VOL. 12 (1953) 



BIOSYNTHESIS OF GLUCOSAMINE 



19 



incubated 3 hours at 30°. The proteins were removed by heat coagulation. Barium 

 acetate was added to the clear liquid and the pH was adjusted to 8. The mixture was 

 centrifuged and the precipitate was washed twice with i ml of water. Three volumes of 

 ethanol were added to the pooled supernatants. The precipitate was redissolved in 10 ml 

 of water, a small precipitate centrifuged off and three volumes of ethanol were again 

 added. The precipitate was then dried with ethanol and ether. Yield, 29 mg. These were 

 dissolved in 2 ml of water. The solution contained 41 /xmoles of total phosphate, 36 

 jLtmoles of reducing substance calculated as glucose, and 5.5 /^imoles of "glucosamine". 

 Direct paper chromatography of this ester mixture in different solvents gave irregular 

 results, so that it was decided to remove the phosphate group. 



0.5 ml of the above solution was made o.oi M in respect to Mg+2, and 10 mg of a 

 kidney phosphatase preparation and a drop of toluene were added. After 16 hours at 

 37°, about 70% of the phosphate was hydrolysed. The mixture was then deproteinized 

 with trichloroacetic acid, washed with ether and used for paper chromatography. A 

 sample of glucosamine-6-phosphate was run simultaneously. One of the solvents used 

 was a mixture of ethyl acetate-pyridine-ammonia^ with which it is possible to separate 

 glucosamine from galactosamine. The other solvent was phenol- water^^ with ammonia. 

 Phenol without ammonia was used with paper which had been immersed in o.i M zinc 

 sulphate and dried in air. This procedure was based on a previous observation which 

 disclosed that zinc ions greatly retard the migration of hexosamines but only have a 

 small influence on other sugars. It was also observed that with an alkaline solvent there 

 was no retardation by zinc ions. 



TABLE III 



PAPER CHROMATOGRAPHY OF THE "GLUCOSAMINE" ESTER AFTER TREATMENT WITH PHOSPHATASE 



Prepared from glucosamine with ATP and hexokinase®. 



The results of the chromatography are shown in Table III. The ex-ester sugar 

 mixture gave spots which migrated like glucose and glucosamine. The position of the 

 substances was revealed with the aniline phthalate reagent^", and that of hexosamines 

 was checked with the modified Elson and Morgan reagent^^. Besides glucose and 

 glucosamine, the ex-ester mixture contained small amounts of another hexose which 

 migrated like mannose. In some cases a very faint spot with the Rgiucose value of fructose 

 was observed. The presence of these sugars is not surprising since the sample of hexose-6- 

 phosphate used was obtained by the action of yeast enzymes. 



Acetylation. As shown in Table IV, Neurospora extracts, when suitably supple- 

 mented, are able to bring about the acetylation of glucosamine. These extracts are also 

 able to catalyze the phosphorylation of glucosamine (Table V) and contain phosphatase. 



References p. 22. 



