ABSTRACT
The mammalian purple acid phosphatases (also called tartrate-resistant acid phosphatases) are expressed primarily in actively resorbing osteoclasts and activated macrophages. The enzymes are characterized by the presence of a binuclear iron center at the active site. Recent studies on transgenic mice lacking purple acid phosphatase implicate the osteoclast enzyme in both bone resorption and bone mineralization. To characterize the mammalian enzymes in more detail, particularly with respect to their substrate specificity at the low pH of the osteoclastic resorptive space (2.5-3), we have purified the recombinant human and mouse enzymes from baculovirus-infected insect cells. The properties of the recombinant mouse enzyme are compared with those of the nonrecombinant enzyme isolated from mouse spleen. The kinetics of hydrolysis of the substrates p-nitrophenyl phosphate, phosphotyrosine, and pyrophosphate and a phosphotyrosyl peptide by the recombinant human and mouse enzymes and the nonrecombinant mouse and pig enzymes were analyzed. For all the enzymes the ratio k(cat)/Km was typically approximately 10(6) M(-1) s(-1) and was higher at pH 2.5 than at 4.9. The increase was attributable to a large decrease in Km at the lower pH value. The results indicate that the enzyme exhibits high catalytic efficiency toward substrates such as pyrophosphate and acidic phosphotyrosine-containing peptides, particularly at low pH values typical of the bone resorptive space. The implications of the results for the physiological function of the enzyme are discussed.
Subject(s)
Acid Phosphatase/biosynthesis , Isoenzymes/biosynthesis , Acid Phosphatase/genetics , Animals , Humans , Isoenzymes/genetics , Kinetics , Mice , Nucleopolyhedroviruses/genetics , Oxidation-Reduction , Peptide Fragments/chemistry , Recombinant Proteins/biosynthesis , Sequence Analysis , Species Specificity , Spodoptera/cytology , Spodoptera/virology , Swine , Tartrate-Resistant Acid PhosphataseABSTRACT
The effect of acute ethanol administration on pentylenetetrazole-induced c-fos expression in rat brain was studied. Pentylenetetrazole induced the rapid and transient expression of c-fos mRNA in rat brain. Maximal induction at a dose of 30 mg/kg was detected within 30 min and persisted for 60 min. Thereafter c-fos gene expression decreased to control levels by 180 min. No increase in c-fos mRNA was evident at doses of pentylenetetrazole < or = 20 mg/kg, whereas maximal elevation was seen at 30 or 40 mg/kg. This action was inhibited by acute ethanol treatment (blood alcohol level > or = 100 mg/dl). Acute ethanol treatment alone had no effect on c-fos gene expression.
Subject(s)
Brain/metabolism , Ethanol/pharmacology , Gene Expression/drug effects , Genes, fos/drug effects , Neurons/metabolism , Pentylenetetrazole/toxicity , Proto-Oncogene Proteins c-fos/biosynthesis , Animals , Brain/drug effects , DNA Probes , Dose-Response Relationship, Drug , Male , Neurons/drug effects , Pentylenetetrazole/antagonists & inhibitors , Rats , Rats, WistarABSTRACT
The effect of acute ethanol administration on pentylenetetrazole-induced c-fos expression in rat brain was studied. Pentylenetetrazole induced the rapid and transient expression of c-fos mRNA in rat brain. Maximal induction at a dose of 30 mg/kg was detected within 30 min and persisted for 60 min. Thereafter c-fos gene expression decreased to control levels by 180 min. No increase in c-fos mRNA was evident at doses of pentylenetetrazole less than or equal to 20 mg/kg, whereas maximal elevation was seen at 30 to 40 mg/kg. This action was inhibited by acute ethanol treatment (blood alcohol level greater than or equal to 100 mg/dl). Acute ethanol treatment alone had no effect on c-fos gene expression.
Subject(s)
Brain/metabolism , Ethanol/pharmacology , Pentylenetetrazole/pharmacology , Proto-Oncogene Proteins/genetics , Proto-Oncogenes/drug effects , RNA, Messenger/genetics , Actins/genetics , Animals , Brain/drug effects , DNA Probes , Dose-Response Relationship, Drug , Gene Expression , Kinetics , Male , Pentylenetetrazole/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins c-fos , RNA, Messenger/drug effects , Rats , Rats, Inbred Strains , Reference ValuesABSTRACT
The relative concentrations of folylpolyglutamates of differing chain length in rat liver and the uptake of exogenous [3H]folic acid (20 microCi, 20 microgram) into liver folylpolyglutamates were examined in rats maintained on (a) standard and folate-supplemented standard diets and (b) semi-defined folate-sufficient and folate-deficient diets. Folylpolyglutamates extracted from liver were cleaved to p-aminobenzoylpolyglutamates which were separated by ion-exchange chromatography. The relative concentrations and ultimate radiolabeling of longer-chain folylpolyglutamates (six, seven and eight glutamate residues) were greatest in the livers of folate-deficient rats, whereas the intermediate-chain folylpolyglutamates (three, four and five glutamate residues) were the greatest portion of total liver folates of folate-supplemented rats. Thus, the length of the polyglutamate chain added to liver folates is inversely related to the total concentration of liver folates. These data suggest that folylpolyglutamate biosynthesis in the liver may be controlled by the liver folate concentrations. In folate insufficiency such a control mechanism would serve to enhance the affinity of folates for folate-dependent enzymes and to conserve the liver folate concentration.
