Subunit size and paracrystal structure of avian liver acetyl-CoA carboxylase.

The subunit molecular weight of chicken liver acetyl-CoA carboxylase has been redetermined by immunoprecipitation and sodium dodecyl sulfate gel electrophoresis. In the presence of parotid trypsin inhibitor, the immunoprecipitate gave a single band corresponding to a molecular weight of 230,000, which was also found to contain bound biotin. From the biotin content of the protomer (1.0 prosthetic group per 480,000 daltons) it appears that it consists of two non identical subunits, both with molecular weights of approximately 230,000.Electron microscopy has been carried out on the active filamentous form of the enzyme and on paracrystals formed under high-salt conditions. These indicate that the filaments are readily distortable helical ribbons, with an approximate axial repeat of 1100 Å, containing eight protomers. The paracrystals are made up of a staggered lateral packing of filaments.     

Acetyl-coenzyme-A carboxylase of Candida Lipolytica. 1. Purification and properties of the enzyme.

Acetyl-coenzyme-A carboxylase has been isolated in homogeneous form from Candida lipolytica. The homogeneity of the enzyme preparation is evidenced by analytical ultracentrifugation, dodecyl-sulfate-polyacrylamide gel electrophoresis and Ouchterlony double-diffusion analysis. The purified enzyme exhibits a specific activity of 8.0 U/mg protein at 25 degrees C and contains 1 mol biotin/263000 g protein. The sedimentation coefficient (S20,W) of the enzyme is 18 S. It has been shown by dodecyl-sulfate-polyacrylamide gel electrophoresis that the enzyme possesses only one kind of subunit with a molecular weight of 230000. This finding, together with the biotin content, indicates that the C. lipolytica enzyme has a highly integrated subunit structure. The C. lipolytica enzyme is very labile, but is stabilized by glycerol. The enzyme is markedly activated by poly(ethyleneglycol), the activation being due principally to a decrease in the Km values for substrates. Even in the presence of this activator, the Km value for acetyl-CoA of the C. lipolytica enzyme is much higher than that of the enzyme from Saccharomyces cerevisiae and animal tissues. The C. lipolytica enzyme, unlike the enzyme from animal tissues, is not activated by citrate.     

Phosphoinositide inhibition of acetyl-CoA carboxylase from rat liver

When purified acetyl-CoA carboxylase was incubated with various phospholipids, the effects on carboxylase activity were quite diverse. Phosphatidic acid, phosphatidylcholine, and phosphatidylinositol were slightly stimulatory, whereas carboxylase was inhibited by polyphosphoinositides in a time- and concentration-dependent manner. Phosphatidylinositol 4,5-bisphosphate (TPI) was the most effective inhibitor; carboxylase activity was inhibited 50% after incubation with 1.5 μm TPI for 30 min. Incubation of carboxylase with citrate reduced the susceptibility to inhibition by TPI. The inhibition was reversed by removal of TPI from the inhibited enzyme. Incubation of TPI with divalent metal cations removed its ability to inhibit carboxylase. Sedimentation studies showed that TPI treatment shifts carboxylase to a less-polymerized form. The K_m for ATP, 24 μm, was not affected by the inhibitor. However, the apparent K_m for acetyl-CoA was decreased from 44 to 11 μm following incubation with TPI. The possibility that polyphosphoinositides may play a role in acetyl-CoA carboxylase regulation is discussed.     

Acetyl-coenzyme-A carboxylase of Candida lipolytica. 2. Regulation of cellular content and synthesis of the enzyme.

The level of acetyl-coenzyme-A carboxylase activity in Candida lipolytica undergoes large variations depending upon the carbon source on which the yeast is grown. Cells grown on n-alkanes or fatty acids exhibit a lower activity level than do cells grown on glucose. Among the n-alkanes and fatty acids tested, n-heptadecane, n-octadecane, oleic acid and linoleic acid reduce the enzyme activity to the lowest levels, which are 16-18% of the activity level in glucose-grown cells. Immunochemical titrations and Ouchterlony double-diffusion analysis with specific antibody as well as kinetic studies have indicated that the observed decrease in the level of acetyl-CoA carboxylase activity is due to a reduction in the cellular content of the enzyme. Furthermore, isotopic leucine incorporation studies with the use of the immunoprecipitation technique have demonstrated that the relative rate of synthesis of the enzyme in oleic-acid-grown cells is diminished to 12% of that in glucose-grown cells. Evidence has also been obtained to support the view that the enzyme in this yeast is not degraded at a rate high enough to contribute to the marked decrease in the cellular content of the enzyme. Thus, it is concluded that the reduction in acetyl-CoA carboxylase content in fatty-acid-grown cells is due to diminished synthesis of the enzyme.     

Reevaluation of properties of acetyl-CoA carboxylase from rat liver

Rat liver acetyl-CoA carboxylase can be rapidly isolated by a new procedure which uses avidin-Sepharose affinity chromatography. The isolated enzyme has Mr = 260,000; none or very little of the proteolytic products of the carboxylase which are formed in conventional purification procedures are found in our preparations. It is apparent that the previously reported subunit of the carboxylase, with Mr = 230,000, is itself the product of proteolysis. The properties of the enzyme produced by our new method are quite different from those of the conventionally prepared enzyme. Our enzyme contains 6 mol of alkali-labile phosphate/mol of subunit, rather than 2 mol; the Km for acetyl-CoA is about 8-fold higher and the specific activity is only about one-fifth of that previously reported. The large amount of phosphate does not appear to cause the low specific activity of the new enzyme preparation, because alkaline phosphatase treatment reduces the number of phosphates/subunit from 6 to 3 mol but does not change the specific activity.     

Subunit structure and autophosphorylation mechanism of casein kinase-TS (type-2) from rat liver cytosol

Type-2 casein kinase-TS (Ck-TS) purified to homogeneity from rat liver cytosol exhibits a molecular mass of 130000 daltons in non-denaturating media and a subunit composition consistent with an alpha 2 beta 2 heterotetramer. The quaternary structure of Ck-TS is not compromised by limited proteolysis with trypsin which converts the 38-kDa alpha subunit into 36-kDa (alpha') and 34-kDa (alpha") derivatives, inducing a parallel decrease of enzymatic activity. Since the 25-kDa beta subunit is unaffected under comparable conditions, the catalytic activity seemingly resides in the alpha subunits. The beta subunit, on the other hand, undergoes a very rapid phosphorylation upon incubation of Ck-TS with ATP/Mg2+: 0.8-1.5 mol P/mol Ck-TS are incorporated within 30 s. Such a fast autophosphorylation is neither prevented nor slowed down by the addition of a large excess of phosphorylatable substrates and takes place through an intra-molecular rather than inter-molecular process. This conclusion is supported by the following data. (a) The autophosphorylation rate is linearly proportional to the concentration of Ck-TS. (b) Thermally inactivated Ck-TS is not phosphorylated by catalytic amounts of active enzyme. (c) Basic polypeptides like protamine and polylysine stimulate the activity of Ck-TS toward phosphorylatable substrates while preventing the autophosphorylation reaction. Since the effectors that inhibit autophosphorylation also induce a remarkable decrease of the Km values for the protein substrates, the possibility is discussed that autophosphorylation might represent a regulatory device by which Ck-TS could be converted into a partially inactivated form exhibiting reduced affinity toward its endogenous targets.     

Regulation of acetyl-coenzyme A carboxylase. I. Purification and properties of two forms of acetyl-coenzyme A carboxylase from rat liver

Acetyl-CoA carboxylase of animal tissues is known to be dependent on citrate for its activity. The observation that dephosphorylation abolishes its citrate dependence (Thampy, K. G., and Wakil, S. J. (1985) J. Biol. Chem. 260, 6318-6323) suggested that the citrate-independent form might exist in vivo. We have purified such a form from rapidly freeze-clamped livers of rats. Sodium dodecyl sulfate gel electrophoresis of the enzyme gave one protein band (Mr 250,000). The preparation has high specific activity (3.5 units/mg in the absence of citrate) and low phosphate content (5.0 mol of Pi/mol of subunit). The enzyme isolated from unfrozen liver or liver kept in ice-cold sucrose solution for 10 min and then freeze-clamped has low activity (0.3 unit/mg) and high phosphate content (7-8 mol of Pi/mol of subunit). Citrate activated such preparations with half-maximal activation at greater than 1.6 mM, well above physiological range. The low activity may be due to its high phosphate content because dephosphorylation by [acetyl-CoA carboxylase]-phosphatase 2 activates the enzyme and reduces its dependence on citrate. Since freeze-clamping the liver yields enzyme with lower phosphate content and higher activity, it is suggested that the carboxylase undergoes rapid phosphorylation and consequent inactivation after the excision of the liver. The carboxylase is made up of two polymeric forms of Mr greater than or equal to 10 million and 2 million based on gel filtration on Superose 6. The former, which predominates in preparations from freeze-clamped liver, has higher activity and lower phosphate content (5.3 units/mg and 4.0 mol of Pi/mol of subunit, respectively) than the latter (2.0 units/mg and 6.0 mol of Pi/mol of subunit, respectively). The latter, which predominates in preparations from unfrozen liver, is converted to the active polymer (Mr greater than or equal to 10 million) by dephosphorylation. Thus, the two polymeric forms are interconvertible by phosphorylation/dephosphorylation and may be important in the physiological regulation of acetyl-CoA carboxylase.     

Subunit structure and peptide mapping of junctional and extrajunctional acetylcholine receptors from rat muscle.

We have purified the junctional acetylcholine receptor from normal rat skeletal muscle and compared its structure with that of the extrajunctional receptor from denervated muscle. The two receptors from leg muscle were distinguished by isoelectric focusing and by reaction with sera from patients with myasthenia gravis. The junctional form of the acetylcholine receptor was purified from normal leg muscle by affinity chromatography on concanavalin A/Sepharose and cobrotoxin/Sepharose followed by sucrose gradient centrifugation. Analysis of radioiodinated receptor by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the subunit structure of the junctional receptor was similar to that previously determined for the extra-junctional form (Froehner, S. C., et al. (1977) J. Biol. Chem. 252, 8589-8596), with major polypeptides, whose apparent molecular weights in 9% polyacrylamide gels were 45 000 and 51 000. In addition, several minor polypeptides were found. When the two receptors were labeled with different isotopes of iodine and run together on a sodium dodecyl sulfate gel, the subunits of one receptor could not be resolved from those of the other. As seen earlier with the extrajunctional form, the affinity alkylating reagent [3H]MBTA labeled the 45 000- and 49 000-dalton polypeptides of the junctional receptor. Peptide mapping showed that the two MBTA binding subunits are structurally related, although they are unrelated to the other polypeptides, and that the 45 000- and 51 000-dalton polypeptides of the junctional receptor were indistinguishable from those of the extrajunctional receptor. In addition, peptide mapping of the four subunits of acetylcholine receptor isolated from Torpedo californica electric organ showed that these four polypeptides appear to be structurally unrelated.     

Subunit structure of bovine milk xanthine oxidase. Effect of limited cleavage by proteolytic enzymes on activity and structure.

Bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) has been purified by a modified method without the use of proteases, and its structure has been analyzed by polyacrylamide gel electrophoresis. Native xanthine oxidase is found to consist of only two polypeptide chains A with molecular weights of 150 000 each. These chains have NH2-terminal methionine. Limited proteolysis with trypsin, chymotrypsin, or subtilisin at pH 8 did not affect molecular weight and activities of the enzyme while each of the A chains was cleaved under these conditions to three fragments C, E, and F with molecular weights of 92 00, 42 000 and 20 000, respectively. These fragments remained bound to each other and were relatively resistant to subsequent proteolysis. The isolation of xanthine oxidase in the presence of pancreatin as described by Hart et al. (1970, Biochem. J. 116, 851) gives partially digested enzyme composed mainly of chains C, E (Mr 35 000) and a small component (Mr approx. 15 0-0). The action of subtilisin on xanthine oxidase at pH 11 resulted in complete digestion of E chains, FAD separation, and total loss of xanthine:oxygen oxidoreductase activity while xanthine:indophenol oxidoreductase activity was relatively little affected. The residual enzyme has a molecular weight of about 200 000, is composed mainly of two C chains (and may probably contain F and/or proteolytic fragments of low molecular weight), contains molybdenum, and does not contain FAD.     

The deoxyribonucleic acid modification enzyme of bacteriophage P1. Subunit structure

The bacteriophage P1 modification enzyme was purified 1400-fold from induced lysogens of a thermoinducible mutant of bacteriophage P1. The most purified fraction, when analysed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate, showed two principal stained bands. The two bands co-sedimented in a glycerol gradient with the modification activity, at a rate which, when compared with the rate of sedimentation of marker proteins, corresponds to a sedimentation coefficient in water of 6S. The mobilities of the bands on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis corresponded to polypeptides of molecular weight 70000 and 45000 and they were present in equimolar amounts. It was concluded that the 6S species of the enzyme is a dimer of unlike subunits.     

Heat activation of rat liver acetyl-CoA carboxylase in vitro.

Acetyl-CoA carboxylase in rat liver homogenates was activated in vitro in a time- and temperature-dependent manner. The activity of acetyl-CoA carboxylase in rat liver preparations was determined in a 1-min assay to preclude the possibility of citrate activation of the enzyme during the assay period. Activation of the enzyme occurred more rapidly in liver preparations continuously maintained at ambient or greater temperatures than in homogenates of liver which had been chilled. High speed supernatant (105,000 X g, 60 min) did not heat-activate, and reconstitution of the heat-activatable 27,000 X g, 20-min, fraction by recombining the high speed pellet with the high speed supernatant only partially restored the heat activatability. Elution of the 105,000 X g supernatant from Sephadex G-25 resulted in an enzyme preparation which was heat-activatable. Addition of boiled 105,000 X g supernatant to the Sephadex G-25-treated enzyme again prevented heat activation. Dilution of the enzyme 5-fold did not prevent heat activation.     

Primary structure of chicken liver acetyl-CoA carboxylase deduced from cDNA sequence

The complete amino acid sequence of acetyl-CoA carboxylase from chicken liver has been deduced by cloning and sequence analysis of DNA complementary to its messenger RNA. The results were confirmed by Edman degradation of peptide fragments obtained by digestion of the enzyme polypeptide with Achromobacter proteinase I or staphylococcal serine proteinase. Chicken liver acetyl-CoA carboxylase is predicted to be composed of 2,324 amino acid residues, having a calculated molecular weight of 262,706. The biotin carboxyl carrier protein domain is located in the middle region of the enzyme polypeptide. The amino-terminal portion of the acetyl-CoA carboxylase has been found to exhibit a homologous primary structure to that of carbamyl phosphate synthetase. Localization of possible functional domains including biotin carboxylase subsite in the acetyl-CoA carboxylase polypeptide is discussed.