Regulation of mitochondrial aconitase by phosphorylation in diabetic rat heart.

Mitochondrial dysfunction and protein kinase C (PKC) activation are consistently found in diabetic cardiomyopathy but their relationship remains unclear. This study identified mitochondrial aconitase as a downstream target of PKC activation using immunoblotting and mass spectrometry, and then characterized phosphorylation-induced changes in its activity in hearts from type 1 diabetic rats. PKCbeta(2) co-immunoprecipitated with phosphorylated aconitase from mitochondria isolated from diabetic hearts. Augmented phosphorylation of mitochondrial aconitase in diabetic hearts was found to be associated with an increase in its reverse activity (isocitrate to aconitate), while the rate of the forward activity was unchanged. Similar results were obtained on phosphorylation of mitochondrial aconitase by PKCbeta(2) in vitro. These results demonstrate the regulation of mitochondrial aconitase activity by PKC-dependent phosphorylation. This may influence the activity of the tricarboxylic acid cycle, and contribute to impaired mitochondrial function and energy metabolism in diabetic hearts.

Exercise restores endothelial function independently of weight loss or hyperglycaemic status in db/db mice.

AIMS/HYPOTHESIS: Exercise ameliorates oxidative stress-mediated diabetic vascular endothelial dysfunction through poorly defined mechanisms. We hypothesised that, in addition to improving metabolic parameters, upregulation of antioxidants such as superoxide dismutase (SOD) mediates exercise-induced reductions of oxidative stress and increased nitric oxide (NO) bioavailability, and also restores vasodilatation. METHODS: Type 2 diabetic db/db and normoglycaemic wild-type mice were exercised at moderate intensity for 1 h a day for 7 weeks, leading to a 10% body weight loss. Sedentary animals or those undergoing a low-intensity exercise regimen causing non-significant weight loss were also used. We examined aortic endothelial cell function, NO bioavailability and various biomarkers of oxidative stress. RESULTS: Moderate-intensity exercise lowered body weight, increased mitochondrial manganese SOD (MnSOD) and both total and phosphorylated (Ser1177) endothelial nitric oxide synthase (eNOS) protein production; it also reduced whole-body (plasma 8-isoprostane) and tissue oxidative stress (nitrotyrosine immunostaining or protein carbonyl levels in the aorta). Low-intensity exercise did not alter body weight; however, it upregulated cytosolic Cu/Zn-SOD instead of MnSOD, and still demonstrated all the above benefits in the db/db aorta. Importantly, both exercise protocols improved endothelial-dependent vasodilatation and NO bioavailability without altering hyperglycaemic status in db/db mice. CONCLUSIONS/INTERPRETATION: Exercise reverses diabetic vascular endothelial dysfunction independently of improvements in body weight or hyperglycaemia. Our data suggest that upregulation of eNOS and specific SOD isoforms could play important roles in improving NO bioavailability, as well as in reversing endothelial dysfunction in type 2 diabetes patients through lifestyle modifications in the management of diabetes.

Regulation of acetyl-CoA carboxylase.

Acetyl-CoA carboxylase (ACC) catalyses the formation of malonyl-CoA, an essential substrate for fatty acid synthesis in lipogenic tissues and a key regulatory molecule in muscle, brain and other tissues. ACC contributes importantly to the overall control of energy metabolism and has provided an important model to explore mechanisms of enzyme control and hormone action. Mammalian ACCs are multifunctional dimeric proteins (530-560 kDa) with the potential to further polymerize and engage in multiprotein complexes. The enzymatic properties of ACC are complex, especially considering the two active sites, essential catalytic biotin, the three-substrate reaction and effects of allosteric ligands. The expression of the two major isoforms and splice variants of mammalian ACC is tissue-specific and responsive to hormones and nutritional status. Key regulatory elements and cognate transcription factors are still being defined. ACC specific activity is also rapidly modulated, being increased in response to insulin and decreased following exposure of cells to catabolic hormones or environmental stress. The acute control of ACC activity is the product of integrated changes in substrate supply, allosteric ligands, the phosphorylation of multiple serine residues and interactions with other proteins. This review traces the path and implications of studies initiated with Dick Denton in Bristol in the late 1970s, through to current proteomic and other approaches that have been consistently challenging and immensely rewarding.

Glycolysis and pyruvate oxidation in cardiac hypertrophy--why so unbalanced?

Cardiac hypertrophy, induced by chronic pressure or volume overload, is associated with abnormalities in energy metabolism as well as characteristic increases in muscle mass and alterations in the structure of the heart. Hypertrophied hearts display increased rates of glycolysis and overall glucose utilization, but rates of pyruvate oxidation do not rise in step with rates of pyruvate generation. Glycolysis and glucose oxidation, therefore, become markedly less 'coupled' in hypertrophied hearts than in non-hypertrophied hearts. Because the pyruvate dehydrogenase complex (PDC) contributes so powerfully to the control of glucose oxidation, we set out to test the hypothesis that the function of PDC is impaired in cardiac hypertrophy. In this review we describe evidence indicating that the alterations in glucose metabolism in hypertrophied hearts cannot be explained simply by changes in PDC expression or control. Additional mechanisms that may lead to an altered balance of pyruvate metabolism in cardiac hypertrophy are discussed, with commentaries on possible changes in pyruvate transport, NADH shuttles, lactate dehydrogenase, and amino acid metabolism.

Long-term treatment with the dipeptidyl peptidase IV inhibitor P32/98 causes sustained improvements in glucose tolerance, insulin sensitivity, hyperinsulinemia, and beta-cell glucose responsiveness in VDF (fa/fa) Zucker rats.

The incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are responsible for >50% of nutrient-stimulated insulin secretion. After being released into the circulation, GIP and GLP-1 are rapidly inactivated by the circulating enzyme dipeptidyl peptidase IV (DP IV). The use of DP IV inhibitors to enhance these insulinotropic hormonal axes has proven effective on an acute scale in both animals and humans; however, the long-term effects of these compounds have yet to be determined. Therefore, we carried out the following study: two groups of fa/fa Zucker rats (n = 6 each) were treated twice daily for 3 months with the DP IV inhibitor P32/98 (20 mg.kg(-1).day(-1), p.o.). Monthly oral glucose tolerance tests (OGTTs), performed after drug washout, revealed a progressive and sustained improvement in glucose tolerance in the treated animals. After 12 weeks of treatment, peak OGTT blood glucose values in the treated animals averaged 8.5 mmol/l less than in the controls (12.0 +/- 0.7 vs. 20.5 +/- 1.3 mmol/l, respectively). Concomitant insulin determinations showed an increased early-phase insulin response in the treated group (43% increase). Furthermore, in response to an 8.8 mmol/l glucose perfusion, pancreata from controls showed no increase in insulin secretion, whereas pancreata from treated animals exhibited a 3.2-fold rise in insulin secretion, indicating enhanced beta-cell glucose responsiveness. Also, both basal and insulin-stimulated glucose uptake were increased in soleus muscle strips from the treated group (by 20 and 50%, respectively), providing direct evidence for an improvement in peripheral insulin sensitivity. In summary, long-term DP IV inhibitor treatment was shown to cause sustained improvements in glucose tolerance, insulinemia, beta-cell glucose responsiveness, and peripheral insulin sensitivity, novel effects that provide further support for the use of DP IV inhibitors in the treatment of diabetes.

Lack of in vivo effect of vanadium on GLUT4 translocation in white adipose tissue of streptozotocin-diabetic rats.

Vanadium treatment, in vivo, corrects the severe hyperglycemia observed in streptozotocin (STZ)-diabetic rats. A number of metabolic effects of vanadium have been demonstrated in vitro and might contribute importantly to normalization of glucose homeostasis. However, many in vitro effects of vanadium occur at concentrations substantially higher than those achieved in vivo. Effects of vanadium on white adipose tissue have been particularly well characterized in vitro. To examine the relationship between in vitro and in vivo actions of vanadium, we examined the effects of vanadium treatment on acute glucose tolerance and adipose tissue GLUT4 control in vivo. In agreement with previous studies, vanadium treatment of STZ-diabetic rats restored normoglycemia with no appreciable restoration of insulin secretion. GLUT4 expression in white adipose tissue was reduced by 22% in STZ-diabetic rats compared with controls. Vanadium treatment did not significantly alter GLUT4 expression in controls, but completely restored normal expression levels in STZ-diabetic rats. In overnight-fasted control animals, GLUT4 translocation to the plasma membrane (PM) was maximally elevated (by 50%) in adipose tissue within 5 to 10 minutes after an intravenous (IV) glucose challenge. No glucose-induced translocation of GLUT4 was detected in diabetic rats, and peak PM GLUT4 content was 40% lower than in controls. Vanadium treatment did not increase peak PM GLUT4 content in either control or diabetic animals in response to a glucose load. Finally, the suppression of whole-body acute glucose tolerance in diabetic animals was only partially normalized by vanadium treatment. We conclude: (1) that concentrations of vanadium effective for maintaining normoglycemia in vivo (typically below 30 micromol/L) promote normal GLUT4 expression, but do not influence the subcellular localization of GLUT4 in white adipose tissue and (2) that in vivo effects of vanadium may not necessarily reflect the actions observed in vitro at supraphysiologic concentrations.

Mechanisms of vanadium action: insulin-mimetic or insulin-enhancing agent?

The demonstration that the trace element vanadium has insulin-like properties in isolated cells and tissues and in vivo has generated considerable enthusiasm for its potential therapeutic value in human diabetes. However, the mechanisms by which vanadium induces its metabolic effects in vivo remain poorly understood, and whether vanadium directly mimics or rather enhances insulin effects is considered in this review. It is clear that vanadium treatment results in the correction of several diabetes-related abnormalities in carbohydrate and lipid metabolism, and in gene expression. However, many of these in vivo insulin-like effects can be ascribed to the reversal of defects that are secondary to hyperglycemia. The observations that the glucose-lowering effect of vanadium depends on the presence of endogenous insulin whereas metabolic homeostasis in control animals appears not to be affected, suggest that vanadium does not act completely independently in vivo, but augments tissue sensitivity to low levels of plasma insulin. Another crucial consideration is one of dose-dependency in that insulin-like effects of vanadium in isolated cells are often demonstrated at high concentrations that are not normally achieved by chronic treatment in vivo and may induce toxic side effects. In addition, vanadium appears to be selective for specific actions of insulin in some tissues while failing to influence others. As the intracellular active forms of vanadium are not precisely defined, the site(s) of action of vanadium in metabolic and signal transduction pathways is still unknown. In this review, we therefore examine the evidence for and against the concept that vanadium is truly an insulin-mimetic agent at low concentrations in vivo. In considering the effects of vanadium on carbohydrate and lipid metabolism, we conclude that vanadium acts not globally, but selectively and by enhancing, rather than by mimicking the effects of insulin in vivo.

Dichloroacetate improves postischemic function of hypertrophied rat hearts.

OBJECTIVES: We sought to determine whether improving coupling between glucose oxidation and glycolysis by stimulating glucose oxidation during reperfusion enhances postischemic recovery of hypertrophied hearts. BACKGROUND: Low rates of glucose oxidation and high glycolytic rates are associated with greater postischemic dysfunction of hypertrophied as compared with nonhypertrophied hearts. METHODS: Heart function, glycolysis and glucose oxidation were measured in isolated working control and hypertrophied rat hearts for 30 min before 20 min of global, no-flow ischemia and during 60 min of reperfusion. Selected control and hypertrophied hearts received 1.0 mmol/liter dichloroacetate (DCA), an activator of pyruvate dehydrogenase, at the time of reperfusion to stimulate glucose oxidation. RESULTS: In the absence of DCA, glycolysis was higher and glucose oxidation and recovery of function were lower in hypertrophied hearts than in control hearts during reperfusion. Dichloroacetate stimulated glucose oxidation during reperfusion approximately twofold in both groups, while significantly reducing glycolysis in hypertrophied hearts. It also improved function of both hypertrophied and control hearts. In the presence of DCA, recovery of function of hypertrophied hearts was comparable to or better than that of untreated control hearts. CONCLUSIONS: Dichloroacetate, given at the time of reperfusion, normalizes postischemic function of hypertrophied rat hearts and improves coupling between glucose oxidation and glycolysis by increasing glucose oxidation and decreasing glycolysis. These findings support the hypothesis that low glucose oxidation rates and high glycolytic rates contribute to the exaggerated postischemic dysfunction of hypertrophied hearts.

Bimodal activation of acetyl-CoA carboxylase by glutamate.

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, an essential substrate for fatty acid biosynthesis and a potent inhibitor of fatty acid oxidation. Here, we provide evidence that glutamate may be a physiologically relevant activator of ACC. Glutamate induced the activation of both major isoforms of ACC, prepared from rat liver, heart, or white adipose tissue. In agreement with previous studies, a type 2A protein phosphatase contributed to the effects of glutamate on ACC. However, the protein phosphatase inhibitor microcystin LR did not abolish the effects of glutamate on ACC activity. Moreover, glutamate directly activated purified preparations of ACC when protein phosphatase activity was excluded. Phosphatase-independent ACC activation by glutamate was also reflected by polymerization of the enzyme as judged by size-exclusion chromatography. The sensitivity of ACC to direct activation by glutamate was diminished by treatment in vitro with AMP-activated protein kinase or cAMP-dependent protein kinase or by beta-adrenergic stimulation of intact adipose tissue. We conclude that glutamate, an abundant intracellular amino acid, induces ACC activation through complementary actions as a phosphatase activator and as a direct allosteric ligand for dephosphorylated ACC. This study supports the general hypothesis that amino acids fulfill important roles as signal molecules as well as intermediates in carbon and nitrogen metabolism.

Multiple-site phosphorylation of the 280 kDa isoform of acetyl-CoA carboxylase in rat cardiac myocytes: evidence that cAMP-dependent protein kinase mediates effects of beta-adrenergic stimulation.

Two major forms of mammalian acetyl-CoA carboxylase (EC 6.4.1.2), ACC-alpha and ACC-beta, have been described and the sequences of the isoforms deduced. ACC-beta is the predominant isoform expressed in heart and skeletal muscles, in which a major role of malonyl-CoA is probably to regulate fatty acid beta-oxidation. The regulatory properties of ACC-beta are incompletely defined but it is known that some cellular stresses lead to inhibition in parallel with the activation of AMP-activated protein kinase (AMP-PK). Here we examine the phosphorylation state of ACC-beta within intact rat cardiac ventricular myocytes. Treatment of myocytes with the beta-adrenergic agonist isoprenaline (isoproterenol) led to increased ACC-beta phosphorylation that was maximal within 2 min and with 50 nM agonist. Effects of isoprenaline were revealed by the incorporation of 32P into ACC in cells incubated with [32P]Pi and also by a marked decrease (approx. 80%) in subsequent phosphorylation in vitro with cAMP-dependent protein kinase (PKA). Analysis of tryptic phosphopeptides revealed that ACC-beta was phosphorylated at multiple sites by incubation in vitro with PKA or AMP-PK. Treatment of myocytes with isoprenaline affected all the major phosphorylation sites of ACC-beta that were recognized in vitro by purified PKA, so that subsequent phosphorylation in vitro was greatly diminished after cell stimulation. beta-Adrenergic stimulation led to decreases in cellular malonyl-CoA concentrations but no changes in kinetic properties of ACC were detected after cell homogenization and partial purification of proteins. The results suggest that: (1) ACC-beta is rapidly phosphorylated at multiple sites within intact cardiac ventricular myocytes after beta-adrenergic stimulation, (2) ACC-beta is phosphorylated in vitro by PKA and AMP-PK at multiple sites, including at least one site accessible to each kinase, as well as kinase-selective sites, and (3) PKA is a physiologically significant ACC-beta kinase.

Cell volume and the metabolic actions of insulin.

Insulin increases the volume of isolated hepatocytes and cells in perfused livers, but effects of the hormone on the volume of fat or muscle cells have not been demonstrated. Exogenous amino acids may stimulate swelling of liver cells and induce insulin-like effects on hepatic protein metabolism; however, swelling of liver cells can be induced by some treatment that do not induce insulin-like metabolic responses. Exogenous amino acids also influence protein metabolism of fat and muscle cells, but no relationship with cell volume has been established and no corresponding effects on metabolism of carbohydrates or lipids have been observed. Three families of mitogen-activated protein kinases are activated after changes in extracellular osmolarity but they appear to play little or no role in the metabolic actions of insulin. Direct evidence against a metabolic role for the extracellular signal-regulated kinases ERK-1 and ERK-2 is discussed. The c-Jun N-terminal kinases (also called stress-activated protein kinases) and the mammalian homologs of the yeast Hog protein kinase are strongly activated by environmental stresses associated with catabolic metabolism. We conclude that cell volume and protein metabolism may be correlated in liver but there is no compelling evidence that the effects of insulin on metabolism of liver, fat, or muscle cells can be accounted for by changes in cell volume. The effects of insulin on cell volume may represent a discrete aspect of the complete physiological response rather than an obligatory intermediate step in metabolic signalling.

Evidence for selective effects of vanadium on adipose cell metabolism involving actions on cAMP-dependent protein kinase.

The insulin-like effects of vanadium in vivo are likely to be achieved at micromolar concentrations. Demonstrated effects of vanadium on adipose tissue of streptozotocin-diabetic rats include inhibition of basal and stimulated rates of lipolysis and effects on fat cell protein phosphorylation. The studies described below examined the effects of vanadium (to a maximum concentration of 0.5 mM) on adipose cells or tissue in vitro. Vanadium, added as a vanadyl-albumin complex or as sodium orthovanadate, produced a marked (greater than 50%) inhibition of isoproterenol-stimulated lipolysis. Inhibition of lipolysis equivalent to that seen with insulin, was achieved with approximately 100 microM vanadium. In contrast, no insulin-like stimulation of de novo fatty acid biosynthesis was observed with vanadium below 0.5 mM. Surprisingly, the antilipolytic effects of vanadium persisted in the presence of cilostamide, an inhibitor of the insulin-sensitive isoform of cyclic nucleotide phosphodiesterase. Studies with purified preparations of the catalytic subunit of cyclic AMP-dependent protein kinase revealed dose-dependent inhibition with vanadyl-glutathione (to a maximum of approximately 40% inhibition). Equivalent inhibition of cyclic AMP-dependent phosphorylation of Kemptide (approximately 50%) was observed upon incubation of freshly-prepared fat-pad supernatant fractions with vanadyl-glutathione. These results suggest that effects of low concentrations of vanadium may be mediated, at least in part, by actions on the catalytic subunit of cyclic AMP-dependent protein kinase.

Vanadate induces normolipidemia and a reduction in the levels of hepatic lipogenic enzymes in obese Zucker rat.

The effects of vanadate administration on the plasma lipids and hepatic lipogenic enzymes were investigated in Zucker (fa/fa) rat, a model for obesity and non insulin-dependent diabetes. These animals were administered sodium orthovanadate through drinking water for a period of four months. The plasma levels of insulin, triacylglycerols and total cholesterol were significantly (p < 0.001) elevated in untreated obese control rats as compared to the lean animals. In the livers of obese rats, the number of insulin receptors decreased by 60% and the activities of lipogenic enzymes acetyl-CoA carboxylase and ATP-citrate lyase increased by 4.7- and 5.6-folds, respectively. The messenger RNA for ATP-citrate lyase as measured by Northern blot analysis showed a parallel increase in obese control rats. Treatment of these rats with vanadate caused 56-77% decreases in the plasma levels of insulin, triacylglycerols and total cholesterol. The insulin receptor numbers in vanadate-treated obese rats increased (119%) compared to levels in untreated obese animals. The elevated activities of acetyl-CoA carboxylase and ATP-citrate lyase observed in livers of obese rats were significantly reduced by vanadate. The messenger RNA for ATP-citrate lyase also decreased in vanadate-treated obese rats back to the lean control levels. This study demonstrates that vanadate exerts potent actions on lipid metabolism in diabetic animals in addition to the recognized effects on glucose homeostasis.

CD14-dependent activation of protein kinase C and mitogen-activated protein kinases (p42 and p44) in human monocytes treated with bacterial lipopolysaccharide.

To investigate mechanisms of mononuclear phagocyte cell signaling, the effects of bacterial LPS on protein kinase activities in normal human peripheral blood monocytes were examined. Incubation of intact monocytes with LPS brought about time- and concentration-dependent increases in myelin basic protein (MBP) phosphotransferase activity in high speed supernatants of cell lysates. Anion-exchange chromatography on Mono Q demonstrated that LPS treatment resulted in two principal peaks of stimulated MBP kinase activity. Evidence was obtained to indicate that the first eluted peak of MBP kinase activity is accounted for by p42 and p44 mitogen-activated protein (MAP) kinases. Thus, 1) MBP kinase activity within peak 1 was quantitatively precipitated by anti-MAP kinase Abs, 2) the enzyme effectively phosphorylated a specific peptide substrate, 3) peak 1 contained proteins of subunit size M(r) 42,000 and M(r) 44,000 that reacted specifically with anti-MAP kinase Abs, and that 4) were recognized by anti-phosphotyrosine Abs only after stimulation of cells with LPS. Studies of the second peak of LPS-stimulated MBP kinase activity indicate that it is an isoform of protein kinase C (PKC) because: 1) enzyme activity was quantitatively immunoprecipitated by anti-PKC Abs, 2) the activity of the enzyme was potently and selectively inhibited by a specific peptide modeled on the autoinhibitory domain of PKC, and 3) the presence of a protein of subunit size M(r) 80,000 recognized by anti-PKC Abs. Because the second peak of MBP kinase activity (like the first) was active in the absence of added calcium and in the presence of 2 mM EGTA, it appears to be a type II, calcium-independent isoform of PKC. Abs to CD14 completely abrogated LPS-induced activation of both Mono Q peaks of MBP phosphotransferase activity. These results indicate that LPS coordinately activates both an apparently calcium-independent PKC and MAP kinase in mononuclear phagocytes and these responses appear to be initiated by signaling through the cell surface receptor, CD14.

Gamma interferon induces rapid and coordinate activation of mitogen-activated protein kinase (extracellular signal-regulated kinase) and calcium-independent protein kinase C in human monocytes.

Gamma interferon plays an important role in regulating the functional properties of mononuclear phagocytes. In the present study, the role of activated protein kinases in the mechanism of action of gamma interferon cell signaling in human peripheral blood monocytes was investigated. Analysis in vitro of 100,000 x g cytosolic fractions from untreated and interferon-treated cells showed that agonist treatment resulted in time- and concentration-dependent increases in phosphotransferase activity when myelin basic protein (MBP) was used as the substrate. Anion-exchange chromatography of high-speed supernatants prepared from detergent extracts of interferon-treated cells revealed two discrete peaks of MBP phosphotransferase activity. Immunoblotting of fractions from these peaks with antiphosphotyrosine antibodies and with antibodies that specifically recognize the family of mitogen-activated protein (MAP) kinases detected a MAP kinase with a subunit M(r) of 42,000 in the earliest-eluting peak (peak 1). Phosphorylation of the 42,000-M(r) protein on tyrosine was observed only after treatment of cells with interferon. The contribution of MAP kinase to the interferon-stimulated activity in peak 1 was confirmed by quantitative immunoprecipitation with anti-MAP kinase and antiphosphotyrosine antibodies. The conclusion that the interferon-activated MBP kinase in peak 1 could be accounted for by an activated MAP kinase was also supported by the finding that fractions from Mono Q peak 1 demonstrated activity towards a MAP kinase-specific substrate. The later-eluting peak of interferon-activated MBP phosphotransferase activity appeared to be accounted for by an activated protein kinase C (PKC). This conclusion is based upon analyses of immunoblotting and immunoprecipitation experiments with antibodies to PKC and was also supported by the observed inhibition of this kinase with a PKC pseudosubstrate peptide. The interferon-stimulated PKC present in Mono Q peak 2 was active in the absence of calcium ions, suggesting that it is a calcium-independent isoform of PKC.

Unique structural features and differential phosphorylation of the 280-kDa component (isozyme) of rat liver acetyl-CoA carboxylase.

Rat liver acetyl-CoA carboxylase (ACC, EC 6.4.1.2) exhibits major and minor subunits (M(r) of 265,000 and 280,000 respectively), the structure and function of which are compared in this study. The two subunits copurified and each contained biotin as demonstrated by avidin reactivity and direct determination of biocytin. In agreement with previous studies, the ACC subunits could be distinguished with specific monoclonal antibodies and differential tissue expression. We now report extensive differences in primary structure revealed by peptide mapping, mass spectrometric analysis of peptides following reverse phase high performance liquid chromatography, and microsequencing of selected peptides. Four peptides derived from the 265-kDa subunit were sequenced and matched sequences within the predicted structure of rat 265-kDa ACC. Although one identical peptide sequence was detected within both subunits (residues 2009-2024 of the 265-kDa subunit), 12 peptides derived from the 280-kDa subunit exhibited entirely novel sequences or matched partially (average 70% identity) with sequences within the 265-kDa subunit. The 280-kDa subunit may also exhibit distinct functional properties, since the initial rate of phosphorylation was at least 10-fold greater than that of the 265-kDa subunit in the presence of cAMP-dependent protein kinase. Two-dimensional mapping demonstrated that the tryptic phosphopeptides released from the two ACC subunits are distinct. These structural studies suggest that the 265- and 280-kDa components (isozymes) of ACC are so distinct they may be encoded by separate genes, while the differential phosphorylation observed in vitro suggests a key role for the 280-kDa subunit in regulating enzyme activity within intact cells.

Analytical and micropreparative peptide mapping by high performance liquid chromatography/electrospray mass spectrometry of proteins purified by gel electrophoresis.

We report the use of microbore reverse-phase high performance liquid chromatography connected on-line to an electrospray mass spectrometer for the separation/detection of peptides derived by proteolytic digestion of proteins separated by polyacrylamide gel electrophoresis. A small fraction (typically 10% of the total) of the peptides eluting from the column was diverted through a flow-splitting device into the ion source of the mass spectrometer, whereas the majority of the peptide samples was collected for further analyses. We demonstrate the feasibility of obtaining reproducible peptide maps from submicrogram amounts of protein applied to the gel and good correlation of the signal detected by the mass spectrometer with peptide detection by UV absorbance. Furthermore, independently verifiable peptide masses were determined from subpicomole amounts of peptides directed into the mass spectrometer. The method was used to analyze the 265-kDa and the 280-kDa isoforms of the enzyme acetyl-CoA carboxylase isolated from rat liver. The results provide compelling evidence that the two enzyme isoforms are translation products of different genes and suggest that these approaches may be of general utility in the definitive comparison of protein isoforms. We furthermore illustrate that knowledge of peptide masses as determined by this technique provides a major advantage for error-free data interpretation in chemical high-sensitivity peptide sequence analysis.

Evidence for a protein regulator from rat liver which activates acetyl-CoA carboxylase.

1. A regulator of acetyl-CoA carboxylase has been identified in high-speed supernatant fractions from rat liver. The regulator was found to activate highly purified acetyl-CoA carboxylase 2-3-fold at physiological citrate concentrations (0.1-0.5 mM). The effects of the regulator on acetyl-CoA carboxylase activity were dose-dependent, and half-maximal activation occurred in 7-8 min at 30 degrees C. 2. The acetyl-CoA carboxylase regulator was non-dialysable and was inactivated by heating or by exposure to carboxypeptidase. The regulator was enriched from rat liver cytosol by first removing the endogenous acetyl-CoA carboxylase and then using a combination of purification steps, including (NH4)2SO4 precipitation, ion-exchange chromatography and size-exclusion chromatography. The regulator activity appeared to be a protein with a molecular mass of approx. 75 kDa, which could be eluted from mono-Q with approx. 0.35 M KCl as a single peak of activity. 3. Studies of the effects of the regulator on phosphorylation or subunit size of acetyl-CoA carboxylase indicated that the changes in enzyme activity are most unlikely to be explained by dephosphorylation or by proteolytic cleavage. 4. The regulator co-migrates with acetyl-CoA carboxylase through several purification steps, including ion-exchange chromatography and precipitation with (NH4)2SO4; however, the proteins may be separated by Sepharose-avidin chromatography, and the association between the proteins is also disrupted by addition of avidin in solution. Furthermore, the binding of the regulator itself to DEAE-cellulose is altered by the presence of acetyl-CoA carboxylase. Taken together, these observations suggest that the effects of the regulator on acetyl-CoA carboxylase may be explained by direct protein-protein interaction in vitro.