A highly purified rat liver protein kinase phosphorylates and inactivates acetyl-CoA carboxylase, and causes rapid inactivation of microsomal HMG-CoA reductase in the presence of MgATP. Both effects are stimulated in an identical manner by AMP, and are greatly reduced by prior treatment of the kinase with purified protein phosphatase. The dephosphorylated kinase can be reactivated in the presence of MgATP, apparently due to a distinct kinase kinase, and this reactivation is stimulated by nanomolar concentrations of palmitoyl-CoA. These results show that a common, bicyclic protein kinase cascade can potently inactivate the regulatory enzymes of both fatty acid and cholesterol biosynthesis.

1. We have purified the AMP-activated protein kinase 4800-fold from rat liver. The acetyl-CoA carboxylase kinase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase kinase activities copurify through all six purification steps and are inactivated with similar kinetics by treatment with the reactive ATP analogue fluorosulphonylbenzoyladenosine.2. The final preparation contains several polypeptides detectable by SDS/polyacrylamide gel electrophoresis, but only one of these, with an apparent molecular mass of-63 kDa, is labelled using [L4C]fluorosulphonylbenzoyladenosine. This is also the only polypeptide in the preparation that becomes significantly labelled during incubation with [y3'P]ATP. This autophosphorylation reaction did not affect the AMP-stimulated kinase activity.3. In the absence of AMP the purified kinase has apparent K , values for ATP and acetyl-CoA carboxylase of 86 pM and 1.9 pM respectively. AMP increases the V,,, 3 -5-fold without a significant change in the K, for either protein or ATP substrates.4. The response to AMP depends on thc ATP concentration in the assay, but at a near-physiological ATP concentration the half-maximal effect of AMP occurs at 14 yM. Studics with a range of nucleoside monophosphates and diphosphates, and AMP analogues showed that the allosteric activation by AMP was very specific. ADP gave a small stimulation at low concentrations but was inhibitory at high concentrations.5. These results show that the AMP-activated protein kinase is the major HMG-CoA reductase kinase detectable in rat liver under our assay conditions and that it is therefore likely to be identical to previously described HMG-CoA reductase kinase(s) which are activated by adenine nucleotides and phosphorylation. The AMP-binding and catalytic domains of the kinase are located on a 63-kDa polypeptide which is subject to autophosphorylation.Cholesterol and fatty acids in mammals are derived either from the diet, or from de nuvu synthesis via branching pathways which diverge from a common pool of cytoplasmic acetyl-CoA. There arc scveral situations in which the two biosynthetic pathways are regulated in parallel. They exhibit a synchronous diurnal rhythm in rat liver [l, 21, a finding which may perhaps be explained by diurnal variation in the insuliniglucagon ratio, since insulin stimulates and glucagon inhibits both pathways in isolated rat hepatocytes and in vivo [3-51. The peak diurnal rates of both paihways are also reduced by 60-70% when rats are fed a diet high in polyunsaturated fatty acids [6], a treatment which also depresses plasma cholesierol and triglyccridc levels 171.The important sites of regulation within the pathways of fatty acid and cholesterol synthesis are believed to be those catalyzed by acetyl-CoA carboxylase and HMG-CoA reductase, respectively, and both enzymes are known to be regu- lated by reversible phosphorylaiion. Acetyl-CoA carboxylase is phosphorylated at multiplc sites by a variety of protein kinases, some of which inactivate the enzyme 181. These protein kinases include a ...

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1. The activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase were measured in muscles from a large number of animals, in order to provide some indication of the importance of the citric acid cycle in these muscles. According to the differences in enzyme activities, the muscles can be divided into three classes. First, in a number of both vertebrate and invertebrate muscles, the activities of all three enzymes are very low. It is suggested that either the muscles use energy at a very low rate or they rely largely on anaerobic glycolysis for higher rates of energy formation. Second, most insect flight muscles contain high activities of citrate synthase and NAD+-linked isocitrate dehydrogenase, but the activities of the NADP+-linked enzyme are very low. The high activities indicate the dependence of insect flight on energy generated via the citric acid cycle. The flight muscles of the beetles investigated contain high activities of both isocitrate dehydrogenases. Third, other muscles ofboth vertebrates and invertebrates contain high activities of citrate synthase and NADP+-linked isocitrate dehydrogenase. Many, if not all, of these muscles are capable of sustained periods of mechanical activity (e.g. heart muscle, pectoral muscles of some birds). Consequently, to support this activity fuel must be supplied continually to the muscle via the circulatory system which, in most animals, also transports oxygen so that energy can be generated by complete oxidation of the fuel. It is suggested that the low activities of NAD+-linked isocitrate dehydrogenase in these muscles may be involved in oxidation ofisocitrate in the cycle when the muscles are at rest. 2. A comparison of the maximal activities of the enzymes with the maximal flux through the cycle suggests that, in insect flight muscle, NAD+-linked isocitrate dehydrogenase catalyses a non-equilibrium reaction and citrate synthase catalyses a near-equilibrium reaction. In other muscles, the enzyme-activity data suggest that both citrate synthase and the isocitrate dehydrogenase reactions are near-equilibrium.The citric acid cycle is the most important metabolic pathway for energy production under aerobic conditions. The hydrogens removed during the cycle are transferred to the electron-transport chain, and the energy released during electron transport is conserved by the formation of ATP. The energy that is formed during the oxidation of glucose or fatty acids in the cycle is important in muscle to support sustained mechanical activity. Nonetheless, a detailed investigation into the maximum activities of the enzymes that catalyse key reactions in the cycle in muscle has not been undertaken. This paper reports the re-

The topology of carnitine palmitoyltransferase I (CPT I) in the outer membrane of rat liver mitochondria was studied using several approaches. 1. The accessibility of the active site and malonyl-CoA-binding site of the enzyme from the cytosolic aspect of the membrane was investigated using preparations of octanoyl-CoA and malonyl-CoA immobilized on to agarose beads to render them impermeant through the outer membrane. Both immobilized ligands were fully able to interact effectively with CPT I. 2. The effects of proteinase K and trypsin on the activity and malonyl-CoA sensitivity of CPT I were studied using preparations of mitochondria that were either intact or had their outer membranes ruptured by hypo-osmotic swelling (OMRM). Proteinase K had a marked but similar effect on CPT I activity irrespective of whether only the cytosolic or both sides of the membrane were exposed to it. However, it affected sensitivity more rapidly in OMRM. By contrast, trypsin only reduced CPT I activity when incubated with OMRM. The sensitivity of the residual CPT I activity was unaffected by trypsin. 3. The proteolytic fragments generated by these treatments were studied by Western blotting using three anti-peptide antibodies raised against linear epitopes of CPT I. These showed that a proteinase K-sensitive site close to the N-terminus was accessible from the cytosolic side of the membrane. No trypsin-sensitive sites were accessible in intact mitochondria. In OMRM, both proteinase K and trypsin acted from the inter-membrane space side of the membrane. 4. The ability of intact mitochondria and OMRM to bind to each of the three anti-peptide antibodies was used to study the accessibility of the respective epitopes on the cytosolic and inter-membrane space sides of the membrane. 5. The results of all these approaches indicate that CPT I adopts a bitopic topology within the mitochondrial outer membrane; it has two transmembrane domains, and both the N- and C-termini are exposed on the cytosolic side of the membrane, whereas the linker region between the transmembrane domains protrudes into the intermembrane space.

The possibility that triacylglycerol (TAG) synthesis occurs on both aspects of the endoplasmic-reticular membrane during the process of incorporation of TAG into secreted very-low-density lipoprotein (VLDL) [Zammit (1996) Biochem. J. 314, 1-14] was investigated by measuring the latency of diacylglycerol acyltransferase (DGAT) in microsomal fractions obtained from rat liver homogenates. Permeabilization of microsomes with taurocholate resulted in the doubling of the activity, indicating that DGAT activities of approximately equal magnitude occur on either aspect of the microsomal membrane. The taurocholate concentrations required for exposure of the latent activity of DGAT were identical with those that resulted in the exposure of marker enzymes for the lumen of the endoplasmic reticulum. Fractionation of the microsomes into smooth and rough populations indicated that the distribution of overt and latent DGAT activities was the same throughout. The possibility that taurocholate effects may result from non-specific activation of the overt enzyme was excluded by employing the channel-forming peptide alamethicin to effect permeabilization, and by varying the mode of delivery of diacylglycerol substrate to the microsomal membranes. Permeabilization using alamethicin gave a slightly higher latent/overt ratio for DGAT. The possible roles of overt and latent DGAT activities in the synthesis and secretion of TAG by the liver are discussed.

PACS 31.15. Qg, 62.20.+e Auxetics (i.e. systems with negative Poisson's ratios) exhibit the unexpected feature of becoming fatter when stretched and narrower when compressed. This property is highly desirable as it imparts many beneficial effects on the material's macroscopic properties. Recent research suggests that in an idealised scenario, systems composed of connected 'rigid squares' can exhibit auxetic behaviour (Poisson's ratio = -1) due to a mechanism involving relative rotation of the squares. This paper shows through force-field based molecular modelling simulations that although 'rotating squares' are responsible for negative Poisson's ratios in various zeolite frameworks, in these real materials, the squares are not rigid and the auxeticity is not as pronounced as in the 'idealised' model. In view of this, a new model system made from connected 'semi-rigid' squares is proposed and analytical equations for the mechanical properties of this new model system are derived and discussed. It will be shown that the Poisson's ratios in this new model are highly dependent on the extent of rigidity of the squares and the direction of loading. It will also be shown that this new model provides a better description for the behaviour of auxetic zeolite frameworks than the original 'rotating rigid squares' model.

The two diacylglycerol acyltransferases, DGAT1 and DGAT2, are known to have non-redundant functions, in spite of catalysing the same reaction and being present in the same cell types. The basis for this distinctiveness, which is reflected in the very different phenotypes of Dgat1 ) ⁄ ) and Dgat2) ⁄ ) mice, has not been resolved. Using selective inhibitors of human DGAT1 and DGAT2 on HepG2 cells and gene silencing, we show that, although DGAT2 activity accounts for a modest fraction (< 20%) of overall cellular DGAT activity, inhibition of DGAT2 activity specifically inhibits (and is rate-limiting for) the incorporation of de novo synthesized fatty acids and of glycerol into cellular and secreted triglyceride to a much greater extent than it affects the incorporation of exogenously added oleate. By contrast, inhibition of DGAT1 affects equally the incorporation of glycerol and exogenous (preformed) oleate into cellular and secreted triacylglycerol (TAG). These data indicate that DGAT2 acts upstream of DGAT1, largely determines the rate of de novo synthesis of triglyceride, and uses nascent diacylglycerol and de novo synthesized fatty acids as substrates. By contrast, the data suggest that DGAT1 functions in the re-esterification of partial glycerides generated by intracellular lipolysis, using preformed (exogenous) fatty acids. Therefore, we describe distinct but synergistic roles of the two DGATs in an integrated pathway of TAG synthesis and secretion, with DGAT2 acting upstream of DGAT1.