Electrophoretic and kinetic studies of human erythrocytes deficient in pyrimidine 5?-nucleotidase

Abstract

A new case of a defect in red cell pyrimidine 5'-nucleotide (P5N) activity was found in a large family from Guadeloupe in the West Indies. The propositus presented a characteristic hemolytic anemia with red cell basophilic stippling, an increased GSH level, and a shift of the peak in absorbance of nucleotide. The enzyme activity from the deficient red cells differed from that of the normal. The P5N activity of the deficient red cells was about 14% that of normal. The electrophoretic pattern of P5N activity of the deficient red cells was distinct from that of the control in terms of its Km and of the effects of pH on its maximum activity and heat stability. The significance of such differences is discussed.

Identification of thymidine nucleotidase and deoxyribonucleotidase activities among normal isozymes of 5'-nucleotidase in human erythrocytes.

“…The variations in pH optima, kinetic constants, thermostability, and electrophoretic migration reported by Rosa et al (12), Fujii et al (13), Shinohara and Tanaka (14), and Ishida et al (15,16) in other deficient cases may not be due to molecular alteration of residual PyrNase as presumed, or to a minor PyrNase isozyme as postulated by Rosa et al (12), but more likely represent characteristics of the normal dNase isozyme no longer obscured by significant PyrNase activity. Their studies employed partially purified enzyme preparations and UMP or CMP as substrate, and they found pH optima variously shifted from the 7.0-8.0 region to approximately pH 5.7-6.1 and Km(UMP) or Km(CMP) increased severalfold, consistent with our findings for the dNase isozyme.…”

Genetic basis of hemolytic anemia caused by pyrimidine 5′ nucleotidase deficiency

“…Blood transfusions are rarely necessary, and splenectomy has generally given little benefit, 1,3,4,25 though experience is limited. The enzyme deficiency has been linked to learning difficulties of variable severity in 7 patients, including 3 Peruvian siblings.…”

Electrophoretic and Kinetic Studies of a New Mutant Red Cell Pyrimidine 5'-Nucleotidase

“…After adjusting the pH of the hemolysate to 6.5, the stroma was removed by centrifugation at 10,000 g for 30 min. The clear supernatant was passed through a DEAE-cellulose (DE-52, Whatman) column balanced with the same hemolyzing buffer as described by Rosa et al [9]. Hemoglobin was elimi nated by washing the column with the starting buffer (pH 6.5) and two bed volumes of a 100 mmol/1 potas sium phosphate buffer pH 6.5 containing I mmol/1 EDTA and 2 mmol/1 2-mercaptoethanol were applied to the top of the column.…”

“…The nonheme proteins (NHP) eluted by this buffer were concentrated by ultrafiltration in a Centriflo® CF-25 system. The con centrated NHP preparation was dialyzed overnight against 200 voi of saline containing 10 mmol/1 TrisHCl, pH 8.0, and 10 mmol/1 MgCfi in order to re move the majority of the phosphate [9].…”

“…Since the first de scription of P5N deficiency by Valentine et al [1] in 1974, more than 33 individuals from 24 separate kindreds have been re ported in the literature [2][3][4][5][6], Molecular char acteristics of the normal enzyme have been studied by several authors in dialyzed hemo- moving band on electrophoresis [4,9,10,12], These molecular properties of deficient P5N mutants have given support to the hy pothesis that the absence of a major isozyme, caused by a genetic defect, might be the main mechanism of the decreased enzyme activity [9,11],…”