Acetyl-CoA acetyltransferase from bovine liver mitochondria. Molecular properties of multiple forms.

Bovine liver mitochondrial acetyl-CoA acetyltransferase (acetyl-CoA:acetyl-CoA C-acetyltransferase, EC 2.3.1.9) has been obtained in three forms designated transferase I, A and B on the basis of their elution positions from chromatography on phosphocellulose. All forms have been shown to have a molecular weight of about 152 000, each being composed of four similar subunits. Amino acid analysis of transferase A and B, the two major forms, revealed a close relationship between both forms with almost identical amino acid composition and arginine as N-terminal residue. The three transferases differ with respect to their redox state and their multiplicity of forms with isoelectric points of 6.9, 7.5 and 8.8, into which the transferases I and A were spontaneously transformed upon isoelectric focusing or rechromatography on phosphocellulose. Transferase B represents a stable enzyme form with an isoelectric point of 8.8. Although the redox state of transferase B can be adjusted to that of transferase A still a difference in charge and in the multiplicity of forms exists, thus indicating different protein states.     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

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.     

The tricarboxylate carrier from rat liver mitochondria. Purification, reconstitution and kinetic characterization

The tricarboxylate carrier from rat liver mitochondria has been purified and reconstituted into phospholipid vesicles. Its activity has been characterized by both a radioactive citrate uptake assay and a coupled enzymatic assay. A Km of 40 microM and a Vmax of 1.56 mumol x min-1 x mg-1 have been determined for the carrier. Cholesterol levels of between 5-10% of total lipid content are shown to cause a decrease in carrier activity.     

Purification and properties of ATPase inhibitor from rat liver mitochondria.

(1) The ATPase inhibitior protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9. (2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F1, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria. (3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg2+. ATP at slightly acidic pH. (4) The inhibitor has a minimal effect on Pi-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimules Pi-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Demonstration of glyoxalase II in rat liver mitochondria. Partial purification and occurrence in multiple forms

Glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6), which has been regarded as a cytosolic enzyme, was also found in rat liver mitochondria. The mitochondrial fraction contained about 10-15% of the total glyoxalase II activity in liver. The actual existence of the specific mitochondrial glyoxalase II was verified by showing that all of the activity of the crude mitochondrial pellet was still present in purified mitochondria prepared in a Ficoll gradient. Subfractionation of the mitochondria by digitonin treatment showed that 56% of the activity resided in the mitochondrial matrix and 19% in the intermembrane space. Partial purification of the enzyme (420-fold) was also achieved. Statistically significant differences were found in the substrate specificities of the mitochondrial and the cytosolic glyoxalase II. Electrophoresis and isoelectric focusing of either the crude mitochondrial extract or of the purified mitochondrial glyoxalase II resolved the enzyme activity into five forms with the respective pI values of 8.1, 7.5, 7.0, 6.85 and 6.6. Three of these forms (pI values 7.0-6.6) were exclusively mitochondrial, with no counterpart in the cytosol. The relative molecular mass of the partially purified enzyme, as estimated by Superose 12 gel chromatography, was 21,000. These results give evidence for the presence of mitochondrial glyoxalase II which is different from the cytosolic enzymes in several characteristics.     

delta-Aminolevulinic acid synthetase from rat liver mitochondria. Purification and properties.

delta-Aminolevulinic acid synthetase has been purified from liver mitochondria of young, uninduced rats. After nonionic detergent solubilization of mitochondrial inner membrane-matrix fractions, the enzyme was purified to a specific activity of approximately 2,000 nmol of delta-aminolevulinic acid formed/h/mg of protein at 30 degrees C, by means of ammonium sulfate precipitation, diethylaminoethyl cellulose chromatography, Sephacryl chromatography, and preparative gel electrophoresis. The purified enzyme preparation thus obtained was apparently homogeneous as judged by its migration as a single band with a molecular weight of 58,000 +/- 6,000 upon electrophoresis in sodium dodecyl sulfate polyacrylamide gels. The native enzyme probably exists as a dimer with a molecular weight of approximately 120,000. A pH optimum of 7.5 and an isoelectric point of 4.5 were also determined. Both monovalent cations and hemin strongly inhibited the activity of the purified enzyme.     

Oxidative properties of swollen rat liver mitochondria.

Rat liver mitochondria undergo extensive swelling when they are incubated in hypotonic sucrose medium containing 5 mm P_i. After 30 min of swelling at 25 °C, a three- to fourfold increase in volume has occurred, accompanied by gross disorganization of the matrix as observed by electron microscopy. Succinate-supported respiration was unchanged, but the respiration of NAD-linked substrates was reduced and there was a complete and irreversible loss of phosphorylation in both cases. β-Hydroxybutyrate-supported respiration was regained completely on addition of NAD to the swollen mitochondria. α-Ketoglutarate- and malate + pyruvate-supported respiration was only partially restored by the addition of NAD. This inhibition of respiration in swollen mitochondria may be due to a disorganization of a putative complex of Krebs cycle enzymes on the inner surface of the inner membrane.     

Molecular properties of multiple forms of acid phosphatase from horse liver.

1. Horse liver acid phosphatase was separated into two partially purified fractions differing in molecular weight (enzyme I about 100 00, enzyme II about 25 000). 2. Enzyme I was separated into several subfractions by DEAE-cellulose chromatography and isoelectric focusing. 3. Molecular weight, sedimentation coefficient and effective molecular radii were determined for acid phosphatases I and II by gel filtration and density-gradient centrifugation.     

Purification and properties of ATPase inhibitor from rat liver mitochondria

(1) The ATPase inhibitor protein has been isolated from rat liver mitochondria in purified form. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis is approximately 9500, and the isoelectric point is 8.9.

(2) The protein inhibits both the soluble ATPase and the particle-bound ATPase from rat liver mitochondria. It also inhibits ATPase activities of soluble F₁, and inhibitor-depleted submitochondrial particles derived from bovine heart mitochondria.

(3) On particle-bound ATPase the inhibitor has its maximal effect if incubated in the presence of Mg²⁺. ATP at slightly acidic pH.

(4) The inhibitor has a minimal effect on P_i-ATP exchange activity in sonicated submitochondrial particles. However, unexpectedly the inhibitor greatly stimulates P_i-ATP exchange activity in whole mitochondria while the low ATPase activity of the mitochondria is not affected. The possible mechanism of action of the inhibitor on intact mitochondria is offered.     

Purification and properties of alpha-aminoadipate aminotransferase from rat liver and kidney mitochondria.

Recently we reported an affinity chromatography method to purify alpha-aminoadipate aminotransferase (AadAT) activity from rat kidney supernatant fraction. Using the same affinity column, we purified AadAT activities from rat kidney and liver mitochondria. The physical and kinetic properties such as pH optima, Km for substrates, molecular weight, subunit structure, isoelectric pH, electrophoretic mobility and inhibition by dicarboxylic acids of mitochondrial AadAT were similar to those of the AadAT from rat kidney supernatant fraction. These results indicate that AadAT from different subcellular fractions is structurally and immunologically identical.     

The multiple forms and kinetic differences of rat colonic beta-N-acetylhexosaminidases

Rat colonic beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) has been separated into three forms by DEAE-cellulose chromatography with an increasing salt gradient. It was not possible to separate the glucosaminidase activity from the galactosaminidase activity by a variety of chromatographic procedues, but the ratio of the two specific activities varied during purification. The pH optima were however identical, for both activities and all three forms. Kinetic measurements including inhibition by substrate analogues showed differences between the two activities as well as among the three forms. A common active site model was inconsistent with the results. Data from mixed substrate experiments were consistent with a model wherein the two activities reside in seperate active sites, each able to be inhibited by the substrate for the other site. The effect of acetate and SH reagents confirmed the two-site model. Treatment with neuraminidase, thimerosal, p-hydroxymercuribenzoate, HgCl2 and AgNO3 or heating at 50 degrees C did not produce any effect on the A form that could be identified as a conversion to the B form. Measurement of the effects on both activities supported the two-site model. It is concluded that the relationship between the A and B forms in the rat colonic mucosa hexosaminidases must be different from that reported for such enzymes from other sources.     

Purification and properties of deoxyadenosine kinase from rat liver mitochondria. I. Purification and physical properties

Deoxyadenosine kinase (ATP: deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76, AdR kinase) from rat liver mitochondria has been partially purified and compared with partially purified AdR kinase from the cytosol of the same biological material. Some physical properties of both enzymes, including molecular weight, gel electrophoresis and gel isoelectric focusing were investigated and considerable differences between these data for mitochondrial and cytosol AdR kinase were found.