Characterization of the reaction mechanism for the XL-I form of bovine liver xenobiotic/medium-chain fatty acid:CoA ligase.

The XL-I form of xenobiotic/medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria and used to determine the reaction mechanism. A tersubstrate kinetic analysis was conducted by varying the concentrations of ATP, benzoate and CoA in turn. Both ATP and benzoate gave parallel double-reciprocal plots against CoA, which indicates a Ping Pong mechanism, with either pyrophosphate or AMP leaving before the binding of CoA. Addition of pyrophosphate to the assays changed the plots from parallel to intersecting; addition of AMP did not. This indicates that pyrophosphate is the product that leaves before binding of CoA. Based on end-product inhibition studies, it was concluded that the reaction follows a Bi Uni Uni Bi Ping Pong mechanism, with ATP binding first, followed in order by benzoate binding, pyrophosphate release, CoA binding, benzoyl-CoA release and AMP release. A similar mechanism was obtained when the ligase was examined with butyrate as substrate. However, butyrate activation was characterized by a much higher affinity for CoA. This is attributed to steric factors resulting from the bulkier nature of the benzoate molecule. Also, with butyrate there is a bivalent cation activation distinct from that associated with binding to ATP. This activation by excess Mg(2+) results in non-linear plots of 1/v against 1/[ATP] for butyrate unless the concentrations of Mg(2+) and ATP are varied together.

Isolation and preliminary characterization of the medium-chain fatty acid:CoA ligase responsible for activation of short- and medium-chain fatty acids in colonic mucosa from swine.

The ATP-dependent activation of short- and medium-chain fatty acids to their respective CoA thioester adducts was investigated in the colonic mucosa from swine. Subcellular fractionation of a homogenate of the mucosa from the entire length of the colon revealed a predominantly mitochondrial localization for activity toward fatty acids ranging from propionate through laurate. These activities could be released from mitochondria in soluble form by freeze-thaw lysis. Purification of these activities revealed that they all appeared to reside with a single enzyme. This suggests that the entire colon contains a single form of medium-chain fatty acid:CoA ligase (MCFA:CoA ligase). The ligase also had activity toward benzoate and salicylate, although this activity was significantly lower than activity toward medium-chain fatty acids. The enzyme had the highest activity at Vmax with butyrate as substrate and had the lowest Km for octanoate. Butyrate and octanoate were mutually inhibitory. Activity toward both substrates was also efficiently inhibited by cyclohexane carboxylate. The molecular weight of the enzyme was estimated by gel filtration chromatography to be ca. 46,500. These data indicate that the colonic MCFA:CoA ligase is significantly different from the hepatic and kidney MCFA:CoA ligases.

Monovalent cation effects on the activity of the xenobiotic/medium-chain fatty acid:CoA ligases are substrate specific.

The effect of monovalent cation on the activity of the XL-I and XL-III forms of xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) was investigated using a variety of different carboxylic acid substrates. With benzoate or p-hydroxybenzoate as substrate, the XL-I ligase was essentially inactive in the absence of monovalent cation. However, with phenylacetic acid and medium-chain fatty acids as substrate, the enzyme retained 3 to 10% activity upon removal of monovalent cation. Further, while Na+ was ineffective with benzoate and p-hydroxybenzoate as substrates, it was effective with other substrates, although still less effective than K+. For XL-III, activity toward benzoate, hydroxybenzoate, and salicylate was insignificant in the absence of monovalent cation, but this rate was 10% of the K(+)-supported rate for hexanoate and 20% for decanoate. Also, with decanoate as substrate, XL-III was activated more by Na+ than by K+. Thus, the nature of the dependence on monovalent cation for activity is substrate-selective. Kinetic analysis of the effect of K+ on the activity of XL-I and XL-III revealed that activation by K+ was not the result of alteration of the affinity of the enzymes for either ATP or the carboxylic acid. For both forms of XM-ligase, K+ was found to enhance the affinity of the enzyme for CoA, regardless of the substrate, although the extent of the enhancement was substrate-specific. In almost all cases there was further activation, even at saturating concentrations of CoA, which indicates an additional effect of monovalent cation on the catalytic rate constant for the reaction. The exception was activation of XL-III activity toward decanoate, which was solely the result of enhanced binding affinity for CoA.

Isolation and sequencing of cDNAs for the XL-I and XL-III forms of bovine liver xenobiotic-metabolizing medium-chain fatty acid:CoA ligase.

The XL-I form of xenobiotic-metabolizing medium-chain fatty acid:CoA ligase was previously purified to apparent homogeneity from bovine liver mitochondria, and the amino acid sequence of a short segment of the enzyme was determined. This sequence was used to develop a probe for screening a bovine cDNA library from which a 1.6 kb cDNA was isolated. This cDNA was sequenced and found to contain the code for the known amino acid sequence. The complete open reading frame was not present in this cDNA, but it was estimated to code for approximately 75% of the XL-I sequence. The XL-III ligase was purified to apparent homogeneity from bovine liver mitochondria. The enzyme eluted from a gel filtration column as a single peak with an apparent molecular weight of ca. 55,000. It ran as a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular weight of 62 kDa. N-Terminal sequence analysis of the enzyme gave no sequence, which indicates a blocked N-terminus. The enzyme was chemically cleaved using CNBr. The resulting peptides were separated by SDS-PAGE. The cleavage pattern revealed two large peptides of ca. 21 and 25 kDa, plus several smaller peptides including a prominent 6 kDa peptide. The N-terminus of the 6, 21, and 25 kDa peptides was sequenced and the 21 and 25 kDa sequences were identical indicating incomplete cleavage. The sequences were used to design probes for screening a bovine liver cDNA library. This resulted in the isolation of a 2,065 bp cDNA. This cDNA was sequenced and found to contain the initiation and termination codons, as well as the requisite amino acid sequences. The open reading frame coded for a 64,922 Da protein. The sequence of XL-III cDNA was markedly different from that of XL-I, indicating the genetic uniqueness of the two ligases. They are, however, 64% homologous, which suggests a common evolutionary origin.

Characterization of the CoA ligases of human liver mitochondria catalyzing the activation of short- and medium-chain fatty acids and xenobiotic carboxylic acids.

Two distinct forms of xenobiotic/medium-chain fatty acid:CoA ligase (XM-ligase) were isolated from human liver mitochondria. They were referred to as HXM-A and HXM-B based on their order of elution from a DEAE-cellulose column. Activity of the two ligases was determined toward 15 different carboxylic acids. HXM-A represented 60-80% of the benzoate activity in the lysate, and kinetic analysis revealed that benzoate was the best substrate (highest V(max)/K(m)). The enzyme also had medium-chain fatty acid:CoA ligase activity. HXM-B had the majority of the hexanoate activity and hexanoate was its best substrate. It was, however, also active toward many xenobiotic carboxylic acids. Comparison of these two human XM-ligases with the previously characterized bovine XM-ligases indicated that they were kinetically distinct. When assayed with benzoic acid as substrate, both HXM-A and HXM-B had an absolute dependence on either Mg(2+) or Mn(2+) for activity. Further, addition of monovalent cation (K(+), Rb(+), or NH(4)(+)) stimulated HXM-A activity by >30-fold and HXM-B activity by 4-fold. For both forms, activity toward straight-chain fatty acids was stimulated less by K(+) than was activity toward benzoate or phenylacetate. A 60 kDa short-chain fatty acid:CoA ligase was also isolated. It had activity toward propionate and butyrate, but not acetate, hexanoate or benzoate. The K(m)(app) values were high but similar for propionate and butyrate (285 microM and 250 microM, respectively) but the V(max)(app) was nearly 6-fold greater with propionate as substrate. While the K(m) values are somewhat high, the enzyme is still more efficient with these substrates than either of the XM-ligases.

Effect of dehydroepiandrosterone (DHEA) on intestinal mucosal immunity in young adult and aging rats.

The present study assesses the effectiveness of oral DHEA on the intestinal mucosal immune response in aging rats. Young adult (6 months) and aging (21 months) female rats received powdered rat chow with or without 0.2% DHEA for 23 days. The animals were immunized intraduodenally with either cholera toxin (CTx) or vehicle alone and boosted two weeks later. Seven days after boosting, serum, bile, small intestinal tissue, and liver were collected for analysis. Anti-CTx IgA antibody titers were measured in serum and bile and the concentration of anti-CTx antibody containing cells (ACCs) in the small intestinal lamina propria and liver were determined by quantitative immunohistochemistry. Intergroup comparisons indicated that there was only one significant difference in serum and none in bile anti-CTx IgA titers between CTx-immunized animals fed DHEA or the diet alone. Immunohistochemical analysis determined that the density and distribution patterns of ACCs within the lamina propria were unaffected by DHEA. Both DHEA-treated and control young immunized animals exhibited similar numbers of ACCs. Only 40% of the aging rats responded to intraduodenal immunization with CTx, as determined by the presence of ACCs in the intestine, regardless of the presence or absence of DHEA in the diet. These data suggest that DHEA in the diet does not enhance the intestinal mucosal immune response to intraduodenal CTx in either young adult or aging rats.

Determination of the sequence of the arylacetyl acyl-CoA:amino acid N-acyltransferase from bovine liver mitochondria and its homology to the aralkyl acyl-CoA:amino acid N-acyltransferase.

The arylacetyl acyl-CoA:amino acid N-acyltransferase was previously purified to homogeneity from bovine liver mitochondria, and partial sequences were obtained for peptides generated by cyanogen bromide cleavage of the enzyme. One of these sequences was used to design an oligonucleotide probe that was utilized to screen a bovine liver cDNA library. Several clones were isolated and sequenced, and the sequence is given. The cDNA contains 346 bases of 5'-untranslated region and 439 bases of 3' untranslated region. The cDNA codes for an enzyme containing 295 amino acid residues. The sequence gives a molecular weight for the enzyme of 38,937, which is larger than that previously estimated for the functional enzyme, which suggests the existence of ca. 5 kDA of signal peptide. The molecular weight of the enzyme was slightly lower than that of the aralkyltransferase, which was previously determined to be 39,229. Comparison of this sequence with that which we previously obtained for the aralkyltransferase indicated that the coding regions were of identical length and that the sequences were 78% homologous. However, the 5' and 3' untranslated regions had less than 29% homology. The derived amino acid sequences were 71% homologous. This high homology indicates a common origin for the two enzymes. There are, however, significant differences in amino acid compositions, and these are discussed.

Characterization of the monovalent and divalent cation requirements for the xenobiotic carboxylic acid: CoA ligases of bovine liver mitochondria.

The XL-I, XL-II and XL-III forms of xenobiotic/medium-chain fatty acid: CoA ligase were found to be inactive toward benzoate in the absence of either monovalent or divalent cations. The absolute requirement for monovalent cation was satisfied by either K+, Rb+, or NH4+. Na+ only supported a very low rate. Varying the nature of the anion had only a minor effect. For XL-I and XI-II, the optimum concentration of K+ was 50 mM; higher (physiologic) concentrations led to a decrease in activity. K+ did not inhibit XL-III. The absolute requirement for divalent cation was satisfied by Mg2+ or Mn2+, or to a lesser extent by Co2+ or Fe2+. For the XL-I and XL-II, excess uncomplexed Mg2+ or Mn2+ decreased the rate; the optimum concentration of Mn2+ was approximately the same as the concentration of ATP in the assay, and the optimum concentration of Mg2+ was approximately double the concentration of ATP in the assay. This is consistent with the concept that the divalent cation is required to complex with ATP and with the known stability constants for the ATP complexes of these two divalent cations. XL-III was not inhibited by uncomplexed divalent cations. Uncomplexed ATP was a moderate inhibitor of XL-I and XL-II, and a weak inhibitor of XL-III. The data indicate that in vivo benzoate conjugation is K+ and Mg2+ dependent, and that the cation effects are complex and differ for XL-I and XL-II as compared with XL-III.

Development of a radiolabeled ATP assay for carboxylic acid:CoA ligases and its use in the characterization of the xenobiotic carboxylic acid:CoA ligases of bovine liver mitochondria.

A radiolabeled ATP assay was developed for measuring carboxylic acid:CoA ligase activity. The assay was designed to measure the formation of [gamma-33P]pyrophosphate from [gamma-33P]ATP in the course of the reaction. The assay was linear with protein concentration, and rates as low as 1 pmol/min were measurable. Rates determined with this assay were in agreement with rates determined with [14C]carboxylic acids. The assay was used to characterize the substrate specificity of the XL-I, XL-II, and XL-III ligases from bovine liver mitochondria. Forty carboxylic acids were tested for activity. The enzymes differed in their substrate specificities with XL-I and XL-II being the most similar and XL-III having the broadest specificity. This study has uncovered 19 new carboxylic acids that are substrates for these enzymes.

Purification and partial sequencing of the XL-I form of xenobiotic-metabolizing medium chain fatty acid:CoA ligase from bovine liver mitochondria, and its homology with the essential hypertension protein.

The XL-I form of xenobiotic-metabolizing medium-chain fatty acid:CoA ligase was purified to apparent homogeneity from bovine liver mitochondria. The procedure gave rise to a 435-fold increase in specific activity, with a yield of 12%. The enzyme eluted from a gel filtration column as a single peak with an apparent molecular weight of ca. 55,000. It ran as a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) which had an apparent molecular weight of 62 kDa. N-Terminal sequence analysis of the enzyme gave no sequence, which indicates a blocked N-terminus. To obtain sequence data, the enzyme was cleaved at methionine residues using CNBr. The resulting peptides were separated by SDS-PAGE. The cleavage pattern revealed two large peptides with molecular weights of ca. 10,000 and 12,000, plus several smaller peptides of lesser intensity. The 10 kDa and 12 kDa peptides were electroblotted onto Trans-Blot, and then sequenced directly from the blot. The N-terminal sequences of these two peptides are presented. When compared with known sequences it was discovered that these two peptides both have high homology with regions of the SA essential hypertension protein. This suggests a role for a carboxylic acid:CoA ligase in the control of high blood pressure.

Effect of bile acids on the growth and differentiation of cultured human keratinocytes.

Sixteen bile acids were tested at a concentration of 50 microM for their effect on growth of preconfluent cultures of proliferating keratinocytes. Monohydroxy bile acids (3-beta-hydroxy-delta 5-cholenate and lithocholate) stopped the accumulation of protein, dramatically decreased DNA content and led to a 90% loss of cell viability. Deoxycholate (DOC) and chenodeoxycholate inhibited protein accumulation and blocked increases in DNA content, without affecting cell viability. DOC had measurable growth-retarding effects at concentrations as low as 15 microM, and lithocholate at 2 microM. The glycine and taurine conjugates of bile acids were significantly less effective inhibitors of growth, as was the sulfate conjugate of lithocholic acid. DOC and chenodeoxycholate at 25-50 microM enhanced the differentiation-specific increase in particulate transglutaminase activity by as much as 80% over 6 days. Lithocholate had a similar effect at 5 microM. Glycine and taurine conjugates of DOC had a similar effect but were less potent; tauroursodeoxycholate had no effect. The data indicate that bile acids, at levels seen in obstructive biliary disease, can lead to a down-regulation of keratinocyte growth and an up-regulation of differentiation.

Determination of the sequence of the aralkyl acyl-CoA:amino acid N-acyltransferase from bovine liver mitochondria.

The aralkyl acyl-CoA:amino-acid N-acyltransferase was previously purified to homogeneity from bovine liver mitochondria. The N-terminal amino-acid sequence and sequences obtained by cyanogen bromide cleavage of the enzyme were used to design oligonucleotide probes that were used to screen a bovine liver cDNA library. Several clones were isolated and sequenced, and the sequence is given. The cDNA contains 126 bases of 5'-untranslated region and 188 bp of 3' untranslated region. The cDNA codes for an enzyme containing 295 amino-acid residues. The sequence gives a molecular weight for the enzyme of 39,229, which is larger than previously estimated. The amino-acid composition of the enzyme, based on this sequence, is in agreement with the previously obtained amino-acid analysis on the purified kidney enzyme.

Interaction of salicylate and ibuprofen with the carboxylic acid: CoA ligases from bovine liver mitochondria.

Neither salicylate nor ibuprofen was a substrate or inhibitor of the long-chain fatty acid:CoA ligase. In contrast, all three xenobiotic-metabolizing medium-chain fatty acid:CoA ligases (XL-I, XL-II, and XL-III) had activity toward salicylate. The K(m) value for salicylate was similar for all three forms (2 to 3 microM), but XL-II and XL-III had higher activity at Vmax. For ibuprofen, only XL-III catalyzed its activation, and it had a K(m) for ibuprofen of 36 microM. Studies of salicylate inhibition of XL-I, XL-II, and XL-III revealed that it inhibited the benzoate activity of all three forms with K1 values of ca. 2 microM, which is in agreement with the K(m) values obtained with salicylate as substrate. Kinetic analysis revealed that salicylate conjugation by all three forms is characterized by substrate inhibition when salicylate exceeds ca. 20 microM. Substrate inhibition was more extensive with XL-I and XL-III. Previous work on the ligases employed assay concentrations of salicylate in the range of 0.1 to 1.0 mM, which are clearly inhibitory, particularly toward XL-I and XL-III. Thus, activity was not properly measured in previous studies, which accounts for the fact that salicylate conjugation was only found with one form, which is most likely XL-II since it has the highest Vmax activity and shows the least amount of substrate inhibition. Studies with ibuprofen indicated that it inhibited XL-I, XL-II, and XL-III, with KI values being in the range of 75-125 microM. The short-chain ligase was inhibited by both salicylate and ibuprofen with KI values of 93 and 84 microM, respectively. It was concluded that pharmacological doses of salicylate, but not ibuprofen, will affect the metabolism of medium-chain fatty acids and carboxylic acid xenobiotics and that the previously described mitochondrial ibuprofen:CoA ligase activity is attributable to XL-III.

Isolation from bovine liver mitochondria and characterization of three distinct carboxylic acid: CoA ligases with activity toward xenobiotics.

A mitochondrial freeze/thaw lysate was fractionated on a DEAE-cellulose column into four distinct acyl-CoA ligase fractions. First to elute was a 50 kDa short-chain ligase that activated only short-chain fatty acids. Next to elute were three ligases that had activity toward both medium-chain fatty acids and xenobiotic carboxylic acids; these were termed xenobiotic/medium-chain ligases (X-ligases) and labeled XL-I, XL-II, and XL-III, respectively, based on order of elution. The molecular weight of X-ligases I, II, and III were ca. 55,000, 55,500 and 53,000, respectively. Form XL-III showed no pH optimum; the rate increased steadily with pH beginning from pH 7.0. XL-I and XL-II showed the same behavior with benzoate as substrate, but with medium-chain fatty acids, both forms had a pH optimum at 8.8. The three X-ligases differed in substrate specificity. XL-I was the predominant nicotinic acid activating form and had the lowest Km for benzoate. Form XL-II was the only form with measurable salicylate activity, although it was extremely low. XL-III was the only 2,4,6,8-decatetraenoic acid activating form and also was the predominant medium-chain fatty acid-activating form. By comparison of substrate specificities, it was concluded that the two previously reported ligase preparations were mixtures of the three forms. When the ligase rates were compared to previously determined N-acyltransferase rates toward benzoyl-CoA and phenylacetyl-CoA, the data showed that ligase activities are 100-fold lower, and thus the ligase is rate limiting for the conjugation of both of these xenobiotics.

Inhibition of bile acid conjugation by cyclosporin A.

Each of the two steps involved in bile acid conjugation was tested in vitro for its sensitivity to inhibition by cyclosporin A (CsA). Bile acid-CoA: glycine/taurine N-acyltransferase, the enzyme which catalyzes the second step, was tested and found to be insensitive to inhibition by 20 microM CsA. Bile acid:CoA ligase, the enzyme which catalyzes the first step, was found to be inhibited by 25% at 10 microM CsA in the standard assay. The inhibition was competitive vs. bile acid and noncompetitive vs. ATP, and uncompetitive vs. CoA. CsA was also found to interfere with the divalent cation requirement of the enzyme at low concentrations of Mg2+ the maximum inhibition was 70%. The maximum inhibition obtainable at physiologic Mg2+ concentration was 40%. The extent of inhibition was presumably limited by the insolubility of CsA. At concentrations of CsA reached in vivo during drug therapy, CsA can be expected to significantly inhibit bile acid conjugation.

Differentiation-induced enhancement of the ability of cultured human keratinocytes to suppress oxidative stress.

Human keratinocytes in culture were harvested at different stages of differentiation. Both the level of antioxidants and the response of cells to oxidative stress were measured as a function of growth and differentiation. As the keratinocyte cultures became confluent and began to differentiate, the cellular levels of glutathione, glutathione peroxidase, glutathione S transferase, and glucose-6-phosphate dehydrogenase increased. This higher level of antioxidants was maintained until the cells began to lose viability. Further, as the keratinocyte cultures began to differentiate, they became more resistant to the toxic effect of cumene hydroperoxide in terms of both of the rate of loss of cell mass and total glutathione and of the rate of decline in the activity of oxidation-sensitive enzymes. To determine how tightly the observed effects are linked to the calcium-dependent aspects of differentiation and to rule out effects related to time in culture, the cells were switched from 1.2 mM Ca++ to 0.03 mM Ca++ to suppress Ca(++)-dependent differentiation. After 4 d, these cells were then treated with 0.5 mM cumene hydroperoxide. The switch to 0.03 mM Ca++ blocked the normal increases in both glutathione peroxidase and glucose-6-phosphate dehydrogenase activities. Further, cells in 0.03 mM Ca++ had reduced resistance to cumene hydroperoxide relative to cells cultured for the same length of time in 1.2 mM Ca++. This indicates that there is a differentiation-associated, Ca(++)-specific increase in both the level of antioxidants and in tolerance to organic hydroperoxides.

Determination of the mechanism of reaction for bile acid: CoA ligase.

The reaction of cholic acid, CoA and ATP to yield cholyl-CoA was investigated by kinetic analysis of the reaction as catalysed by guinea pig liver microsomes. The enzyme has an absolute requirement for divalent cation for activity so all kinetic analyses were carried out in excess Mn2+. A trisubstrate kinetic analysis was conducted by varying, one at a time ATP cholate and CoA. Both ATP and cholate gave parallel double reciprocal plots versus CoA, which indicates a ping-pong mechanism with either pyrophosphate or AMP leaving prior to the binding of CoA. Addition of pyrophosphate to the assays changed the parallel plots to intersecting ones; addition of AMP did not. This indicates that pyrophosphate is the first product. The end-product, AMP, was a competitive inhibitor versus ATP, as was cholyl-CoA at saturating concentrations of cholate. Both AMP and cholyl-CoA were uncompetitive inhibitors versus CoA. Based on this information, it was concluded that the reaction follows a bi uni uni bi ping-pong mechanism with ATP binding first, and with the release of the final products, AMP and cholyl-CoA, being random. CoA showed substrate inhibition at high but non-saturating concentrations and this inhibition was competitive versus ATP, which is consistent with the predicted ping-pong mechanism. The ability of cholyl-CoA, but not cholate or CoA, to bind with high affinity to the free enzyme was suggestive of a high affinity of the enzyme for the thioester link.

Dual role of divalent cations in the bile acid:CoA ligase catalyzed reaction.

The role of divalent cations in the bile acid:CoA ligase catalyzed reaction of cholic acid, CoA and ATP to yield cholyl-CoA was investigated using guinea pig liver microsomes as the source of enzyme. EDTA treatment completely eliminated activity indicating an absolute requirement for divalent cation for enzyme activity. Analysis of this requirement revealed that it was twofold. First, the data suggested that ATP which was not complexed with a divalent cation did not appreciably bind to the enzyme and thus a divalent cation complex of ATP is the form of ATP that is the substrate for the enzyme. Further, this was shown to be the basis for the absolute requirement for divalent cation in the reaction. In addition, analysis revealed that there is a secondary site which binds divalent cations with relatively low affinity, and results in a rate enhancement. Binding at this secondary site is estimated to increase the rate by greater than 60%.

Characterization of the acyl-CoA:amino acid N-acyltransferases from primate liver mitochondria.

The acyl-CoA:amino acid N-acyl-transferases were partially purified from human liver mitochondria. The aralkyl transferase (ArAlk) had glycine conjugating activity toward the following compounds: benzoyl-CoA > butyryl-CoA, salicylyl-CoA > heptanoyl-CoA, indoleacetyl-CoA. Its kinetic properties and responses to salt were very similar to those of bovine ArAlk. Further, its molecular weight was found to be similar to that of the bovine enzyme, in contrast to reports from other laboratories. Thus, it was concluded that the human and bovine ArAlk are not significantly different. The human arylacetyl transferase (AAc) had glutamine conjugating activity toward phenylacetyl-CoA, but only 3-5% as much activity toward indoleacetyl-CoA or 1-naphtylacetyl-CoA, respectively. While this was similar to the bovine AAc, the two forms differed in several respects. First, the human liver AAc was insensitive to salts. Second, glycination of phenylacetyl-CoA by human AAc could only be detected at a high concentration of glycine (50 mM), and the rates were < 2% of the rate of glutamination. In contrast, glycine conjugation predominates with bovine AAc. Kinetic analysis of the glutamination of phenylacetyl-CoA by human AAc revealed a KD for phenylacetyl-CoA of 14 microM and a Km for glutamine of 120 mM. These values indicate that the human AAc is not more efficient at glutamination than the AAc from bovine liver. An AAc was purified from rhesus monkey liver and found to have similar kinetic constants to the human form. This indicates that nonprimate enzymes do not have a defect in glutamine conjugation.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical modification of bile acid: CoA ligase.

The effect of chemical modification of bile acid:CoA ligase on its enzymatic activity was examined. Reagents which modify tyrosine and carboxyl groups did not affect the activity of either the purified enzyme or the enzyme in its native microsomal environment. The modification of arginine residues with either diacetyl or phenylglyoxal resulted in a loss of activity for both the purified and microsomal forms of the enzyme. ATP was able to protect the enzyme from inactivation. Neither cholate nor CoA were able to alter the time-course of inactivation. The sulfhydryl reagent N-ethylmaleimide (MalNEt) produced a biphasic effect on both the purified and microsomal forms of the enzyme. At short reaction times ligase activity increased, but further reaction lead to nearly complete inactivation. With the purified enzyme, ATP increased the extent of activation by MalNEt and decreased the rate of inactivation. With microsomes, ATP did not affect the extent of activation by MalNEt, but did slow the rate of inactivation. For both the purified and microsomal forms cholate provided no protection. Treatment of both forms of the enzyme with the sulfhydryl reagent iodoacetic acid produced a similar biphasic activation/inactivation of the ligase. It was hypothesized that modification of a fast-reacting cysteine leads to activation while a slower-reacting cysteine leads to inactivation. This latter cysteine appeared to be in the ATP-binding site on the enzyme.