorithine to rat liver ornithine decarboxylase

Inactivation of rat liver ornithine decarboxylase by incubation with [5-14C]-α-difluoromethylornithine resulted in the covalent binding of radio-activity to the enzyme. The extent of binding correlated with the degree of inactivation and with the amount of enzyme present. The labeled protein eluted as a single peak which coincided exactly with the active enzyme when chromatographed on Sephadex G-200 and ran as a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at a position corresponding to a M.W. of about 55,000. The stoichiometric binding of [5-14C]-α-difluoromethylornithine therefore provides a convenient method for quantitating ornithine decarboxylase protein and for determining the purity of preparations of the enzyme. Assuming that 1 molecule of the drug is needed to inactivate each sub-unit, it was calculated that after stimulation with thioacetamide ornithine decarboxylase represents about 0.00014% of the liver soluble protein.

Effect of diabetes on guanine nucleotide exchange factor activity in skeletal muscle and heart.

The present study examined the effects of diabetes and insulin treatment of diabetic rats on the activity of the protein synthesis initiation factor, the guanine nucleotide exchange factor. In extracts from gastrocnemius and psoas muscles from two-day diabetic rats, guanine nucleotide exchange factor activity was reduced to 80% and 67% of control values, respectively. Insulin treatment (2 h) restored guanine nucleotide exchange factor activity to control values in both muscles. In contrast, guanine nucleotide exchange factor activity was unchanged in extracts from either soleus muscle or heart from diabetic rats compared to controls. Also, insulin treatment did not increase guanine nucleotide exchange factor activity in extracts from soleus and heart. The results suggest that the diabetes-induced impairment in peptide-chain initiation in fast-twitch skeletal muscle (i.e. gastrocnemius and psoas) is related to an inhibition of guanine nucleotide exchange factor activity and that slow-twitch muscle is spared from the effect on initiation due to the preservation of guanine nucleotide exchange factor activity.

Purification and characterization of eukaryotic translational initiation factor eIF-2B from liver.

Eukaryotic initiation factor (eIF)-2B was purified to greater than 95% homogeneity from both rat and bovine liver. The purified protein consisted of five nonidentical subunits with apparent molecular weights ranging from 30.9 to 89.1 kDa. The holoprotein was characterized in terms of its Stokes radius and frictional coefficient. The isoelectric points for the beta-, gamma-, and epsilon-subunits were found to be 6.4, 6.9, and approximately 6.0, respectively; the alpha- and delta-subunits did not focus well because their isoelectric points as predicted by the nucleotide sequences of cDNAs for the two proteins are greater than 8.5. The purified protein was used as antigen to generate monoclonal antibodies to the epsilon-subunit. The eIF-2B epsilon monoclonal antibodies and monoclonal antibodies to the alpha-subunit of eIF-2 were then used to directly quantitate the amounts of eIF-2B and eIF-2 in rat liver and rat reticulocytes. The ratio of eIF-2B to eIF-2 was found to be approx. 0.6 and 0.3 in liver and reticulocytes, respectively, supporting the proposition that phosphorylation of only part of the total cellular eIF-2 could potentially sequester all of the eIF-2B into an inactive eIF-2.eIF-2B complex. The purified protein was also used as substrate in protein kinase assays. Extracts of rat liver were shown to contain protein kinase activity directed toward the epsilon-subunit, but no other subunit of eIF-2B. Overall, the studies presented here are the first to show a direct quantitation of eIF-2 and eIF-2B in different tissues. They also provide evidence that the epsilon-subunit of eIF-2B is the only subunit of eIF-2B that is phosphorylated by protein kinase(s) present in extracts of rat liver.

Maintenance of protein synthesis in hearts of diabetic animals

Abstract Skeletal muscle of diabetic animals contained increased numbers of ribosomal subunits and decreased polysomes. However, numbers of polysomes and subunits in heart muscle 5 diabetic rats were the same as controls. Incorporation of14C-phenylalanine into protein of diabetic hearts occurred at the control rate. When control hearts were perfused, in vitro , with medium containing glucose and normal plasma levels of amino acids, polysomes decreased and subunits increased. These changes were prevented by addition of either insulin or palmitate suggesting that these factors facilitated peptide-chain initiation in heart muscle.

Studies on ornithine decarboxylase activity in the isolated perfused rat liver.

Abstract l -Ornithine decarboxylase ( l -ornithine carboxy-lyase, EC 4.1.1.17) activity was studied in the isolated perfused rat liver. Activity was lost rapidly on perfusion in the presence of cycloheximide to block protein synthesis but the decline was retarded in the presence of high levels of amino acids. An even more rapid loss of activity was produced by the addition of 5 mM putrescine to the perfusion medium. In this case, activity was lost with a half-time of 15 min. Perfusion for 3 h in a recirculating system in the presence of 10 times normal plasma levels of amino acids led to a 4–5-fold increase in ornithine decarboxylase activity during the final hour of perfusion. This effect was also seen in perfusion of livers from hypophysectomized or adrenalectomized rats. Growth hormone produced an additional increase of activity during a 3 h perfusion. Although amino acids were required for the increased activity, a similar increase could not be produced by addition of l -ornithine or dihydro- l -orotate to the perfusion medium. Since putrescine levels fell during the first 2 h of perfusion it is suggested that the increase of ornithine decarboxylase activity was in response to the lowered putrescine levels. Addition of putrescine to the perfusion medium at any time produced a rapid revesal of the increase in activity. After perfusion in the presence of putrescine a non-diffusible inhibitor of ornithine decarboxylase was found in liver extracts. These data indicate the value of the isolated perfused liver in studies of the control of onithine decarboxylase and strongly support the hypothesis that putrescine levels may play a key role in regulating ornithine decarboxylase activity and polyamine synthesis.

Inhibition of microsomal calcium sequestration causes an impairment of initiation of protein synthesis in perfused rat liver

The present study examined the effect of 2,5-di-(tert-butyl)-hydroquinone (tBuHQ), an inhibitor of liver microsomal calcium sequestration, on initiation of protein synthesis in perfused rat liver. Perfusion of livers with a concentration of tBuHQ previously shown to completely inhibit microsomal calcium sequestration in isolated hepatocytes caused a 50% inhibition of protein synthesis. The inhibition was characterized by an increase in liver content of free ribosomal particles and a decrease in polysomes indicating that peptide-chain initiation was slowed relative to elongation. Furthermore, the inhibition was associated with a 7.5-fold increase in the proportion of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2) present in the phosphorylated form and a reduction in the activity of eukaryotic initiation factor 2B (eIF-2B) to 37% of the control value. The results suggest that protein synthesis in rat liver is regulated directly by changes in intracellular calcium concentration through a mechanism involving modulation of the phosphorylation state of eIF-2 alpha.

Liver as a source of transformed cell growth factor

Abstract Isolated rat hepatocytes release an acidic glycoprotein(s) that can selectively promote the growth of transformed cells. This factor has a molecular weight of 60 000–70 000 D. Liver microsomal and cytosol fractions contain two species of stimulatory activity—44 000 and 3 500 D. Mitochondrial and nuclear fractions contain only the lower molecular weight factor.

Effect of methylglyoxal bis(guanylhydrazone) on S-adenosylmethionine decarboxylase in the isolated perfused rat liver☆

S-Adenosylmethionine decarboxylase (E.C. 4.1 .I SO) is a key enzyme in the biosynthetic pathway leading to polyamines [l-7] . The possibility that information as to the biological role of polyamines might be obtained by inhibition of the activity of this enzyme was raised by the discovery that methylglyoxal bis(guanylhydrazone) (MGBG) is a very potent but reversible inhibitor of mammalian Sadenosylmethionine decarboxylases [ 1 ] . However studies of the effects of this inhibitor in vivo have revealed that its administration leads to a large increase in the amount of S-adenosylmethionine decarboxylase activity present in various tissues when extracts are assayed removal of any residual inhibitor [2-51. This finding coupled with the toxicity and rate of excretion of the drug has prevented the use of MGBG to investigate the role of polyamines in intact animals, although studies of the effects of MGBG on cells in culture have proved more rewarding [S-7]. The mechanism by which a large increase in the activity of an enzyme inhibited by MGBG is produced by administration of the drug is of considerable interest particularly since MGBG is a useful antileukaemic agent [8]. The present paper reports studies of the