ABSTRACT
The purine phosphoribosyltransferases of Crithidia fasciculata were identified and some of their properties described. The organism possesses three separate enzymes for the production of AMP, IMP, and GMP. The evidence for this comes from the observed differences in elution patterns from gel filtration columns, differences in heat sensitivity, and especially the clear separation of hypoxanthine phosphoribosyltransferase from guanine phosphoribosyltransferase by affinity chromatography on GMP-agarose. APRTase is activated most efficiently by Zn++, whereas HPRTase and GPRTase are activated most effectively by Co++. In no case did the product mononucleotides produce strong inhibition of the transferase activities.
Subject(s)
Adenine Phosphoribosyltransferase/metabolism , Crithidia/enzymology , Hypoxanthine Phosphoribosyltransferase/metabolism , Pentosyltransferases/metabolism , Animals , Cell-Free System , Cobalt/pharmacology , Enzyme Activation , Hot Temperature , Magnesium/pharmacology , Manganese/pharmacology , Substrate Specificity , Zinc/pharmacologyABSTRACT
The capacity for the rapid transport of purine bases by Crithidia fasciulata is found only in cells starved for purines. Cells grown in complete medium transport poorly. Rapid transport capability appears and then disappears during growth of purine-depleted cultures. This rapid transport appears to occur by a process of mediated diffusion. Two mechanisms are involved, one of low velocity and high affinity, the other of high velocity and low affinity. Accumulation of the bases within the cell occurs by their rapid conversion to ribonucleotides by phosphoribosyltransferases.
Subject(s)
Adenine/metabolism , Eukaryota/metabolism , Guanine/metabolism , Hypoxanthines/metabolism , Adenine Nucleotides/metabolism , Animals , Azides/pharmacology , Biological Transport , Cyanides/pharmacology , Dinitrophenols/pharmacology , Guanine Nucleotides/metabolism , KineticsABSTRACT
The adenine analog 4-aminopyrazolo(3,4-d)pyrimidine inhibits the growth of the kinetoplastid (trypanosomatid) flagellate Crithidia fasciculata. This inhibition is partially overcome only by adenine (of a number of purines tested), with an inhibition index of 0.025. More effective reversal of inhibition is obtained with any of a number of naturally occurring pyrimidine compounds, up to a concentration of 0.18 mM. Higher concentrations of pyrimidines or addition of guanine, as well as adenine and uracil, to the medium increases inhibition. The analog (presumably as the ribonucleotide) was found not to be inhibitory to any enzyme of the pyrimidine biosynthetic pathway that could be tested. It is suggested that the analog competes with adenine for adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.7), is converted to a ribonucleotide, and is incorporated into nucleic acid.
Subject(s)
Adenine/analogs & derivatives , Eukaryota/growth & development , Adenine/pharmacology , Adenine Phosphoribosyltransferase/metabolism , Animals , Dose-Response Relationship, Drug , Eukaryota/drug effects , Eukaryota/enzymologyABSTRACT
A study of the enzymes of the orotate biosynthetic pathway in the kinetoplasid flagellate Crithidia fasciculata has revealed a number of differences between them and those of other organisms, either prokaryotic or eukaryotic. Carbamyl phosphate synthesis could not be demonstrated in cell-free extracts. However, the incorporation of both CO2 and the ureide carbon of citrulline into pyrimidines occurs in growing cells, the latter predominating over the former. The aspartate transcarbamylase of the flagellate has properties which are similar to those of this enzyme as it occurs in mammals rather than other microorganisms. Two enzymes, dihydroorotate synthetase and dihydroorotate hydrolase, are present, the former being responsible for the conversion of carbamylasparate to dihydroorotate. Dihydroorotate hydroxylase, a soluble enzyme requiring a reduced pteridine as a cofactor, converts dihydroorotate to orotate. The hydroxylase is inhibited by orotate, but not by pyrimidine or purine ribonucleotides. Thus orotate serves to control its own biosynthesis.
