l6 L. F. LELOIR, C. E. CARDINI VOL. 12 (l953) 



The synthesis of glucosamine-6-phosphate can also be brought about by a mechanism 

 different from reaction a. Thus Harpur and Quastel^ discovered that glucosamine is 

 phosphorylated by ATP in the presence of brain extracts, and from further studies by 

 Brown^ and Grant and Long', it has been concluded that the phosphorylation is 

 catalysed by hexokinase and that the reaction product is glucosamine-6-phosphate. 

 It is difficult to decide whether this synthesis is a physiological process or simply an 

 unspecific effect. 



Similar events have been found to occur with galactosamine. Liver and yeast 

 extracts containing galactokinase were found to phosphorylate galactosamine to a prod- 

 uct which appears to be galactosamine-i-phosphate^. In this case, as with glucosamine, 

 the corresponding hexose inhibits the phosphorylation of the hexosamine. 



The formation of glucosamine by a process such as reaction a would explain the 

 results of Topper and Lipton^, who found that in Streptococcus the glucosamine formed 

 from glucose-i-^^C contained nearly all the label in the i-position. 



methods 



A 7ialyHcal. The following methods were used: BlixI'^ for glucosamine. Kunitz and McDonaldi* 

 for protein. Glutamate was estimated with ninhydrin after paper chromatography with phenol^^. 

 .\mide nitrogen by estimation of the ammonia liberated after heating eleven minutes at ioo° in i A'' 

 acid^'. Ammonia by distillation in Conway unitsi* and nesslerization. 



For the estimation of acetylglucosamine the method of Morgan and ElsonI^ was slightly 

 modified in order to make it less sensitive to buffers and to reduce the time needed for colour develop- 

 ment. The /j-dimethylamino benzaldehyde (DAB) reagent was prepared by adding 0.5 g of DAB to 

 10 ml of concentrated HCl and completing to 100 ml with glacial acetic acid. The analytical procedure 

 was as follows: the neutralyzed unknowns and standards containing 0.1-0.5 //moles of acetyl 

 glucosamine were taken to 0.5 ml with water. After adding o.i ml of i M sodium carbonate the tubes 

 were heated 5 minutes in a boiling water bath. After cooling 2.5 ml of the DAB reagent were added 

 and mixed immediately with a suitably glass rod. The optical density at 544 m/j, was measured after 

 3 to 5 minutes with a Beckman spectrophotometer. The colour increases during 2 minutes and begins 

 te decrease slowly after 3 minutes. If the time elapsing between the addition of the DAB reagent and 

 the colorimetric reading is equal in all the samples a good proportionality between concentration of 

 acetylglucosamine and optical density is obtained. 



Preparation of the enzyme. A wild type Neurospora crassa E-5297a was grown for three days on 

 "minimal medium"^* at 30° under forced aeration. The mycelium was separated by filtration, washed 

 with water, lyophylized and stored over calcium chloride in an evacuated desiccator at 5°. Extraction 

 of the dried mycelium was effected by homogenizing 0.8 g in 16 ml of water at 0°, followed by cen- 

 trifugation. The supernatant containing about 40 mg of protein per ml is referred to as crude extract. 

 Partial purification was carried out as follows: 6.5 ml of acetone were added to 13 ml of the crude 

 extract at 0°. The inactive precipitate was centrifuged off at 0°. To the supernatant 3.9 ml of acetone 

 were added. The precipitate was separated by centrifugation, washed three times with acetone and 

 dried in an evacuated desiccator. The yield was about 60 mg of a white powder. 



The formation of "glucosamine" was found to be greater in the presence of 8-hydroxyquinoline, 

 and this fact was attributed to protection of the enzyme from metal inactivation. Therefore, 8- 

 hydroxyquinoline was added to the acetone used in the purification (about 10 mg %) and the buffer 

 (pH 6.5) used for dissolving the enzyme was saturated with 8-hydroxyquinoline. 



The enzymein solution was found to lose activity in a few hours at 5''' and in a few days at — 10° 



The ratio: //moles of glucosamine formed/mg protein per hour, was about 0.04 for the crude 

 enzyme and usually about 0.3 for the acetone fractionated enzyme. 



Acetylation experiments. The enzyme preparation used was a crude extract which had been 

 dialyzed about two hours against running water. The enzyme system was similar to that used by 

 Kaplan and Lipmann^^. The Co.^. solution was an aqueous extract of rat liver. In every case controls 

 in which the reaction was stopped at time = o were run simultaneously. The reaction was stopped by 

 immersing the tubes in boiling water followed by centrifugation. 



Acetylglucosamine was estimated in the supernatant as described above. In some cases the 

 phosphoric esters were precipitated by adding 0.3 ml of 5% zinc sulphate and 0.3 A'^ barium hydroxide 

 until the suspension gave a rose colour with phenolphthalein. 



References p. 22. 



