COMMUNICATIONS TO THE EDITOR
4758
Vol. 81
The authors thank Dr. P. L. Pauson for advice, and J. F. T.-B. thanks Sheffield University and the Imperial Chemical Industries Ltd. for a Research Fellowship.
hexoses were used as phosphate acceptors, with P H T and PNH2, the corresponding hexose-6phosphate was isolated and identified chromatographically using the solvent systems of Mortimer.2 DEPARTMF~NT OF CHEMISTRY UNIVERSITY OF SHEFFIELD J. F.TILNEY-BASSETT P H T showed neither a divalent metal requireDEPARTMENT OF CHEMISTRY ment nor a dependence on added nucleoside diUNIVERSITY OF MANCHESTER 0. s. M I L L S phosphates and treatment of the partially purified ENGLAXD enzyme with charcoal (pH 5.5), or with Dowex-I RECEIVED J L L Y 20, 1959 or with Versene (pH 7.5) failed to remove any cofactors participating in the reaction. Furthermore, A NEW ENZYMATIC SYNTHESIS O F HEXOSE when P H T was coupled with glucose-6-phosphate PHOSPHATES' dehydrogenase no T P N H formation was observed if Sir: PNHz was replaced by adenosine triphosphate An enzyme which catalyses the formation of (ATP). When crystalline yeast hexokinase rehexose-6-phosphate and ammonia from potassiuni placed P H T in the coupled system T P N H formation phosphoramidate (PNH2) and hexose (reaction I) was observed only if ATP was added. The enzyme preparation used in these studies has been obtained from extracts of succinate grown E . coli. The enzyme, presently named phosphor- was contaminated with PNHz hydrolase activity, amidic hexose transphosphorylase (PHT) , has been making stoichiometric measurements of NH3 in purified about 60-fold by fractionation with pro- reaction 1 unreliable. Further work on the puritamine sulfate, ammonium sulfate and diethyl- fication and characterization of PHT is in progress. aminoethyl cellulose. The formation of P32labeled (2) D. C. Nortimer, C a n . J . Chcm., SO, 653 (1952). organic phosphate esters from the free sugar and (3) R. A . Smith and D. J. Burrow, Biochim. el Biophys. Acto, 34, 274 (1959). labeled PNHz was used as an enzyme rate assay. OF CHEMISTRY The results shown in Table I indicate that P H T DEPARTMENT AT Los ANGELES catalyzed a phosphoryl transfer from PNHz to UKIVERSITPOF CALIFORNIA, ROBERTS A. SXIIH several hexoses, although the rate differed con- LOSANCELES21, CALIFORNIA RECEIVED JUNE 12, 1959 siderably with different hexoses. Neither pentoses nor nucleosides were phosphorylated by partially purified PHT. THE EFFECT OF PRESSURE ON SEDIMENTATION TABLE I FORMATION 01' HEXOSE PHOSPHATES FROM PHOSPHORAMIDATE
The reaction mixture contained it1 1 iul. 2-a1ni110-2metliyl-1,3-propanediol buffer, PH 8.0, 100 pinoles; PNHz, 9 Mmoles (2160 c.p.m. per pmolej; carbohydrate, as shown, 10 Mmoles; enzyme cn. 0.9 mg. protein (obtained from a protamine treated extract of E . coli by precipitation with (?;H4)2SO( a t 68 to 78% saturation). Reaction was incubated a t 37", and stopped by addition of 0.5 ml. of 125; trichloroacetic acid and boiled for two minutes to hydrolyze remaining PSH?. Organic substrate
Total incorporationa c.p.m./15 min.
PXHt utilizationb pmoles/l5 min.
o-Fructose 3810 1.85 D-Fructosec 3030 I .40 1.-Sorbose 2480 1.15 D-Glucose IS00 0.80 n-Glucosamine 980 I I , 4%; D-Galactose :i 70 0.20 Organic and inorganic phosphates separated by method of S. 0. Kielson and A. L. Lehninger, J . B i d . Chein., 215, 555 (1955). * Measured as inorganic phosphate by method o f C. H. Fiske and Y . SubbarRow, ibid., 66, 375 (1926). Enzyme pretreated with charcoal a t p H 5.5, 0.7 rng. of protein in assay.
The P H T reaction could be coupled to the reaction catalyzed by glucose-&phosphate dehydrogenase, when glucose was used as the phosphate acceptor. Thus, the rate of the over-all reaction could be followed by reduced triphosphopyridine nucleotide (TPNH) formation and furthermore glucose-6-phosphate could be assumed as the product of transphosphorylation reaction. When individual ( 1 ) This investigation was supported in part b y grants from t h e Williams-Waterman Fund a n d t h e United States Public Health Service.
RATE
Sir: Recently, Fujital has derived relationships relating the boundary position to time for a monodisperse species in a sector-shaped cell, when the sedimentation coefficient depends on both pressure and concentration. The boundary position y* = [ r ~ ' r o )the 2 , dilution ClCO and the re(or concentration) factor e* duced time, 7 = 3u2Sof,are related as showri in eq. (1). =1
where wz is a pressure dependeiice parameter' mid a is a concentration dependence parameter.' We have solved Fujita's system of Equatioiis (69) through (Z),11. 3603 of reference ( l ) , by the use of Runge-Kutta integration combined with trial and error iteration, using a Bendix C-15D digital computer programmed in pseudocode. A range of a froni 0.1 to 1.O was covered, and of m from 0.1 t o 0.9. For flotation, negative values of r were used. In the case of flotation, the reference pressure is the pressure a t the cell bottom !see reference ( I ) ) for definition of symbols. I t is the purpose of this communicatioii to show that a simple relation between boundary position and reduced time can be developed, which fits the exact solution of this system of equations to a very good approximation. 0 t h and DesreuxZdeveloped a relationship which is essentially equivalent to letting 8* = 1 'y*in Equation (1). 1) 11. Fujita, THISJoux~;.\L, 73,3.30Y (1956). (2) J. 0 t h and V. Desreux, Bull. soc. Chim. Beiges, 63, 133 (1954)
