Differential regulation by triiodothyronine of substrate-specific uridinediphosphoglucuronate glucuronosyl transferases in rat liver

Hepatic uridinediphosphoglucroonate glucuronosyl transferase (UDPglucuronyltransferase, EC 2.4.1.17) functionally heterogeneus; 4-nitrophenol and bilirubin are representative subtrates for two separated from of the enzyme. UDPglucuronyltransferase activity for bilirubin and 4-nitrophenol was separated from solubilized rat liver microsomes by DEAE-cellulose chromatography and corresponding enzymes were purified. A radioimmunoassay was developed using a rabbit antiserum against purified rat 4-nitrophenol-specific UDPglucuronyltransferase, which precipitated enzyme activities toward both 4-nitrophenol and bilirubin. After treatment with triiodothyronine(T₃) (0.55 mg/kg body weight), hepatic microsomal UDPglucuronyltransferase activity for 4-nitropheelos was increased 400% as compared to controls; the enzyme activity for bilirubin was decreased by 80%; the changes in the substrate-specific enzyme activities were reflected in the enzymatically active fractions separated after DEAE-cellulose chromatography. The changes in enzyme activities paralleled changes in the concentrations of the two corresponing UDP glucuronyltransferase proteins in the chromatographic fractions, as measured by radioimmunoassay. The results indicate that the opposite effects of T₃ on the two forms of UDPglucuronyltransferase activity is due to its differential effect on corresponding enzyme proteins.     

Multiple forms of pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase from wheat seedlings. Regulation by fructose 2,6-bisphosphate

Two forms of pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase have been isolated from wheat seedlings. One of these enzymes, termed PFP-1, has been purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of two different polypeptide chains of Mr = 67,000 (alpha) and 60,000 (beta). PFP-1 has been assigned a molecular structure consisting of alpha 2 beta 2 based on an estimated Mr of 234,000 for the native enzyme. PFP-2, the other form of phosphotransferase, has also been purified extensively. Preliminary data suggest that the active form of PFP-2 is probably a dimer of a polypeptide chain of Mr = 60,000. Immunological studies indicate that the two enzyme preparations share common antigenic determinants. The two forms of enzyme have very similar kinetic properties. The phosphotransferases are activated by fructose 2,6-bisphosphate (Fru-2,6-P2) which lowers the Km of the enzymes for fructose 6-phosphate but not that for PPi. Interestingly, PFP-1 is significantly more active than PFP-2 in the absence of Fru-2,6-P2. Also, PFP-1 exhibits a greater affinity (Ka = 7 nM) than PFP-2 (Ka = 26 nM) for the activator. Based on kinetic, immunological, and physicochemical parameters, it is suggested that the two enzymic forms are related in that they share the same catalytic moiety, i.e. the 60,000-dalton or beta subunit. The beta subunit when in complex formation with the alpha subunit, as in PFP-1, becomes more active in the absence of Fru-2,6-P2 as well as exhibits a greater sensitivity toward the effector.     

Regulation of microsomal enzymes by phospholipids VI. Abnormal enzyme-lipid interactions in liver microsomes from gunn rats

The rates of synthesis of some glucuronides by liver microsomes from the Gunn strain of rat are abnormally low, but previous investigators of the activity of the p-nitrophenol metabolizing form of UDPglucuronyltransferase (UDPglucuronate glucuronyltransferase, EC 2.4.1.17) have reported normal levels of activity in these animals. Data presented in this paper indicate, however, that this enzyme is abnormal in Gunn rats. Thus, treatment of liver microsomes from normal Wistar rats with phospholipase A (EC 3.1.1.4) or Triton X-100 increases the activity of the p-nitrophenol metabolizing form of UDPglucuronyltransferase 10- and 20-fold, respectively, but these agents do not alter activity in microsomes from homozygous Gunn rats. Similarly, phospholipase A and Triton X-100 activate the o-aminophenol and o-aminobenzoate metabolizing forms of UDPglucuronyltransferase in microsomes from normal rats, but are without effect on the enzyme in microsomes from Gunn rats. In contrast, the rates of synthesis of o-aminophenyl- and o-aminobenzoylglucuronides are increased several fold by addition of diethylnitrosamine to microsomes from Gunn rats indicating that the maximum potential activities of UDPglucuronyltransferases are constrained in liver microsomes from both normal and Gunn rats.These data indicate that assays of UDPglucuronyltransferase in native microsomes are not sufficient for delineating the full extent of the defect in the Gunn rat, that there are defects in the function of at least two proteins in liver microsomes from these animals, and that there are abnormal interrelations between some forms of microsomal UDPglucuronyltransferase and their phospholipid environments.     

Cholesterol-dependent modification of microsomal dynamics and UDPglucuronyltransferase kinetics

The effect of both in vitro incorporation and removal of cholesterol in guinea pig liver microsomes on the lipid composition, dynamic properties of the membrane, and kinetic constants of UDPglucuronyltransferase was studied. No significant changes either in the fatty acid composition or in the distribution of phospholipid classes were observed upon cholesterol incorporation and removal. Lateral and rotational mobility measured by the efficiency of pyrene excimer formation and fluorescence of 1,6-diphenylhexatriene decreased with cholesterol incorporation and increased in parallel to cholesterol removal. These changes were associated with alterations in the kinetic properties of UDPglucuronyltransferase. Whereas Vmax increased, the Km of the different steps of the reaction decreased with cholesterol incorporation. The negative homotropic effect and apparent cooperativity of UDP-glucuronic acid decreased when cholesterol was incorporated and increased after cholesterol removal. Moreover, the UDP-N-acetylglucosamine-dependent activation of the enzyme decreased in correlation with an increase of cholesterol concentration in microsomes. It has been demonstrated that both the shift of the non-Michaelian kinetics of the enzyme to Michaelian and the decrease of the UDP-N-acetylglucosamine-dependent activation of the enzyme are evoked by a change of the physical state of the UDPglucuronyltransferase milieu from a gel phase to a liquid-crystalline phase. Therefore, we must admit that cholesterol incorporation in the microsomes while producing an increased packing of the bulk lipids would also cause the separation of more fluid phospholipids, which increase the proportion of molecules in the liquid-crystalline state within the enzyme environment.     

Purification and characterization of membrane-bound and cytosolic forms of diacylglycerol kinase from rat brain

Two different types of diacylglycerol kinase (DGK) have been purified 10,455-fold (DGK I) and 7,410-fold (DGK IV) from the cytosol and membrane fractions of rat brain, respectively. The cytosolic DGK was purified by successive chromatographies on Affi-Gel Blue, Q-Sepharose F.F., Mono Q, hydroxylapatite, and ATP-agarose. The membrane-bound DGK was purified from the 2 M NaCl extract of membranes by chromatography on Affi-Gel Blue, phenyl-Superose, hydroxylapatite, and ATP-agarose. The resultant preparations contained homogeneous enzymes with a Mr of 110,000 (DGK I) and 150,000 (DGK IV) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These enzymes both phosphorylate 1,2-dioleoyl glycerol at rates of 11.5 mumol/min/mg protein for DGK I and 5.2 mumol/min/mg protein for DGK IV. Both enzymes require divalent cations and ionic detergents for activity. Magnesium is the most potent cation for both enzymes, but Ca2+ was also found to be fairly effective. Manganese is less effective than Mg2+ or Ca2+. Anionic detergents such as sodium deoxycholate or sodium cholate stimulate the activities of both enzymes, although DGK IV is stimulated more markedly than DGK I at lower concentrations. The optimal pH for the two enzymes was found to be the same, pH 7.4. Some phospholipids such as phosphatidylserine and phosphatidylinositol elevate the kinase activities of these kinases even in the absence of detergents. DGK IV is activated more significantly than DGK I by low amounts of phospholipids. The two enzymes also show structural differences. DGK I and DGK IV give different peptide maps after digestion with Staphylococcus aureus V8 protease or alpha-chymotrypsin. The results suggest that these enzymes are different forms of DGK and may be involved in different biological processes.     

Purification, Properties, and Localization of Two Carbonic Anhydrases from Amaranthus cruentus Leaves

The method has been developed for obtaining two purified forms of carbonic anhydrase (CA, A and B forms) from amaranth (Amaranthus cruentus L.) leaves. The method includes precipitation with ammonium sulfate, fractionation by ion-exchange chromatography on DEAE Sephadex A-50, gel filtration on AcA-34 ultragel, and ion-exchange chromatography on DEAE cellulose. The molecular weights of A and B forms were different and equaled to 151 and 251 kD, respectively. The results suggest that SH groups and zinc play important roles in the catalytic activity of both CA forms. Both forms exhibited a high hydratase activity and did not represent allosteric enzymes. However, the catalytic properties of A form, evaluated from the pH dependence of kinetic parameters, differed from those of B form, which was apparently caused by dissimilar structures of these forms. Furthermore, the A form was localized in chloroplast membranes of bundle sheath cells, whereas B form was a soluble enzyme located in the cytoplasm of mesophyll cells.     

Purification and characterization of a multienzymatic complex in sugarcane, a C4 plant

We have discovered a multienzymatic complex in fresh young sugarcane leaves. This complex is constituted of three enzymes: PEPcase, NADP-MDH and malic enzyme. After successive molecular sieving chromatography, we have obtained a highly purified sample of the complex which has a molecular weight of 711 kDa. Its functional interest has been evaluated by comparing the kinetic properties of the enzymes in their free forms to those in their complexed form. We show that the association of the three enzymes leads to important changes in their respective kinetic properties.     

Quinoprotein D-glucose dehydrogenase of the Acinetobacter calcoaceticus respiratory chain: membrane-bound and soluble forms are different molecular species

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases, while other oxidative bacteria contain the membrane-bound enzyme exclusively. The two forms of glucose dehydrogenase were believed to be the same enzyme or interconvertible forms. Previously, Matsushita et al. [(1988) FEMS Microbiol. Lett 55, 53-58] showed that the two enzymes are different with respect to enzymatic and immunological properties, as well as molecular weight. In the present study, we purified both enzymes and compared their kinetics, reactivity with ubiquinone homologues, and immunological properties in detail. The purified membrane-bound enzyme had a molecular weight of 83,000, while the soluble form was 55,000. The purified enzymes exhibited totally different enzymatic properties, particularly with respect to reactivity toward ubiquinone homologues. The soluble enzyme reacted with short-chain homologues only, whereas the membrane-bound enzyme reacted with long-chain homologues including ubiquinone 9, the native ubiquinone of the A. calcoaceticus. Furthermore, the two enzymes were distinguished immunochemically; the membrane-bound enzyme did not cross-react with antibody raised against the soluble enzyme, nor did the soluble enzyme cross-react with antibody against the membrane-bound enzyme. Thus, each glucose dehydrogenase is a molecularly distinct entity, and the membrane-bound enzyme only is coupled to the respiratory chain via ubiquinone.     

Properties of membrane-bound and solubilized forms of alkaline phosphatase from human liver

To determine whether the properties of alkaline phosphatase in human liver are altered by releasing the enzyme from its native environment, we studied the membrane-bound and purified forms, and the enzyme released by applying phosphatidylinositol-specific phospholipase-C. The bound enzyme had the lowest affinities for eight substrates and the competitive inhibitor phenylphosphonate. The Ki for inorganic phosphate was lower with the bound enzyme than with the other forms, whereas the values for uncompetitive inhibitors were the same with all three. Phenylglyoxal reacted with essential residues of arginine at similar rates with the bound and purified enzymes, whereas essential cations were more readily removed and replaced in the bound and released forms. Arrhenius plots of the bound enzyme revealed two breaks, with activation energy above the second break similar to that of the purified enzyme. Activity of the bound enzyme increased when the membrane was perturbed by butanol and assayed below 30 degrees C. These experiments demonstrate that, even though binding of alkaline phosphatase to the plasma membrane is not essential for catalytic function, the properties of the enzyme in the membrane are different from those of the soluble form.     

Phospholipid dependence of UDP-glucuronyltransferase.

Very extensive hydrolysis of phospholipids with pure Bacillus cereus phospholipase C at 5 degrees C greatly inhibited the maximum demonstrable rate of glucuronidation of p-nitrophenol by UDPglucuronyltransferase in guinea pig liver microsomes. Lysophosphatidylcholine restored much of the inhibited activity but non-phospholipid surfactants or hydrolysis of diglycerides failed to reactivate. Phospholipid depletion likewise inhibited o-aminophenol glucuronidation and phospholipids restored activity. It is concluded that glucuronyltransferase specifically requires phospholipids for optimal activity. It seems unlikely that these phospholipids only serve to dissolve aglycones, or that they are direct physiological regulators of the transferase. Instead, a permissive role is ascribed to phospholipids, allowing glucuronyltransferase to be regulated by other means.     

Glutathione S-transferase in the insect Apis mellifera macedonica kinetic characteristics and effect of stress on the expression of GST isoenzymes in the adult worker bee

The glutathione S-transferase present in the adult worker bee Apis mellifera macedonica was purified and analyzed for its physicochemical and kinetic properties. The enzyme is heterodimeric with subunit molecular masses of 29 and 25 kDa, respectively. Two main isoenzymes with distinct kinetic properties are present, with isoelectric points of 7.40 for the alkaline and 4.58 for the acidic forms, respectively. The two enzymes are induced independently by factors such as insecticide treatments and environmental conditions, including low temperatures or starvation.     

Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase

Hormonal inhibition of adenylate cyclase is mediated by a guanine nucleotide regulatory protein (Ni) which is different from the one which mediates hormonal stimulation. There is substantial evidence that the active component of Ni (termed alpha i can be ADP-ribosylated by a toxin from Bordetella pertussis. We have found that in bovine cerebral cortex there are three proteins of similar molecular weight (39,000-41,000) which are modified by pertussis toxin. We have purified these proteins and have resolved the 41,000-dalton protein from the 40,000/39,000-dalton doublet. All three forms of pertussis toxin substrate can be isolated in free form or together with a 36,000 beta component. We have also purified this beta component. ADP-ribosylation of the three pertussis toxin substrates is greatly enhanced by the addition of the purified beta component. This makes possible an assay of beta subunit activity based on its interaction with alpha i. The three forms of pertussis toxin substrate which we have purified differ in two functions: susceptibility to ADP-ribosylation and GTPase activity. The 41,000-dalton protein is more readily ADP-ribosylated by pertussis toxin than the smaller forms. The 39,000-dalton protein has GTPase activity with a low Km (0.3 microM) for GTP. The GTPase activity can be doubled by phospholipids. The GTPase activity of the 41,000-dalton protein is almost undetectable. It is not yet known what the relationship of the forms is to each other. The smaller forms may be derived from the larger by proteolysis or it may be intrinsically different. It remains to be shown whether one of the forms represents a different type of regulatory protein which transmits a hormonal signal to effectors other than adenylate cyclase.     

Comparison of phospho- and dephospho-ATP citrate lyase

The dephospho- form of rat liver citrate lyase has been prepared by treating purified [³²P]-ATP citrate lyase with a partially purified phosphatase. A comparison of the properties of the phospho- and dephosphoenzyme has been performed. The pH optima were the same for both forms of the enzyme in four different buffer systems although the optimum values varied identically for both enzyme forms with the buffer. Both the phospho- and dephosphoenzymes show the same kinetic properties except for the K_m observed for ATP in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer system where it was 54 μm for the phosphoenzyme and 292 μm for the dephosphoenzyme. The present study also indicates that both enzymes are cleaved by trypsin and lysosomal proteases in a similar manner. Both forms of the enzyme tend to associate with mitochondria to the same extent and both enzymes have identical temperature stability curves.     

The interaction of RNA polymerase II with non-promoter DNA sites.

Various complexes formed between purified RNA polymerase II and simian virus 40 DNA have been characterized with respect to rates of formation, rates of dissociation, and initial velocity of RNA synthesis. Two different types of complexes can form on intact DNA templates. One of these is formed rapidly, but is quite labile; the other forms more slowly, but is moderately stable once formed. The introduction of a single strand break into DNA leads to rapid and stable complex formation, and thus is expected to create the favored binding site. The observed properties of these complexes provide a general framework for describing the interactions of RNA polymerase II at non-promoter DNA sites. This framework appears to be similar to that established for Escherichia coli RNA polymerase interactions, suggesting that the fundamental mode of non-promoter DNA binding is similar for the bacterial, plant, and mammalian enzymes.     

Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism

Two forms of cytochrome P-450 were purified to apparent homogeneity from several different preparations of human liver microsomes. One form, designated P-450DB, had relatively high catalytic activity towards the drugs debrisoquine, sparteine, bufuralol (both the (+)- and (-)-isomers), encainide, and propranolol and appears to be the enzyme involved in the polymorphic distribution of oxidative activities towards these substrates in humans. The other form, designated P-450PA, had relatively high phenacetin O-deethylase activity and appears to be involved in the variation of this activity among humans. Polyclonal antibodies raised to the two enzymes were specific for the antigens as judged by immunoelectrophoresis and immuno-inhibition studies. The two enzymes and their activities were distinguished by chromatographic separation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, amino acid composition, immuno-inhibition studies, and steady-state kinetic assays. Immunochemical studies suggest that each form represents only a small fraction of the total cytochrome P-450 in human liver microsomes. These biochemical studies provide a basis for better understanding the mechanisms which underlie genetic polymorphisms involving P-450 cytochromes in humans.     

Properties and Partial Purification of Choline Acetyltransferase from the Nematode Caenorhabditis elegans

We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW approximately 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the "native" small form (MW approximately 71,000). We have purified the small form of the enzyme greater than 3,300-fold by a combination of gel filtration, ion-exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 mumol/min/mg protein, and is free of acetylcholinesterase and acetyl-CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half-maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration.     

Isolation of multiple forms of indanol dehydrogenase associated with 17 beta-hydroxysteroid dehydrogenase activity from male rabbit liver

Seven multiforms of indanol dehydrogenase were isolated in a highly purified state from male rabbit liver cytosol. The enzymes were monomeric proteins with similar molecular weights of 30,000-37,000 but with distinct electrophoretic mobilities. All the enzymes oxidized alicyclic alcohols including benzene dihydrodiol and hydroxysteroids at different optimal pH, but showed clear differences in cofactor specificity, steroid specificity, and reversibility of the reaction. Two NADP+-dependent enzymes exhibited both 17 beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes and 3 alpha-hydroxysteroid dehydrogenase activity for 5 beta-androstan-3 alpha-ol-17-one. Three of the other enzymes with dual cofactor specificity catalyzed predominantly 5 beta-androstane-3 alpha,17 beta-diol dehydrogenation. The reverse reaction rates of these five enzymes were low, whereas the other two enzymes, which had 3 alpha-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes or 3(17)beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes, highly reduced 3-ketosteroids and nonsteroidal aromatic carbonyl compounds with NADPH as a cofactor. All the enzymes exhibited Km values lower for the hydroxysteroids than for the alicyclic alcohols. The results of kinetic analyses with a mixture of 1-indanol and hydroxysteroids, pH and heat stability, and inhibitor sensitivity suggested strongly that, in the seven enzymes, both alicyclic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities reside on a single enzyme protein. On the basis of these data, we suggest that indanol dehydrogenase exists in multiple forms in rabbit liver cytosol and may function in in vivo androgen metabolism.     

Purification and properties of five different forms of human procarboxypeptidases

Three different procarboxypeptidases A and two different procarboxypeptidases B have been isolated for the first time, in a pure and native state, from human pancreatic extracts. These proteins were purified in one or two quick steps by anion-exchange HPLC. All these forms have been biochemically characterized. Two of the procarboxypeptidases A, the A1 and A2 forms, are obtained in a monomeric state while the other, the A3 form, is obtained as a binary complex of a procarboxypeptidase A with a proproteinase E. This complex is stable in aqueous buffers at various ionic strengths and develops carboxypeptidase A and proteinase E activities in the presence of trypsin. The A1 and A2 forms show clear differences in electrophoretic mobility in SDS/polyacrylamide gels, isoelectric point, proteolytic activation process with trypsin and susceptibility to thermal denaturation. In contrast, these properties are similar in the A1 and A3 (binary complex) forms. On the other hand, with respect to the properties listed above, the B1 and B2 forms differ from each other mainly in isoelectric point. An overall comparison of the above properties reveals the unusual character of the A2 form, midway between the other A and B forms. N-terminal extended sequence analysis carried out on these proenzymes confirm that they constitute different isologous forms.     

The phosphoglucose isomerases of the bloodstream forms of Trypanosoma brucei and Trypanosoma vivax.

1. The phosphoglucose isomerases (PGI's) of the bloodstream forms of Trypanosoma brucei and T. vivax have been purified some 150-fold, using cellulose ion-exchange chromatography, gel filtration and isoelectric focussing. 2. The two trypanosome enzymes showed many similarities in kinetic properties, but differed from each other somewhat in thermal stability and in isoelectric point. 3. Both trypanosome enzymes differ from PGI's from other sources in having a higher Ki for the competitive inhibitor 6-phosphogluconate.     

Rat heart glycogen phosphorylase II is genetically distinct from phosphorylase I

Previous studies in our laboratory have shown that rat heart glycogen phosphorylase (1,4-alpha-D-glucan: orthophosphate alpha-D-glucosyltransferase, EC 2.4.1.1) separates into two forms upon ion-exchange chromatography. Both forms could be shown to have the same subunit Mr and to incorporate one molecule of phosphate per subunit. The studies reported here were done to check whether both forms are native isoenzymes and, further, which form might represent the heart-specific phosphorylase. Firstly, the iso-electric points of the purified enzymes are compared with those associated with phosphorylase activity in crude extracts from rat heart. Two out of four major bands coincided with the bands of purified phosphorylase Ib and IIb (isoelectric points: 5.5 and 6.25), indicating apparent identity. Secondly, antibodies to rat skeletal muscle phosphorylase reacted with rat heart phosphorylase I, whereas phosphorylase II was neither inhibited nor precipitated by the antibody. Thirdly, peptide maps obtained after proteolytic digestion of SDS-denatured phosphorylase I and II showed different patterns. In addition to the kinetic differences between these two forms reported earlier, phosphorylase IIa was inhibited by glucose 6-phosphate, whereas phosphorylase Ia was not. These results suggest that phosphorylase II is a heart-specific isoenzyme which is presumably encoded by a different gene.