Chlorogenic acid and hydroxynitrobenzaldehyde: new inhibitors of hepatic glucose 6-phosphatase.

We have studied the interactions of chlorogenic acid (CHL) and 2-hydroxy-5-nitrobenzaldehyde (HNB) with the components of the rat hepatic glucose 6-phosphatase (Glc-6-Pase) system. CHL and HNB are competitive inhibitors of glucose 6-phosphate (Glc-6-P) hydrolysis in intact microsomes with Ki values of 0.26 and 0.22 mm, respectively. CHL is without effect on the enzyme of fully disrupted microsomes or the system inorganic pyrophosphatase (PPiase) activity. HNB is a potent competitive inhibitor of the system PPiase activity (Ki = 0.56 mm) and a somewhat weaker noncompetitive inhibitor of enzyme activity (Ki = 2.1 mm). These findings indicate CHL binds to T1, the Glc-6-P transporter, and HNB inhibits through interaction with both T1 and T2 the phosphate (Pi)-PPi transporter. Binding of CHL and HNB is freely reversible. However, the inhibition of both PPiase and Glc-6-Pase by HNB becomes irreversible following incubation of HNB-exposed microsomes with 2.5 mm sodium borohydride, indicating that inhibition involves the formation of a Schiff base. The presence of CHL effectively protects T1, but not T2, against the irreversible inhibition by HNB. In contrast, PPi and Pi are effective in protecting T2, but not T1. This is the first report describing an effective inhibitor of the system PPiase activity (T2). CHL is the most specific T1 inhibitor described to date.

Chlorogenic acid and synthetic chlorogenic acid derivatives: novel inhibitors of hepatic glucose-6-phosphate translocase.

The enzyme system glucose-6-phosphatase (EC 3.1.3.9) plays a major role in the homeostatic regulation of blood glucose. It is responsible for the formation of endogenous glucose originating from gluconeogenesis and glycogenolysis. Recently, chlorogenic acid was identified as a specific inhibitor of the glucose-6-phosphate translocase component (Gl-6-P translocase) of this enzyme system in microsomes of rat liver. Glucose 6-phosphate hydrolysis was determined in the presence of chlorogenic acid or of new synthesized derivatives in intact rat liver microsomes in order to assess the inhibitory potency of the compounds on the translocase component. Variation in the 3-position of chlorogenic acid had only poor effects on inhibitory potency. Introduction of lipophilic side chain in the 1-position led to 100-fold more potent inhibitors. Functional assays on isolated perfused rat liver with compound 29i, a representative of the more potent derivatives, showed a dose-dependent inhibition of gluconeogenesis and glycogenolyosis, suggesting glucose-6-phosphatase as the locus of interference of the compound for inhibition of hepatic glucose production also in the isolated organ model. Gl-6-P translocase inhibitors may be useful for the reduction of inappropriately high rates of hepatic glucose output often found in non-insulin-dependent diabetes.

Kinetic studies of human and rat neutrophil lysoPAF acetyltransferase using lysoPAF and dansyllysoPAF as substrates.

Enzyme kinetic studies of lysoPAF acetyltransferase from microsomal preparations of human and rat neutrophils were carried out using lysoPAF or dansyllysoPAF as substrate. With the human enzyme, incomplete conversion of the substrate into the product was observed at 37 degrees C with both substrates. The acetyltransferase was inactivated at 37 degrees C in the absence of substrate with a half-life of 7.5 min. However, the initial rate of product formation under the assay conditions was linear up to 10 min. Both enzymes were optimally active at 40 microM concentration with either substrate, but enzyme activity was inhibited at higher substrate levels. At a constant substrate concentration (40 microM), the Km (microM) and Vmax (nmol product/min/mg protein) values for the human acetyltransferase, with respect to acetyl-CoA were 132 and 23.1, respectively, with lysoPAF as substrate, and 105 and 26.7, respectively, when dansyllysoPAF was used. The Km and Vmax values for the rat enzyme were 105 and 6.5, respectively, with lysoPAF as substrate, and 120 and 5.4, respectively, when dansyllysoPAF was used. Under our standard conditions, lysoPAF required 1 mg of BSA per mL in the assay, whereas full activity of both enzymes was seen with dansyllysoPAF even in the absence of BSA. The results show that dansyllysoPAF can replace lysoPAF in the assay without any significant changes in kinetic parameters.

Fluorophore-labeled ether lipids: substrates for enzymes of the platelet-activating factor cycle in peritoneal polymorphonuclear leukocytes.

Cell-free preparations of ionophore-stimulated peritoneal rat polymorphonuclear neutrophils (PMNs) incubated with 1-(N-dansyl-11-amino-1-undecyl)-sn-glycerol-3-phosphorylcholine (dansyllyso-PAF) converted this fluorescent lyso ether lipid into two different classes of products. In the absence of acetyl-CoA 1-(N-dansyl-11-amino-1-undecyl)-2-long chain acyl-sn-glycerol-3-phosphorylcholine (dansylalkyl-2-acyl-GPC) was the only identified new fluorescent phospholipid. In the presence of acetyl-CoA an additional new product, 1-(N-dansyl-11-amino-1-undecyl)-2-acetyl-sn-glycerol-3-phosphorylcholine (dansyl-PAF), was formed. The formation of dansyl-PAF in PMN homogenates was only transient with a maximum after about 4 min. When PMN homogenates were incubated with dansyl-PAF the formation of dansyllyso-PAF was observed prior to the formation of dansyl-2-acyl-GPC. Thus, our data indicate that enzymatically formed dansyl-PAF is completely remodeled into dansylalkyl-2-acyl-GPC by the sequential action of PAF acetylhydrolase and CoA-independent transacylase. These results demonstrate that peritoneal rat PMNs contain lyso-PAF acetyltransferase, PAF acetylhydrolase, and CoA-independent transacylase and that fluorophore-labeled ether lipids provide an easy means to assay enzymes which catalyze important enzymatic reactions involved in the biosynthesis and remodeling of platelet-activating factor.

Improved screening for beta-lactam antibiotics. A sensitive, high-throughput assay using DD-carboxypeptidase and a novel chromophore-labeled substrate.

The very sensitive and specific method for the detection of beta-lactam antibiotics using DD-carboxypeptidase (DDCase) from Actinomadura strain R39 has been improved to meet the requirements of a high-throughput beta-lactam screening from culture broths of microorganisms. The method is based on a novel chromophor-labeled substrate N alpha-acetyl-N epsilon-4-(7-nitrobenzofurazanyl)-L-lysyl-D-al anyl-D-alanine (ANLA2) which is converted by DDCase into ANLA1 with only one D-alanine residue left. Both compounds are intensely yellow as well as highly fluorescent and can be separated by thin-layer chromatography. This allows easy determination of residual DDCase activity after reaction with beta-lactams by simple visual inspection of chromatograms. Also, many assays can be run at a time without sophisticated instrumentation. Details of the method as well as some results of a beta-lactam screening performed with this type of assay are described.

Mode of action of the macrolide-type antibiotic, chlorothricin. Effect of the antibiotic on the catalytic activity and some structural parameters of pyruvate carboxylases purified from rat and chicken liver.

The macrolide-type antibiotic chlorothricin inhibits pyruvate carboxylases purified from rat liver, chicken liver and Azotobacter vinelandii. Under standard assay conditions the concentration of chlorothricin required for half-maximal inhibition of oxalacetate synthesis is 0.26 mM (rat liver), 0.12 mM (chicken liver), and 0.5 mM (Azobacter vinelandii). Inhibition by chlorothricin appears non-competitive in character when measured as a function of the concentration of the substrates of the pyruvate carboxylase reaction as well as of CoASAc and Mg2+. This pattern of inhibition suggests that this antibiotic interacts at unique sites on chicken and rat liver pyruvate carboxylase which are distinct from both the catalytic and activator sites. Interaction of chlorothricin with the two vertebrate liver pyruvate carboxylases differs from the effect exerted by this antibiotic on pyruvate carboxylase purified from Azotobacter vinelandii. A sigmoidal relationship between initial velocity and inhibitor concentration is observed for the vertebrate enzymes under most conditions whereas a hyperbolic profile characterizes the concentration dependence of inhibition of the Azotobacter vinelandii enzyme by chlorothricin. In the case of rat liver pyruvate carboxylase chlorothricin does not alter the extent of cooperativity in the relationship between initial rate and CoASAc concentration. However, a small but significant increase of the Hill coefficient from a value of 2.7 in the absence of antibiotic to that of 3.3 in the presence of 0.5 mM chlorothricin is observed for chicken liver pyruvate carboxylase. Chlorothricin decreases the rate of inactivation observed when rat liver pyruvate carboxylase is incubated with trinitrobenzenesulfonate and when chicken liver pyruvate carboxylase is incubated at 2 degrees C. The maximal decrease in inactivation observed in the presence of saturating concentrations of antibiotic is 50% for cold inactivation of the chicken liver enzyme and 60% for inactivation of the enzyme from rat liver by trinitrobenzenesulfonate. In both cases a sigmoidal relationship is observed between inactivation rate and chlorothricin concentration. These data as well as the initial rate studies suggest that multiple interacting sites for this antibiotic are present on the vertebrate liver pyruvate carboxylases. The occupancy of these sites appears to cause significant distortion of both the catalytic and the activator sites.

Chlorothricin, and inhibitor of porcine-heart malate dehydrogenases, discriminating between the mitochondrial and cytoplasmic isoenzyme.

The macrolide-type antibiotic chlorothricin was found to inhibit both the mitochondrial and the cytoplasmic form of pig heart malate dehydrogenase. Steady-state kinetic measurements revealed that in the direction of oxalacetate reduction chlorothricin is competitive with respect to NADH and non-competitive with respect to oxalacetate. Both the variation of initial velocity with NADH concentration in the presence of antibiotic, and, at several fixed levels of NADH, the variation of initial velocity with chlorothricin concentration deviates from the classical Michaelis-Menten relationship for the two isoenzymes. Since, despite the very similar kinetic behaviour of the mitochondrial and cytoplasmic species of malate dehydrogenase, the concentration of chlorothricin required for half-maximal inhibition of the two enzymes differs by more than a factor of 10 (the mitochondrial isoenzyme being more susceptible to inhibition), it is concluded that the NADH binding sites of the mitochondrial and cytoplasmic form of malate dehydrogenase from pig heart are different.