i8 



L. F, LELOIR, C. E. CARDINI 



VOL. 12 (1953) 



ence of the substrates was still active after 3 hours at 30°. An experiment was 

 therefore carried out in order to ascertain which of the substrates exerted a stabi- 

 lizing action. Samples of the enzyme were preincubated 30 minutes at 30° with or 

 without substrate, and then the enzyme system was completed. 



The glucosamine formed in one hour was as follows (the amount formed during 

 preincubation was subtracted) : 



Preincubated without substrate 0.50 



Preincubated with glutamine 0.76 



Preincubated with hexose-6-phosphate 0.70 



No preincubation 0.80 



Thus both substrates, and specially glutamine, exerted a considerable stabilizing 

 action. 



Specificity. Glucose-6-phosphate could be replaced by fructose-6-phosphate, but not 

 by any of the following substances: maltose, glucose, mannose, fructose, fructose-1,6- 

 diphosphate, glucose-i,6-diphosphate, a-galactose-i-phosphate, fructose-i-phosphate, 

 glucose-2-phosphate, xylose-5-phosphate, dihydroxyacetone or glyceraldehyde. 



The enzyme preparation was found to contain considerable amounts of the enz3'me 

 which catalyzes the interconversion of fructose-6-phosphate into glucose-6-phosphate. 

 The activity of this isomerase was estimated by measuring the disappearance of fructose 

 phosphate with Roe's^^ method. It was found that under the conditions used for 

 measuring glucosamine formation the equilibrium values for the glucose-fructose esters 

 was attained in about 5 minutes. Therefore, it has not been possible to decide whether 

 the reactant is glucose-6-phosphate or fructose-6-phosphate. 



"Glucosamine" was formed when glucose-i-phosphate was used instead of hexose-6- 



phosphate with the crude enzyme, but not 

 with the purified preparations. Under the 

 conditions of the test and with the purified 

 enzyme the phosphoglucomutase activity 

 was very weak. 



The substances which were tested with 

 negative results as possible substitutes for 

 glutamine were the following : asparagine, 

 glutamic and aspartic acids, argininc, put- 

 rescine, urea, ammonium acetate, alanine, 

 glycine, butyramide, serine, cysteine, ly- 

 sine, ornithine, valine, leucine and citrul- 

 line. Pairs such as ammonium salts with 

 ATP, asparagine and glutamate, etc., also 

 gave negative results. 



pH optimum. As shown in Fig. 2, the 

 reaction has a sharp pH optimum at pH 

 6.4-0.8. 



Study of the "glucosamine'' ester. 100 

 jLtmoleseach of glutamine and hexosemono- 

 phosphate plus 50 mg of enzyme in 10 ml of 

 0.025 ^^ tris-acetate buffer (pH 6.4) were 



l-"ig. 2. pH optimum curve. System composed of 

 2 //moles each of hexose-6-phosphatc plus i mg 

 of enzyme and o.i ml of 0.1 M phosphate or tris- 

 hydroxymethylaminoethanc acetate buffers. In- 

 cubated 2 hours at 30'. The pH was determined 

 on aliquots with a glass electrode. 



References p. 22. 



