STUDIES ON RAT KIDNEY GAMMA-GLUTAMYLCYSTEINE SYNTHETASE AND EFFECT OF DIETARY NUCLEOTIDES ON THE IMMUNE FUNCTION OF MICE
SCHANDLE, VICKY BLAKESLEY
Doctor of Philosophy
An improved purification scheme for rat kidney (gamma)-glutamylcysteinesynthetase ((gamma)GCSase) was developed. Such a preparation yields only onemajor band and one minor band on polyacrylamide gel electrophoresis.Further, the preparation is devoid of contaminating adenylate kinaseactivity. The K(,eq) for the following reaction was estimated to be 400 atpH 8.2: Glu + (alpha)AB ('M('2+)) (gamma)Glu(alpha)AB + ADP + P(,i) The reaction mechanism of (gamma)GCSase was studied using isotope exchange at equilibrium techniques and inhibition studies. Of the four isotope exchanges measured ((alpha)AB()(gamma)Glu(alpha)AB, Glu()(gamma)Glu(alpha)AB, ATP()ADP and P(,i)()ATP) only the ATP()ADP exchange did not show a saturable effect when substrate concentrations were elevated to three times their Km values. Inhibition studies using L-methionine-S-sulfoximine (MSOX) yielded results which showed MSOX was competitive with respect to Glu, noncompetitive with respect to (alpha)AB and uncompetitive with respect to ATP. Results from both experiments are consistent with a random BC-random RQ reaction mechanism. Only ATP and ADP bind oligatorally to free enzyme. The immune function of mice was studied using two inbred strains of mice maintained on one of three diets: rodent chow, nucleotide-free (NF) diet or nucleotide-free + RNA (NFRNA) diet. Immune response towards allogeneic challenges was measured in vivo using either free allografting of caridao tissue or allogeneic spleen cell injections (popliteal lymph node weight gain assay). In both allogeneic model systems, mice maintained on the NF diet exhibited diminished immune response to the challenges relative to the responses shown by the two control groups (chow and NFRNA). A syngeneic model was also tested. A lymphoid leukemia was injected subcutaneously into the flanks of mice, and tumor growth and recipient mouse mortality were monitored. Mice maintained on a NF diet survived longer than mice in the two control groups. Tumor growth in mice fed a NF diet was slower than tumor growth in mice fed either control diet.