Otto Walaas

Insulin increases membrane protein kinase C activity in rat diaphragm

Calcium/phospholipid‐dependent protein kinase activity (protein kinase C) was identified in rat diaphragm membrane and cytosol fractions by means of in vitro phosphorylation either of histones or of a specific 87 kDa protein substrate, combined with phosphopeptide‐mapping techniques. Both insulin and tumor‐promoting phorbol ester treatment of the diaphragm preparations led to increased protein kinase C activity in the membrane fractions. In contrast to the phorbol ester, however, insulin did not induce a concomitant decrease in cytosolic activity, indicating that translocation of the enzyme had not taken place. Thus, insulin appears to increase specifically membrane protein kinase C activity in rat skeletal muscle, possibly through a mechanism not identical to that induced by phorbol esters.

Spectrophotometric and electron-spin resonance studies of complexes of catecholamines with Cu(II) ions and the interaction of ceruloplasmin with catecholamines

Abstract The complex formation of catecholamines with Cu(II) ions, and their interaction with the copper-containing enzyme ceruloplasmin, have been investigated, with special emphasis on complex formation in the absence of oxygen. In argon, stable 1:2 copper(II)ion-catecholamine complexes with definite optical properties have been observed. The electron-spin resonance (ESR) absorption spectra of these complexes in solution have been recorded. They exert the same characteristics as described for Cu(II) complexes with small molecules in general. Introduction of oxygen initiates the transformation of the catecholamines to the corresponding indolic structures, the adrenochrome, and the noradrenochrome, with characteristic optical properties. The ESR absorption spectra of the complexes are retained in the oxygen-containing solution, but the hyperfine structure is diminished. The interaction of catecholamines with ceruloplasmin in the absence of oxygen is characterized by the disappearance of the 605 mμ absorption band. At the same time the ESR signal of ceruloplasmin completely disappears. Introduction of oxygen reactivates the enzyme and promotes oxidation of the ligand. Simultaneously the ESR absorption spectrum of the enzyme partly reappears. A cupric-cuprous couple has been indicated for the mechanism of enzyme action. The mechanism of electron transfer during oxidation of catecholamines by the model complexes and by ceruloplasmin has been discussed.

Oxidation of reduced phosphopyridine nucleotides by p-phenylenediamines, catecholamines and serotonin in the presence of ceruloplasmin.

Abstract Oxidation of reduced diphosphopyridine nucleotide and triphosphopyridine nucleotide has been obtained by a system containing ceruloplasmin and an appropriate substrate. Among the substrates investigated, p -phenylenediamine, N,N -dimethyl- p -phenylenediamine, and 3,4-dihydroxyphenylhydroxyethylamine (noradrenaline) gave the highest rate of oxidation. The N -methyl-substituted 3,4-dihydroxyphenylhydroxyethylamine (adrenaline) showed definitely lower activity, and 5-hydroxyptamine (serotonin) was slightly active. It is indicated that two steps are involved in the reaction. First, free radicals of the substrates are formed as a result of ceruloplasmin activity. Secondly, the radicals oxidize reduced phosphopyridine nucleotides by acting as “one-electron acceptor” agents. These conclusions are based upon experiments where the absorption spectra of substrates and ceruloplasmin during enzymic activity have been investigated. It has been demonstrated that the intensity of the absorption band with maximum at 605 mμ in ceruloplasmin is decreased by the substrates. This indicates a reduction of the Cu 2+ atoms of the enzyme during catalytic activity.

A stimulatory effect of insulin on phosphorylation of a peptide in sarcolemma-enriched membrane preparation from rat skeletal muscle

It has been well established that insulin stimulates glycogen and lipid synthesis by influencing protein phosphorylation of some of the enzymes involved in these processes. Three different mechanisms for the insulin effect on phosphorylation of enzymes in the cytosol have been proposed. These involve: (i) Decreased cyclic AMP-dependent protein kinase activity [ 1,2] . (ii) Inhibition of a newly discovered protein kinase which is independent of cyclic AMP (3). (iii) Activation of protein phosphatase [4] . An insulin effect by any of these mechanisms would lead to dephosphorylation of certain metabolic enzymes. On the other hand we have previously demonstrated that insulin promotes increased turnover of 32P in y-phosphate of ATP in muscle [S] . In light of these observations we have now investigated the possibility that insulin might influence protein phosphorylations at the level of the plasma membrane. For this purpose we have studied a sarcolemma enriched membrane preparation from rat skeletal muscle. This membrane preparation has been isolated by a mild procedure where treatment with high salt concentrations has been avoided. Protein phosphorylation of this membrane preparation has been investigated and it has been demonstrated that insulin specifically itimulates phosphorylation of a membrane peptide of lower molecular weight. 2. Methods

The influence of sodium, potassium and lithium on the response of glycogen synthetase I to insulin and epinephrine in the isolated rat diaphragm

Abstract 1. 1. Intact rat hemidiaphragms were incubated in media of different ionic composition in the absence and the presence of hormones i.e. insulin and epinephrine. After incubation, glycogen synthetase activity of diaphragm extracts was determined in the absence (I-form) and the presence of glucose 6-phosphate (D-form). 2. 2. Incubation of the diaphragms in media of high K+ concentration led to decreased glycogen synthetase I levels. The response of the synthetase system to insulin (conversion of the D-form to the I-form) was reduced under these conditions. Furthermore, the conversion of the I-form to the D-form of glycogen synthetase promoted by epinephrine was also reduced. 3. 3. Incubation of the diaphragm in lithium-containing media increased glycogen synthetase I. Insulin and lithium exerted additive effects in promoting D to I conversion of the synthetase. 4. 4. The response of glycogen synthetase of the diaphragm to insulin (conversion of the D-form to the I-form) was markedly reduced when (Na+-K+)-activated ATPase was blocked by 0.2 mM ouabain in the incubation medium. 5. 5. The insulin effect on glycogen synthetase was absent when active Na+ and K+ transport was blocked by incubation in a medium lacking K+. This was shown by: (a) incubation of the diaphragm in a K+-free medium to which tetraphenylboron, a K+-chelating agent, was added. (b) removal of K+ by preliminary soaking of the diaphragm in a medium containing Dowex 50 WNa. 6. 6. The response of glycogen synthetase to epinephrine was not affected by incubation of the diaphragm in a K+-free medium.

Interaction of dimethyl-p-phenylenediamine with ceruloplasmin

The stoichiometric interaction of dimethyl-p-phenylenediamine (DPD) with ceruloplasmin-Cu2+ has been investigated by spectrophotometric and electron paramagnetic resonance technique. Intermediates in the reaction sequence, DPD → (DPD)+ → (DPD)2+ → further oxidation products, were characterized. Cerulo-plasmin-Cu2+ was rapidly reduced by DPD as well as by the monoradical ion (DPD)+ but not by (DPD)2+. The two oxidation products (DPD)+ and (DPD)2+ were reversibly reduced by NADH2. The stoichiometry of the reactions confirmed that DPD is oxidized by one electron transfer to ceruloplasmin-Cu2+ in two subsequent steps. Calculations of some of the rate constants were done. The “association constant” k1 for DPD with ceruloplasmin was 750m−1 sec−1, and k′1 for (DPD)+ 800m−1 sec−1 at 10 °. The rate constant for the reoxidation of ceruloplasmin-Cu+ was 0.037 sec−1. When DPD was oxidized by ceruloplasmin, the formation of a relatively stable protein-bound radical was indicated. Kinetic studies of the oxidation of DPD by ceruloplasmin in catalytical amounts were done. The results suggested two different types of active sites or oxidizing units in ceruloplasmin.

Phosphorylation of multiple sites in a 15 000 dalton proteolipid from rat skeletal muscle sarcolemma, catalyzed by adenosine 3′,5′-monophosphate-dependent and calcium / phospholipid-dependent protein kinases

This study reports a partial characterization of a 15,000 dalton (15 kDa) proteolipid present in rat skeletal muscle sarcolemma. The proteolipid is phosphorylated by both cyclic AMP-dependent and calcium/phospholipid-dependent protein kinases, displays an isoelectric point (pI) of 5.9, and can be extracted from sarcolemma by acidified chloroform/methanol (2:1) or non-ionic detergents. Phosphoamino acid analysis and tryptic fingerprinting of the phosphorylated proteolipid indicate that both cyclic AMP- and calcium/phospholipid-dependent protein kinases predominantly phosphorylate serine residue(s) on a single tryptic peptide. Additivity experiments and thermolytic fingerprinting demonstrate a minimum of two distinct phosphorylation sites on the proteolipid, the phosphorylation of which is independently catalyzed by cyclic AMP-dependent and calcium/phospholipid-dependent protein kinases in vitro. This sarcolemma proteolipid, which appears to be identified to a sarcolemma protein previously reported to be phosphorylated upon addition of insulin in a GTP-dependent manner (Walaas, O., Walaas, E., Rye-Alertsen, A. and Horn, R.S. (1979) Mol. Cell. Endocrinol. 16, 45-55), therefore represents a possible membrane target for those neuronal and hormonal stimuli which can regulate cyclic AMP-dependent or calcium/phospholipid-dependent protein kinase activities in skeletal muscle.

The effect of insulin and guanosine nucleotides on protein phosphorylations by sarcolemma membranes from skeletal muscle

In a previous report we have shown that insulin increases the phosphorylation of an endogenous protein of mol. wt. 16 000 daltons in sarcolemma membranes. In the present work we have demonstrated that phosphorylations of exogenous histones by the sarcolemma membranes are also increased by insulin. These results indicate that insulin activates a cyclic-AMP-independent protein kinase in sarcolemma membranes. The stimulatory effect of insulin on protein phosphorylations is increased by GTP and its analogue GMP-P(NH)P. The insulin effect was increased 3--4-fold by micromolar concentrations of GTP. The effect by the analogue GMP-P(NH)P was somewhat less. In the absence of insulin guanosine nucleotides had no effect on phosphorylation of the proteins. The results suggest that GTP is a modulator in the activation of a sarcolemma membrane protein kinase by insulin.

ADP-ribosylation of sarcolemma membrane proteins in the presence of cholera toxin and its influence on insulin-stimulated membrane protein kinase activity

We have reported that insulin stimulates cyclic AMP-independent protein kinase activity in sarcolemma membranes El]. This insulin effect was enhanced by PM levels of GTP [2]. The suggestion was made that a GTP-binding protein was involved in the hormonal control of this protein kinase. To gain support for this assumption we have investigated protein kinase activity after preincubation of sarcolemma in the presence of cholera toxin and NADf. The A1 fragment of cholera toxin is known to catalyze protein ADP-ribosylation from NAD+ [3] and the wellknown stimulation of adenylated cyclase by cholera toxin has been attributed to ADP-ribosylation of a membrane protein of MI 42 000 [4,5]. It is indicated that this membrane protein is identical with the GTPbinding protein involved in control of adenylate cyclase activity [6]. Here we demonstrate ADPribosylation of a sarcolemma membrane protein of MI 56 000. This results in inhibition of cyclic AMPindependent protein kinase activity and abolishes the stimulatory effect by insulin on this enzyme.

Insulin and phorbol ester stimulate phosphorylation of a 15 000 dalton membrane protein in rat diaphragm in a similar manner

The effects of insulin on the phosphorylation of a 15 kilodalton (kDa) membrane protein in rat diaphragm in situ have been investigated. Incubation of the diaphragm with insulin or tumor-promoting phorbol ester increased the 32P-labelling of the 15 kDa protein at serine residues by 50 +/- 8% and 64 +/- 11%, (mean +/- S.E.), respectively. Thermolytic peptide mapping of the 15 kDa protein after insulin treatment of the diaphragm yielded two major phosphopeptides, one of which was absent from digests from control diaphragms. The same two phosphopeptides were identified after incubation of the diaphragm with phorbol ester and after phosphorylation of sarcolemma in vitro with [gamma-32P]ATP and protein kinase C. Additional experiments indicated that pretreatment of diaphragms with insulin or phorbol ester both increased the state of phosphorylation of the 15 kDa sarcolemma protein on phosphorylation sites regulated by protein kinase C. The stimulatory effect of insulin was decreased by staurosporine or by preincubation of the diaphragms with phorbol esters. These results indicate that the insulin-induced increases in protein kinase C activity previously found in rat diaphragm (Walaas et al. (1987) FEBS Lett. 220, 311-318) may be involved in insulin-mediated regulation of phosphorylation of the 15 kDa protein in situ.