Further studies on glucosamine 6-phosphate synthesis by rat liver enzymes.

Summary of relative aminotransferase activities obtained with 1.7 to 2.3 M ammonium sulfate precipitates In Nos. 1 to 3, controls were incubated without glutamine and this substrate added just before the trichloroacetic acid addition. Controls were stopped at zero time in Nos. 4 and 5. Except where noted otherwise, the hexose-6-P concentration was 10 mM. Treatment of enzyme before assay 1. Dialyzed for 2.5 hours at 4” 2. Dialyzed for 2.3 hours at 4” 3. Dialyzed for 1 hour at 4” a. * b. c.t 4. Undialyzed precipitate al- lowed to stand in pres- ence of glutamine at 38” for 0 hours 3 hours 5. Undialyzed precipitate al- lowed to stand overnight at 0” in presence of glu- tamine ncuba- tion time ?nin 90 60 120 120 120 120 90 90 90 Hemsamine formed I I - ‘ru&xe- 6-P mwle/ml 0.02 0.23 0.31 0.19 0.29 0.12 0.38 0.07 0.20 - _- P - I Glucose- 6-P i?d&d 0.09 Ratio, ructose-6-P/ &mse-6-P 0.32 0.50 0.40 0.53 0.28 0.22 0.72 0.62 0.47 0.55 0.43 0.50 0.76 0.22 0.32 0.51 0.39 * The hexose-6-P concentration was 5 mM. t The enzyme concentration was one-half that in No. 3b were run to eliminate any errors caused by the higher blanks given by fructose-6-P in the Blix-Elson-Morgan method. When such preparations were tested for phosphohexoisomerase activity (4), sufficient enzyme was found to cause complete equi- libration in 30 minutes. It therefore appears likely that the higher activities with glucose-6-P resulted from its ability to protect the aminotransferase from inactivation during short time required before sufficient glucose-6-P was formed from fruc- tose-6-P when the latter was used as substrate. Presumably fructose-6-P affords little or no protection. Experiments with galactose-6-P support this hypothesis. Thus, with an enzyme preparation with a fructose-6-P to glucose-6-P ratio of 0.5, addi- tion of galactose-6-P (40 mM) had no effect on hexosamine for- mation from glucose-6-P but raised that from fructose-6-P to the glucose-6-P level. In the presence of only galactose-6-P plus glutamine, the enzyme system formed negligible hexosamine in this experiment. It has not been shown whether the protective effect of galactose-6-P is due to this ester itself or the small amounts of glucose-6-P present in the preparations (>2% by measurement with glucose-6-P dehydrogenase). The small quantities of hexosamine which were formed from galactose-6-P were shown to be glucosamine-6-P, and most likely were formed from the glucose-6-P in preparation. Nature of Glucose-6-P-protective Inactivation-Our evidence in- dicates that inactivation of the aminotransferase by liver ex- tracts, which is inhibited by glucose-6-P, enzymatic. The degree of inactivation increased with time of incubation and, since the aminotransferase activity of Fraction A was found to be stable at 38”, this inactivation could not have been due to heat denaturation. If rat liver was frozen immediately on Dry- Ice before homogenization and centrifugation, only small traces of aminotransferase activity were found in the 18,000 X g super- natant, even with glucose-6-P present during the procedure (Table V). Presumably the freezing and thawing process re- leased some inactivating agent. It was possible to determine the intracellular localization of inactivators with the use of Frac- tion A as the aminotransferase and a homogenate of unfrozen liver as the source of the inactivators. The results in Fig. 2 show that inactivation of the aminotransferase occurred upon “prein- cubation” with either the 2,000