Ammoniagenesis catalyzed by hippurate-activated gamma-glutamyltransferase in the lumen of the proximal tubule. A microperfusion study in rat kidney in vivo.
gamma-Glutamyltransferase (gamma-GT) is located in the brushborder membrane of the proximal tubule where the catalytic site of the enzyme faces the lumen. The (phosphate-independent) glutaminase activity of gamma-GT in vitro is activated by hippurate. In order to investigate glutamine deamidation in the tubule lumen in vivo, 14C-L-glutamine-containing solutions were continuously microperfused through sections of the proximal convoluted tubule in vivo and in situ. D-aspartate and L-phenylalanine (10 mmol/l, each) were added to the perfusate in order keep the reabsorption of L-glutamine as such low and to block reabsorption of any glutamate possibly formed, respectively. Intraluminal formation of glutamate from glutamine in the absence of hippurate is small. In presence of 10 mmol/l hippurate, 5%-70% of the recovered 14C-activity was 14C-glutamate at an initial 14C-L-glutamine concentration of 1 mmol/l. The respective absolute rate (+/- SEM) of glutamate formation, i.e., 36 +/- 5 pmol X s-1 X m-1, was increased 1.4-fold at an initial L-glutamine concentration of 3 mmol/l, but dropped to one third at initially 0.3 mmol/l. A rough estimate of the apparent kinetic constants resulted in a Km of 0.58 (0.19-0.97) mmol/l and a Vmax of 56 (40-93) pmol X s-1 X m-1. Deamidation of glutamine occurred also in the absence of L-phenylalanine. Acivicin (AT 125), a gamma-GT inhibitor, completely blocked glutamate formation. Endogenous hippurate concentrations determined by free flow micropuncture and HPLC were 0.16 mmol/l in the late proximal convolution, 0.6 mmol/l in the early distal convolution, and 4.9 mmol/l in the final urine.(ABSTRACT TRUNCATED AT 250 WORDS)
D000596:Amino Acids / Q000502:physiology; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D005971:Glutamates / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism*; D006626:Hippurates / Q000494:pharmacology*; D007687:Kidney Tubules, Proximal / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D000596:Amino Acids; D005971:Glutamates; D006626:Hippurates; D005973:Glutamine; D000641:Ammonia; D005723:gamma-Glutamyltransferase