Insulin stimulates the translocation of protein kinase C in rat adipocytes

Insulin-induced changes in protein kinase C were examined in cytosol and membrane fractions of rat adipocytes enzymatically after Mono Q column chromatography and by immunoblotting. During a 5-20 min period of insulin treatment, cytosolic protein kinase C decrease by approximately 50%, whereas membrane protein kinase C increased nearly 2-fold. These findings suggest that insulin stimulates the translocation of protein kinase C in rat adipocytes.     

Insulin stimulates nitric oxide production in rat adipocytes

In adipocytes, insulin regulates the activity of different protein kinases (PI3K/Akt, MAPK, PKC) and protein phosphatases (PP-1, PP-2A). Since these enzymes are implicated in the regulation of NOS activity which is present in adipose tissue, we tested the effects of insulin on white adipocyte NOS activity. Exposure of adipocytes to insulin resulted simultaneously in NOS activity stimulation and Akt activation with maximal effect observed at 1 nM. Higher concentrations of insulin induced a progressive decline of NOS activity. In the presence of wortmannin, a PI3K inhibitor, 1 nM insulin failed to stimulate NOS activity. Insulin (1 nM)-stimulated NOS activity was also abolished by U0126, an inhibitor of p42/p44 MAPK activation, and by 1 microM okadaic acid (OA), which inhibits both PP-1 and PP-2A but not by 1 nM OA which inhibits only PP-2A. Moreover, inhibition of cPKC allowed a high (1 microM) insulin concentration to stimulate NOS activity. These results (i) demonstrate that insulin activates NO production in adipocytes through both PI3K/Akt and MAPK/PP-1 activation and (ii) suggest that PP-1 activation protects NOS against the inhibitory effect of cPKC activation.     

Insulin stimulates turnover of phosphatidylcholine in rat adipocytes

The present study investigated the effect of insulin on phosphatidylcholine turnover in rat adipocytes labelled to equilibrium with [¹⁴C]-choline. Insulin induced a rapid turnover of this major phospholipid that was maximal by 1 min and transient in nature. Following a 1 min stimulation of the cells with insulin at a maximally effective concentration (7 nM), a 4–6% decrease in the percentage of total cellular choline associated with this phospholipid was observed. This reflected a significant transient increase in the percentage of total cellular choline associated with phosphorylcholine, which together with diacylglycerol are the phospholipase C cleavage products of phosphatidylcholine. These effects were observed over a physiological range of insulin concentrations. No effect of insulin on any other choline phospholipid or metabolite (sphingomyelin, lysophophatidylcholine, glycerophosphocholine or choline) was seen. These results suggest that insulin stimulates a phospholipase C-mediated turnover of phosphatidylcholine in rat adipocytes. The rapid nature of this turnover suggests a potential role in signal transduction.     

Sphingosylphosphorylcholine stimulates mitogen-activated protein kinase via a Ca2+-dependent pathway

In cultured porcine aortic smooth muscle cells,sphingosylphosphorylcholine (SPC), ATP, or bradykinin (BK) induced arapid dose-dependent increase in the cytosolicCa²⁺ concentration([Ca²⁺]_i)and also stimulated inositol 1,4,5-trisphosphate(IP₃) generation. Pretreatmentof cells with pertussis toxin blocked the SPC-induced IP₃ generation and[Ca²⁺]_iincrease but had no effect on the action of ATP or BK. In addition, SPCstimulated the mitogen-activated protein kinase (MAPK) and increasedDNA synthesis, whereas neither ATP nor BK produced such effects. Boththe SPC-induced MAPK activation and DNA synthesis were pertussis toxinsensitive. SPC-induced MAPK activation was blocked by treatment ofcells with the phospholipase C inhibitor, U-73122, or the intracellularCa²⁺-ATPase inhibitor,thapsigargin, but not by removal of extracellular Ca²⁺. Lysophosphatidic acidinduced cellular responses similar to SPC in a pertussistoxin-sensitive manner in terms of[Ca²⁺]_iincrease, IP₃ generation, MAPKactivation, and DNA synthesis. Platelet-derived growth factor (PDGF)also induced a[Ca²⁺]_iincrease, MAPK activation, and DNA synthesis in the same cells; however, the PDGF-induced MAPK activation was not sensitive to pertussis toxin and changes in[Ca²⁺]_i.SPC-induced MAPK activation was inhibited by pretreatment of cells withstaurosporine, W-7, or calmidazolium. Our results suggest that, inporcine aortic smooth muscle cells, MAPK is not activated by theincrease in[Ca²⁺]_iunless a pertussis toxin-sensitive G protein is simultaneously stimulated, indicating the role ofCa²⁺ in pertussis toxin-sensitiveG protein-mediated MAPK activation.

     

Insulin stimulates phosphatidylinositol-3-kinase activity in rat adipocytes.

Phosphatidylinositol (PtdIns) 3-kinase is thought to participate in the signal transduction pathways initiated by the activation of receptor tyrosine kinases including the insulin receptor. To approach the physiological relevance of this enzyme in insulin signaling, we studied the activation of PtdIns-3-kinase in adipocytes, a major insulin target tissue for glucose transport and utilisation. To analyze possible interactions of the enzyme with cellular proteins, immunoprecipitations with the following antibodies were performed: (a) anti-phosphotyrosine antibodies, (b) two antibodies to the 85-kDa subunit of PtdIns-3-kinase (p85) and (c) an antibody to the 185-kDa major insulin receptor substrate (p185). We show that in cell extracts from adipocytes exposed to insulin, and after immunoprecipitation with an anti-phosphotyrosine antibody and an antibody to p85, we are able to detect a PtdIns-3-kinase activity stimulated by the hormone. Similarly, after immunoprecipitation with an antibody to p185, an increase in the PtdIns-3-kinase activity could be demonstrated. Taken together these results suggest that, upon insulin stimulation of fat cells, PtdIns-3-kinase itself is tyrosine phosphorylated and/or associated with an insulin receptor substrate, such as p185, which could function as a link between the insulin receptor and PtdIns-3-kinase. The PtdIns-3-kinase was activated within 1 min of exposure to insulin, and the half-maximal effect was reached at the same concentration, i.e. 3 nM, as for stimulation of the insulin receptor kinase. Subcellular fractionation showed that PtdIns-3-kinase activity was found both in the membranes and in the cytosol. Further, immunoprecipitation with an antibody to p85, which possesses the capacity to activate PtdIns-3-kinase, suggests that the presence of the enzyme in the membrane may be due to an insulin-induced recruitment of the PtdIns-3-kinase from the cytosol to the membrane. Finally, we used isoproterenol, which exerts antagonistic effects on insulin action. This drug was found to inhibit both the PtdIns-3-kinase and the insulin receptor activation by insulin, suggesting that the activation of the PtdIns-3-kinase was closely regulated by the insulin receptor tyrosine kinase. The occurrence of an insulin-stimulated PtdIns-3-kinase in adipocytes leads us to propose that this enzyme might be implicated in the generation of metabolic responses induced by insulin.     

Mn2+/Mg2+-dependent pyruvate kinase from a d-lactic acid-producing bacterium Sporolactobacillus inulinus: characterization of a novel Mn2+-mediated allosterically regulated enzyme

Sporolactobacillus inulinus has attracted scientific and commercial interest due to its high efficiency in d-lactic acid production. Pyruvate kinase (PYK) is one of the key regulatory points in glycolysis, and well-activated PYK can improve d-lactic acid production. A novel Mn²⁺/Mg²⁺-dependent PYK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. Kinetic characterization demonstrated that the S. inulinus PYK had drastically higher activity and affinity toward substrates in the presence of Mn²⁺ compared to those of the common PYK cofactor Mg²⁺, and the circular dichroism spectra of the S. inulinus PYK suggested a Mn²⁺-mediated allosteric activation. The S. inulinus PYK was also allosterically regulated by ribose-5-phosphate or AMP activation and inorganic phosphate or ATP inhibition. The inhibition could be marked reduced or fully eliminated in the presence of activators. The result of fermentations by S. inulinus Y2-8 showed that the extracellular-added MnSO₄ and KH₂PO₄ significantly affected glycolysis flux and d-lactic acid production, which is consistent with the allosteric regulation of Mn²⁺ and inorganic phosphate on PYK. The sophisticated regulatory role of PYK would establish the foundation of substantial disturbance or restructuring of cellular metabolism for improving the S. inulinus d-lactic acid production.     

Insulin receptor kinase following internalization in isolated rat adipocytes

We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization.     

Insulin stimulates the activity of a protamine kinase in isolated rat hepatocytes

Treatment of isolated rat hepatocytes with 10-100 nM insulin for 5-10 min increased by about 2-fold the activity of a protamine kinase which exhibited properties similar to those of a protamine kinase from bovine kidney (Damuni, Z., Amick, G. D., and Sneed, T. R. (1989) J. Biol. Chem. 264, 6412-6416). Half-maximal increase in protamine kinase activity occurred at about 1 nM insulin. This effect of insulin was detected only when 25 mM NaF or 50 mM KPO4 were included in the homogenization buffers and was not prevented by preincubation of the hepatocytes with 10 microM cycloheximide. Insulin stimulation of protamine kinase was maintained following chromatography of extracts on protamine-agarose, DEAE-cellulose, and Sephacryl S-200 gel filtration. The apparent Mr of the protamine kinase from control and insulin-treated hepatocytes was 45,000 as estimated by gel permeation chromatography. Experiments utilizing partially purified protamine kinase from control and insulin-treated hepatocytes indicated that insulin did not affect the apparent Km for protamine, Mg2+, or ATP, but increased the Vmax for the protamine kinase reaction by 1.6-2-fold. Incubation with the catalytic subunit of protein phosphatase 2A completely inactivated the protamine kinase from control and insulin-treated cells. The results indicate that the insulin-stimulated increase in protamine kinase activity may be due to a covalent modification, possibly phosphorylation, of the protamine kinase.     

Insulin receptor serine kinase activation by casein kinase 2 and a membrane tyrosine kinase

The insulin receptor (IR) tyrosine kinase can apparently directly phosphorylate and activate one or more serine kinases. The identities of such serine kinases and their modes of activation are still unclear. We have described a serine kinase (here designated insulin receptor serine (IRS) kinase) from rat liver membranes that co-purifies with IR on wheat germ agglutinin-agarose. The kinase was activated after phosphorylation of the membrane glycoproteins by casein kinase-1, casein kinase-2, or casein kinase-3 (Biochem Biophys Res Commun 171:75–83, 1990). In this study, IRS kinase was further characterized. The presence of vanadate or phosphotyrosine in reaction mixtures was required for activation to be observed. Phosphoserine and phosphothreonine are only about 25% as effective as phosphotyrosine, whereas sodium fluoride and molybdate were ineffective in supporting activation. Vanadate and phosphotyrosine support IRS kinase activation by apparently inhibiting phosphotyrosine protein phosphatases present among the membrane glycoproteins. IR -subunit, myelin basic protein, and microtubule-associated protein-2 are good substrates for IRS kinase. The kinase prefers Mn²⁺ (K_a=1.3 mM) as a metal cofactor. Mg²⁺ (K_a=3.3 mM) is only 30% as effective as Mn²⁺. The kinase activity is stimulated by basic polypeptides, with greater than 30-fold activation achieved with polylysine and protamine. Our results suggest that both serine/threonine and tyrosine phosphorylation are required for activation of IRS kinase. Serine phosphorylation is catalyzed by one of the casein kinases, whereas tyrosine phosphorylation is catalyzed by a membrane tyrosine kinase, possibly IR tyrosine kinase. (Mol Cell Biochem121: 167–174, 1993)     

Purification and characterization of a cytosolic insulin-stimulated serine kinase from rat liver.

A cytosolic insulin-sensitive serine kinase has been purified to apparent homogeneity in parallel from livers of control or acutely insulin-treated rats. The kinase is labile and requires rapid purification for stability. The kinase migrates as a band of apparent Mr = 90,000 on denaturing gels and elutes as a monomer on Superose 12 gel filtration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation, the 90-kDa band presumed to be the kinase shows kinase activity toward myelin basic protein in situ. Substrates of the kinase include Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), ribosomal protein S6, S6 peptide, a proline-rich peptide substrate, microtubule-associated protein 2, and myelin basic protein. The kinase also phosphorylates histones H1 and H2B, but does not autophosphorylate to a significant stoichiometry. The activity of the kinase is inhibited by fluoride, glycerophosphate, p-nitrophenyl phosphate, p-nitrophenol, heparin, quercetin, poly-L-lysine, and potassium phosphate, but is unaffected by calcium, cAMP, spermine, protein kinase inhibitor peptide, phorbol myristate acetate, calcium plus phosphatidylserine, or vanadate. The kinase will utilize magnesium (10 mM) as well as manganese (1 mM) as a cofactor for maximal phosphotransferase activity. The kinase is not detected by immunoblotting with antibodies directed against protein kinase C or type II S6 kinase. Taken together, these properties distinguish this kinase from other insulin-sensitive kinases that have been described previously. The purified kinase from livers of insulin-treated rats shows a 5-20-fold higher specific activity compared to enzyme prepared from control rats, suggesting a covalent modification as the mechanism of activation. Incubation of purified, insulin-stimulated kinase with purified phosphatase 2A leads to deactivation of the kinase activity, and the phosphatase inhibitor nitrophenyl phosphate blocks this deactivation. The insulin-activated kinase fails to immunoblot with anti-tyrosine phosphate antibodies. Taken together, these results indicate that insulin activates this novel cytosolic protein kinase by a mechanism that causes its phosphorylation on serine or threonine residues.     

Insulin and glucose modulate protein kinase C activity in rat adipocytes

In the presence of 1 mM glucose, insulin (10 ng/ml) increases both catalytic and receptor-binding properties of adipocyte cytosolic protein kinase C (PKC). Preincubation of adipocytes with 10 mM glucose raises basal PKC catalytic activity and prevents further stimulation of this enzyme by insulin. The effect of hyperglycemia is likely to be mediated by direct conversion of glucose into diacylglycerol. Thus, an incorporation of 14C-glucose into diacylglycerol is enhanced 10-fold in the presence of 10 mM glucose. These observations indicate that, in normal adipocytes, both insulin and glucose activate PKC; hyperglycemia eliminates the ability of insulin to stimulate this enzyme, thereby interfering with insulin action.     

Insulin receptor kinase activity in rat adipocytes is decreased during aging

The tyrosine kinase activity of the insulin receptor derived from rat adipocyte plasma membranes was examined during aging. In the absence of insulin, autophosphorylation and histone H2B phosphorylation activities, measured with equal numbers of insulin receptors, were comparable among 3- and 24-month-old rats. In contrast, insulin-stimulated kinase activity was significantly reduced in the old animals. We have also found that the insulin dependent phosphorylation of a putative endogenous substrate of 60 kDa was drastically reduced in old animals. These results suggest that the decrease in kinase activity in old rats could be related with the insulin resistance of aging.     

A novel Ca2+-dependent protein kinase from Paramecium tetraurelia

The ciliated protozoan Paramecium tetraurelia contained two protein kinase activities that were dependent on Ca2+. We purified one of the enzymes to homogeneity by Ca2+-dependent affinity chromatography on phenyl-Sepharose and ion exchange chromatography. The purified enzyme contained polypeptides of 50 and 55 kDa, with the 50-kDa species predominant. From its Stokes radius (32 A) and sedimentation coefficient (3.9 S), we calculated a native molecular weight of 51,000, suggesting that the active form is a monomer. Its specific activity was 65-130 nmol X min-1 X mg-1 and the Km for ATP was 17-35 microM, depending on the exogenous substrate used. Kinase activity was completely dependent upon Ca2+; half-maximal activation occurred at approximately 1 microM free Ca2+ at pH 7.2. Phosphatidylserine and diacylglycerol did not stimulate activity, nor did the addition of purified Paramecium calmodulin. The enzyme phosphorylated casein and histones, forming primarily phosphoserine and phosphothreonine, respectively. It also catalyzed its own phosphorylation in a Ca2+-dependent reaction; the half-maximal rate of autophosphorylation occurred at approximately 1-1.5 microM free Ca2+, and both the 50- and 55-kDa species were autophosphorylated. After separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation in situ, the 50-kDa protein retained its Ca2+-dependent ability to phosphorylate casein, suggesting that Ca2+ interacts directly with this polypeptide. This was confirmed by direct binding studies; when the enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis transferred to nitrocellulose, and renatured, there was 45Ca2+-binding in situ to both the 50- and 55-kDa polypeptides. The Paramecium enzyme appears to be a new and unique type of Ca2+-dependent protein kinase.     

Identification and biochemical characterization of a unique Mn2+-dependent UMP kinase from Helicobacter pylori

Uridine monophosphate (UMP) kinase converts UMP to the corresponding UDP in the presence of metal ions and ATP and is allosterically regulated by nucleotides such as UTP and GTP. Although the UMP kinase reported to date is Mg²⁺-dependent, we found in this study that the UMP kinase of Helicobacter pylori had a preference for Mn²⁺ over Mg²⁺, which may be related to a conformational difference between the Mn²⁺-bound and Mg²⁺-bound UMP kinase. Similar to previous findings, the UMP kinase activity of H. pylori UMP kinase was inhibited by UTP and activated by GTP. However, a relatively low GTP concentration (0.125 mM) was required to activate H. pylori UMP kinase to a level similar to other bacterial UMP kinases using a higher GTP concentration (0.5 mM). In addition, depending on the presence of either Mg²⁺ or Mn²⁺, a significant difference in the level of GTP activation was observed. It is therefore hypothesized that the Mg²⁺-bound and Mn²⁺-bound H. pylori UMP kinase may be activated by GTP through different mechanisms.     

Insulin stimulates the phosphorylation of calmodulin in intact adipocytes

Phosphorylation of cellular proteins is known to play an important role in mediating the metabolic effects of insulin in target cells. Here we show that exposure of intact adipocytes to physiological concentrations of insulin results in phosphorylation of the calcium receptor protein, calmodulin. The identity of the phosphorylated protein as being calmodulin in intact cells was demonstrated by two-dimensional electrophoresis, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7)-affinity chromatography, and positive staining with the Ca2+ binding protein stain Stains All. Phosphorylation of calmodulin occurred at physiological insulin concentrations with maximum stimulation (608 +/- 114% over basal) at 50 microunits/ml (3.3 X 10(-10) M) insulin. The 32Pi incorporated into calmodulin was stable to base, indicating that phosphotyrosine was involved and thus implicating the insulin-receptor tyrosine kinase as being responsible for its phosphorylation. The phosphorylation of calmodulin may represent an important component of the mechanism for intracellular signaling not only for insulin, but potentially for other physiological regulators of cellular metabolism.     

Mn2+-dependent endonuclease activity of chromatin

The endogenous endonuclease activity of chromatin in isolated rat liver nuclei in the presence of Mn2+, Mg2+ and Ca2+ + Mg2+ was studied. The existence of a Mn2+-dependent endonuclease activity not coupled with the Ca2+, Mg2+-dependent endonuclease was demonstrated, which was weaker than the former one in isolated cell nuclei but higher than in the preparation of Ca2+, Mg2+-dependent nuclease obtained by gel filtration through Toyopearl HW 60F. The Mn2+-dependent splitting of chromatin predominantly occurs at linker DNA of distal parts of chromatin loops. A split-off of purified DNA was more universal than in the presence of Ca2+, Mg2+-dependent endonuclease; the hydrolysis rate of native and denaturated DNA appeared to be the same.     

Ca2+-dependent protein kinase C isoforms induce cholestasis in rat liver

Bile secretion is regulated by different signaling transduction pathways including protein kinase C (PKC). However, the role of different PKC isoforms for bile formation is still controversial. This study investigates the effects of PKC isoform selective activators and inhibitors on PKC translocation, bile secretion, bile acid uptake, and subcellular transporter localization in rat liver, isolated rat hepatocytes and in HepG2 cells. In rat liver activation of Ca(2+)-dependent cPKCalpha and Ca(2+)-independent PKCepsilon by phorbol 12-myristate 13-acetate (PMA, 10nmol/liter) is associated with their translocation to the plasma membrane. PMA also induced translocation of the cloned rat PKCepsilon fused to a yellow fluorescent protein (YFP), which was transfected into HepG2 cells. In the perfused liver, PMA induced marked cholestasis. The PKC inhibitors G?6850 (1 micromol/liter) and G?6976 (0.2 micromol/liter), a selective inhibitor of Ca(2+)-dependent PKC isoforms, diminished the PMA effect by 50 and 60%, respectively. Thymeleatoxin (Ttx,) a selective activator of Ca(2+)-dependent cPKCs, did not translocate rat PKCepsilon-YFP transfected in HepG2 cells. However, Ttx (0.5-10 nmol/liter) induced cholestasis similar to PMA and led to a retrieval of Bsep from the canalicular membrane in rat liver while taurocholate-uptake in isolated hepatocytes was not affected. G?6976 completely blocked the cholestatic effect of Ttx but had no effect on tauroursodeoxycholate-induced choleresis. The data identify Ca(2+)-dependent PKC isoforms as inducers of cholestasis. This is mainly due to inhibition of taurocholate excretion involving transporter retrieval from the canalicular membrane.     

Sennidin stimulates glucose incorporation in rat adipocytes

A novel small molecule compound which exerts insulin mimetic is desirable. Dozens of natural products that have quinone, naphthoquinone, or anthraquinone structure, were tested by a glucose incorporation assay. We found that sennidin A, anthraquinone derivative, stimulated glucose incorporation to near level of maximal insulin-stimulated and sennidin B, a stereoisomer of sennidin A, also stimulated, but the activity of sennidin B was lower than sennidin A. Sennidin A-stimulated glucose incorporation was completely inhibited by wortmannin. Sennidin A did not induce tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1), but induced phosphorylation of Akt and glucose transporter 4 (GLUT4) translocation. Our results suggest that in rat adipocytes, sennidin A stimulates glucose incorporation in the phosphatidylinositol 3-kinase (PI3K)- and Akt-dependent, but in the IR/IRS1-independent manner.     

Ca2+-dependent binding of cytosolic components to insulin-secretory granules results in Ca2+-dependent protein phosphorylation

Interaction of Cu(II) and Gly-His-Lys, a growth-modulating tripeptide from plasma, was investigated by 13C- and 1H-n.m.r. and e.p.r. spectroscopy. The n.m.r. line-broadening was interpreted in terms of major and minor species formed as a function of pH. The results indicate that the n.m.r. line-broadening is due to the presence of minor species in rapid exchange and not due to the major species in solution, which has a large tau M. It is concluded that the technique of 13C- and 1H-n.m.r. line broadening, caused by paramagnetic Cu(II) ion, should be undertaken with caution, since the method may not be useful for obtaining structural information on the major species. The e.p.r. spectra over a wide pH range are almost entirely due to similarly co-ordinating species. Starting at pH 5.5, the narrowest absorption near 340 mT shows superhyperfine structure, which comes out sharply in the pH region 6.0-9.6. The spectra in this pH range showed the seven lines of nitrogen superhyperfine splitting, indicating clearly the co-ordination of three nitrogen atoms to Cu(II). The e.p.r. parameters in the medium pH range, A parallel = 19.5 mT and g parallel = 2.21, fit well with the contention that Cu(II) is ligated to Gly-His-Lys through one oxygen atom and three nitrogen atoms in a square-planar configuration.