A Study of some Enzymes in Extracts of a Non-Capsulated Strain of Pneumococcus Concerned with Uridine Pyrophosphoglycosyl Metabolism

Abstract

Disruption of cells in a Mickle disintegrator in the presence of n-octanol is described as a method for obtaining extracts from the non-capsulated strain R 19 of streptococcus pneumoniae [Diplococcus pneumoniae], derivedfrom a strain of capsular type II. Such extracts were fractionated with ammonium sulphate in the presence of Celite and the distribution in the various fractions so obtained of a number of enzymes concerned with uridine pyrophosphoglycosyl metabolism was studied. Celite in the presence of ammonium sulphate was effective in removing organic pyrophosphatases, phosphatases acting upon uridine pyrophosphoglycosyl compounds and a reduced tri-phosphopyridine nucleotide (TPNH) oxidase. A reduced diphosphopyridine nucleotide (DPNH) oxidase was concentrated by fractionation with ammonium sulphate and the uridyltransferases acting upon uridine pyrophosphoglucose (UPPG) and uridine pyrophosphoacetylglucosamine (UPPAG) were obtained as partially separated entities. Uridine pyrophosphogalactose (UPPgalactose) and guanosine pyrophosphomannose (GPPM) were not pyrophosphorylysed by the unfractionated material or by any of the fractions obtained. UPPgalactose-4-epimerase and phosphogalactosyl uridyl transferase were present in the extracts under study, these enzymes being involved in the conversion of galactose-1-phosphate to glucose-1-phosphate. Such extracts also converted mannose to glucose phosphate without the participation of UPPG. Evidence is provided for the presence of a diphosphopyridine (DPN) linked UPPG dehydrogenase which is capable of forming uridine pyrophosphoglucuronic acid (UPPGA) from UPPG. The UPPGA so formed was separated by ion-exchange chromatography. The extracts used did not catalyse the epimerization of UPPGA to uridine pyrophosphogalacturonic acid (UPP galacturonic acid) as do extracts of a strain of capsulated type I pneumococcus. An examination of the cellular content of uridine nucleotides in relation to the bacterial growth curve showed that UPPGA did not accumulate in significant amounts until the organisms of this strain had reached the stationary phase.