ENZYMATIC SYNTHESIS OF GLUCOSAMINE 6-PHOSPHATE IN RAT LIVER*

BY BURTON M. POGELL AND ROSA M. GRYDERt

(From the Wilmer Ophthalmological Institute of The Johns Hopkins University and Hospital, Baltimore, Maryland)

(Received for publication, March 7, 1957)

Leloir and Cardini in 1953 described an enzyme from Neurospora crassa which formed glucosamine from n-hexose 6-phosphate and n-glutamine (2). Formation of glucosamine by cell-free extracts of streptococci in the presence of glucose, adenosine triphosphate, and glutamine was also re- ported by Lowther and Rogers (3). Although no similar system was described in mammalian tissues, evidence obtained in vivo from several laboratories has clearly established that the carbon chain of glucose is incorporated intact into the glucosamine portion of the mucopolysaccha- rides of mammals and microorganisms (4-7). In the present communi- cation, an enzyme system from rat liver homogenates is described which forms Gm6P’ by the following reaction: n-Glucose 6-phosphate + n-glutamine + n-glucosamine g-phosphate + n-glutamate

Results

Preliminary studies with a number of combinations of possible glucosa- mine precursors in rat liver homogenates yielded only traces of color forma- tion as measured by the Blix-Elson-Morgan method (8). Both G6P and glutamine appeared to be required for this small synthesis. A marked increase in hexosamine formation was observed, however, when the super- natant fluid was tested after centrifugation at 18,000 X g for 90 minutes. In one experiment, the amount of hexosamine formed in 2 hours from 0.2 ml. of extract increased from 0.08 to 0.24 pmole when the particles were removed. No synthesis was found in any of the particulate fractions.

Further experiments with the high speed supernatant fluid revealed that G6P plus glutamine gave the highest synthesis of hexosamine, and no cofactors have yet been found. The effect on hexosamine synthesis in supernatant fluid of varying amounts of G6P or n-glutamine in the pres-

* This investigation was supported by a research grant (No. B-141) from the Na- tional Institute of Neurological Diseases and Blindness, National Institutes of Health, United States Public Health Service. A preliminary note has appeared (1).

t Postdoctoral Research Fellow of the National Institutes of Health. 1 The following abbreviations are employed: Gm6P for n-glucosamine g-phosphate,

G6P for n-glucose g-phosphate, F6P for n-fructose 6-phosphate, and GlP for n-glu- cose l-phosphate.

701

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702 SYNTHESIS OF GLUCOSAMINE 6-PHOSPHATE

ence of constant amounts of the other is shown in Table I. Saturation was found with 10 pmoles per ml. of G6P and 15 to 20 pmoles per ml. of gluta- mine. A pH optimum between 7.4 and 8.0 was observed with the crude supernatant liquid, and a pH optimum of 7.5 with the partially purified enzyme (Fig. 1). Linearity of hexosamine formation with respect to time was observed for 2 hours with this fraction (Fig. 2).

Partial Purification-the of the major difficulties encountered in study- ing this aminotransferase has been its great instability. Dialysis overnight at 4” completely destroyed the activity. Even storage of the supernatant fluid at -20” overnight caused a large diminution of enzyme activity.

TABLE I

Effect of [Glucose 6-Phosphate] and [Glutamine] on Glucosamine Formation in Crude Rat Liver High Speed Supernatant Fluid

Incubation for 1 hour at 38”. Samules hydrolyzed for I hour at 100” in 1 N HCl - before analysis by Blix method.

Experiment No. Glucose &phosphate Glutamine Glucosamine formed

pmoles per ml. pmoles per ml. pmole per ml.

0 15.0 0.02 2.5 15.0 0.14 5.0 15.0 0.21 7.5 15.0 0.24

10.0 15.0 0.26 5.0 0 0.06 5.0 5.0 0.16 5.0 10.0 0.22 5.0 15.0 0.21 5.0 20.0 0.32

Preincubation of the fresh enzyme at 38” for 60 minutes or heating in a water bath to a temperature of 49” reduced the activity to one-half of its initial value. All activity was lost when larger scale homogenization was tried at full speed in a Waring blendor. This instability of the enzyme is discussed further under “Substrate protection of enzyme activity.”

A quick partial purification was obtained as follows: Livers from ex- sanguinated rats were homogenized for 2 to 3 minutes in 2 ml. of 0.154 M

KC1 (containing 0.001 M sodium ethylenediaminetetraacetate, pH 7) per gm. of tissue. Foaming was avoided by running the Waring blendor at low speed. After centrifugation at 18,000 X g for 90 minutes, solid am- monium sulfate was added to the supernatant fluid to a concentration of 1.7 M (9), and the resulting precipitate formed removed by centrifugation and discarded. The supernatant fluid from this step was raised to 2.3 M

ammonium sulfate by addition of the solid, and this precipitate was stored

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B. M. POGELL AND R. M. GRYDER 703

at -20”. The aminotransferase activity was stable for several days in this form. Immediately before use, the enzyme was dissolved in water and dialyzed for 2 hours against 0.0005 M sodium phosphate, pH 7.4. The specific activity (micromoles per hour per mg. of protein) of the super- natant fluid after storage overnight was 0.015 and that of the 1.7 to 2.3 M

ammonium sulfate fraction 0.027 to 0.050, representing a 2- to 3-fold puri- fication.

Xpecijicity of L-Glutamine As Amino Donor-The specificity of the par- tially purified enzyme for L-glutamine is shown in Table II. This was the

1.6 -

I.4 -

I.2 -

o-

A . 0

1 PI 6.0 7.0 8.0 9.0

PH

FIG. 1 TIME IN HOURS

FIG. 2 FIG. 1. pH optimum of aminotransferase. Buffers : 0, tris (hydroxymethyl) a

aminomethane-maleate; l , tris(hydroxymethyl)aminomethane; A, phosphate. Concentration of buffers was 0.05 M.

FIG. 2. Time-course of hexosamine formation at 38”.

only compound found to give significant, hexosamine synthesis in the pres- ence of G6P. When NH4+ was added to the high speed supernatant fluid in concentrations from 0 to 20 pmoles per ml., no increase in hexosamine formation was observed. Addition of adenosine triphosphate and NH,+ caused a slight increase in hexosamine synthesis, presumably by synthesis of glutamine from endogenous glutamate.

Specificity of G6P As Xugar Donor-In the presence of L-glutamine, almost equal amounts of hexosamine were synthesized by the high speed supernatant fluid with either G6P, FBP, or GlP as substrates. Addition of 5 pmoles of sodium ethylenediaminetetraacetate had no effect on syn- thesis from G6P, but, lowered that from GlP by 47 per cent,, presumably by chelating endogenous Mg++ and thus lowering phosphoglucomutase

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704 SYNTHESIS OF GLUCOSAMINE 6-PHOSPHATE

activity. The ratio of hexosamine synthesis from F6P and G6P was close to unity in the crude high speed supernatant fraction, but decreased with purification. These results (Table III) point to G6P as a more direct pre-

TABLE II

Specijkity of Amino Donor in Glucosamine g-phosphate Synthesis

Incubation for 2 hours at 38”. Amino compounds were present at concentrations of 15 @moles per ml. Samples were hydrolyzed for 1 hour at 100’ in 1 N KC1 before analysis by Blix method.

Amino donor Hexosamine formed

jLmole per ml.

L-Glutamine...................................... 0.53 None............................................. 0.05 L-Asparagine...................................... 0.05 L-Glutamate...................................... 0.00 NH~Cl..........................................,. 0.02 L-Glutamate + NH&l............................ 0.01

TABLE III

Relative Enzyme Activity during Purification with Glucose B-Phosphate and Fructose &Phosphate As Substrates

In Experiment 1 samples were hydrolyzed for 1 hour at 100” in 1 N HCl before analysis by the Blix method. This was omitted in Experiment 2. [G6P] and [F6P] were identical in analysis of each fraction (10 to 12.5 pmoles per ml.).

Experi- ment No

la b

c

2a b

Liver preparation

Crude supernatant fluid Dialyzed ammonium sulfate ppt.

(1.7-2.3 M)

Lyophilized, dialyzed ammonium sulfate ppt. (1.7-2.3 M)

Crude supernatant fluid Ammonium sulfate ppt.

1.5-2.0 M

2.0-2.3 “

Incubation time

-

/

min.

92 120

240

120

120 120

G6P _-

pm& per ml.

0.24 0.53

0.30

0.20

0.51 0.19

~mole her ml.

0.21 0.37

0.17

0.16

0.25 0.08

F6P Ratio, =P

0.88 0.70

0.57

0.80

0.49 0.43

cursor of Gm6P than F6P. Synthesis from F6P with the ammonium sul- fate-precipitated enzyme (1.7 to 2.3 M) varied from 67 to 84 per cent of that obtained with G6P over the concentration range from 4 to 15 pmoles per ml. A plateau was reached at 11 pmoles per ml. with both substrates. The data in the second experiment of Table III were obtained on fraction-

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B. M. POGELL AhrD R. M. GRYDER 705

ation of the enzyme in the presence of G6P, which was found to have a pronounced stabilizing effect on the enzyme (see below). These values are therefore only maximal ratios, since there was some G6P present in the enzyme fractions analyzed.

Substrate Protection of Enzyme Activity-As mentioned earlier, our stud- ies on this enzyme were greatly hindered by its instability. Leloir and Cardini (2) reported that G6P and glutamine both exerted some protective effect on the similar enzyme in Neurospora. When this effect was tested on the undialyzed ammonium sulfate fraction of liver, both substrates were found to protect the enzyme. After 30 minutes preincubation of the en- zyme at 38”, 47 per cent of the activity disappeared, whereas, with G6P or glutamine present, 91 and 84 per cent, respectively, of the original ac- tivity was found.

This experiment was repeated over varying time intervals with a di- alyzed ammonium sulfate precipitate, and almost complete protection was found over a 2 hour preincubation period with G6P added (Fig. 3). The protective effect of glutamine, however, had disappeared. These results also indicated that G6P might provide protection from loss of activity even in the cold. The enzyme preparation which had been allowed to stand at 4” after dialysis with no G6P had much lower activity, even with no preincubation, than the material to which G6P was added. When different concentrations of G6P were tested, maximal protection during preincubation was found at about 10 pmoles per ml., the same concentration which gave maximal aminotransferase activity.

The apparent protection of enzyme by G6P at 4” was further tested by addition of G6P to the original homogenization medium. These results (Fig. 4) indicate that consistently higher activities occurred in homogenates made in the presence of GBP. Addition of F6P gave only slightly higher activity, providing additional evidence that G6P is a more immediate precursor for the aminotransferase than F6P. 0.25 M sucrose gave no additional activity when used for homogenization, and neither glucose nor ,%glycerophosphate gave protection when preincubated with the enzyme.

Hexosamine Product-A typical large scale preparation of the products was obtained as follows: 500 Kmoles of G6P, 1000 pmoles of glutamine, and the dialyzed ammonium sulfate fraction from twelve rat livers were incu- bated in a final volume of 50 ml. in phosphate buffer of pH 7.4 for 4 hours at 38”. The reaction was terminated by addition of an equal volume of 0.4 N trichloroacetic acid. Analysis of the supernatant fluid after hydroly- sis for 2 hours in 1 N HCl indicated formation of 94 pmoles of hexosamine. In two other large scale preparations, syntheses of 118 and 257 pmoles of hexosamine product in 5.5 and 6.75 hours, respectively, were indicated by the Blix-Elson-Morgan color reaction without acid hydrolysis.

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706 SYNTHESIS OF GLUCO~AMINE ~-PH~~PHATIG

When this material was fractionated with barium and alcohol (lo), the hexosamine was quantitatively recovered as the water-soluble, alcohol- insoluble Ba++ salt. This compound gave a color spectrum in the Blix test which was identical with that of n-glucosamine (Fig. 5). When this product was placed on a Dowex 50 ion exchange column (Hf form) at

9 ADDITIONS

I [1: 0.6 2 ,

G6P (~~+M/ML)

y 0.5

I- 4

h , F6P (12.5)1M/d NONE

G6P (12Sj~M/w)

F 6P (12.5,uMh) NONE

3 0.2 -I G6f’ (IO.O)iM/d

: 0.1 G6P ( 2.5~Mh.x)

i? NONE v ‘*.-

’ ‘0 25 50 75’-’ I

100 125 0 1.0 2.0

PREINCUBATION TIME (MINUTES) MICROMOLES HEXOSAMINE FORMED

FIG. 3 FIG. 4

FIG. 3. Effect of preincubation at 38” on enzyme activity in presence and absence of substrates. 0, enzyme alone; l , enzyme plus n-glutamine (15 pmoles per ml.) ; A, enzyme plus G6P (10 pmoles per ml.). After preincubation, G6P added to con- centration of 10 pmoles per ml. and L-glutamine to 15 pmoles per ml. in all the tubes and incubation continued for 2 hours. The results are expressed as micromoles of hexosamine formed per hour.

FIG. 4. Aminotransferase activity with G6P and F6P included in homogenization medium. 2.5 gm. of rat liver homogenized in 5 ml. of isotonic KC1 (containing 0.001 M sodium ethylenediaminetetraacetate, pH 7) plus concentrations of G6P and F6P indicated. The samples were centrifuged for 90 minutes at 18,000 X g and 0.3 to 0.4 ml. of supernatant fluids were analyzed. Final [G6P] was 10 to 12.5 pmoles per ml. and [glutamine] 15 pmoles per ml. 2 hour incubation at 38”. The results are ex- pressed as micromoles of hexosamine formed per 30 minutes per gm. of wet weight of tissue.

pH 2, it could be completely eluted with distilled water. Synthetic crystal- line Gm6P behaved similarly. It could not be eluted with 0.0005 M phos- phate buffer of pH 7, but was eluted with distilled water of pH 4.5.

The hexosamine product was found to be hydrolyzed by a microsomal preparation from rat liver with a pH optimum of 6 to 7. After such treat- ment, the product could no longer be eluted from the Dowex 50 column with distilled water. Maley and Lardy (11) have reported that such G6Pase preparations hydrolyze Gm6P. Ascending paper chromatog- raphy in a 75: 30 mixture of 95 per cent ethanol and ammonium acetate

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B. M. POGELL AND R. M. GRYDER 707

(1 M, pH 3.8) (12) for 17 hours at room temperature gave the following RF values: G6P, 0.32; synthetic Gm6P, 0.28; concentrated hexosamine prod- uct after elution from Dowex 50 with water, 0.29.

The hexosamine product was further identified by hydrolysis with wheat germ acid phosphatase (from Worthington Biochemical Corporation). The pH optimum for phosphate liberation from Gm6P was 5.0 to 5.5. 72 pmoles of compound after elution from Dowex 50 with distilled water were incubated for 2.5 hours at pH 5.5 in citrate buffer in the presence of 111

WAVE LENGTH IN MJJ

FIG. 5. Absorption spectra of hexosamine product and n-glucosamine. Product hydrolyzed for 4 hours in 1 N HCl before Blix test. 0, product; 0, o-glucosamine (0.4 pmole).

mg. of phosphatase. The reaction was stopped by addition of trichloro- acetic acid and the filtrate dried by lyophilization after removal of the acid by ethyl ether extraction. This material was then adjusted to pH 2 with HCI and placed on a 10 X 1 cm. Dowex 50 column (H+). Upon gradient elution with a mixer volume of 100 ml. and 1 N HCl in the reser- voir, two major peaks appeared (Fig. 6). The initial small peak is proba- bly hexose 6-phosphate. The compound in the first major peak when analyzed by paper chromatography agreed in RF value with Gm6P. The second major peak was dried by lyophilization and analyzed by paper chromatography. The results (Table IV) show that the only hexosamine detectable was glucosamine. In the third experiment, the compounds

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708 SYNTHESIS OF GL~CO~AMINE &PHOSPHATE

were first converted to pentoses by reaction with ninhydrin as described by Stoffyn and Jeanloz (16).

FIG. 6. Dowex 50 chromatography of products after hydrolysis with wheat germ acid phosphatase. 2 ml. fractions were collected and 0.1 ml. of each analyzed by Dische-Borenfreund method (13). Non-deaminated blanks were not included. Fraction 28 contained 5.5 ml.

TABLE IV

Identijication of Glucosamine in Dephosphorylated Product by Paper Chromatography

FRACTION NUMBER

Solvent

1. n-Butanol-pyridine-water (3:2:1.5) (de- scending) (14) *

2. Ethyl acetate-pyridine-ammonia-water (10:5:3:3) (descending) (15)

3. n-Butanol-ethanol-water (4:l:l) (de- scending) (16)

-

)-Glucos- amine-

HCl

0.87

0.83

0.81

Substance

-Galactosamine- HCl

R glucose

0.71

0.60 (Trailing)

R lyxose

0.99

* The numbers in parentheses represent bibliographic references

0.85

0.84

0.82

D-

Lrabinose

0.83

These data provide strong evidence that the product of the aminotrans- ferase reaction is Gm61’ and most probably of the D configuration, since D-G~P was the substrate and the D form is the enantiomorph of glucosa- mine found in mammalian tissues.

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33. M. POGELL AND R. M. GRYDER 709

Amino Acid Product-A similar large scale preparation was analyzed for amino acids by paper chromatography after extraction of the trichloro- acetic acid with ether. These results (Table V) show that glutamic acid

TABLE V

Paper Chromatographic IdentiJication of Gluta?nic Acid As Product of Reaction Ascending chromatography in all cases.

,

Solvent I Reaction mixture

1. Methanol, 94% (17)*

2. Methanol-water-pyridine (20:5: 1) (18)

3. Phenol-water (100:20) (19)

4. 2-Butanone-propionic acid- water (15:5:6) (20)

RF of substance

x-Glutamine L-Glutamic acid

Initial

0.14

0.32

0.55

0.33

0.23

0.44

0.39

0.45

0.14

0.31 0.43 (trace) 0.57 0.41 (trace) 0.33

* The numbers in parentheses represent bibliographic references.

Final

0.14 0.23 0.31 0.44 0.56 0.41 0.32 0.44

TABLE VI

Aminotransjerase Activity of Mammalian Tissues The results are expressed as micromoles of product formed per hour per gm. of wet

weight of tissue. Supernatant fluids were analyzed after 90 minutes centrifugation at 18,000 X g.

Tissue Hexosamine formation Tissue Hexosamine

formation

Rat liver 1.70-5.10 Beef lung 1.29

“ kidney cortex 1.16 Rabbit intestine 1.10 Calf lung 1.00 Rat t#rachea 0.80

“ intestine 0.38

Rat lung 0.34 Calf intestine 0.06 Beef liver 0.14 Calf “ 0.11 Rabbit liver 0.00 Pigeon “ 0.00

is the only new amino acid which appears during this reaction. An ex- periment run with the partially purified enzyme revealed no formation of glutamate unless G6P was present, thus establishing negligible glutaminase activity under the conditions employed. Since the substrate was L-gluta- mine, the product is probably also of the L configuration.

Occurrence of Amino&a&erase in Other Mammalian l&sues-The rela- tive activities of this enzyme in several mammalian tissues are summarized

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710 SYNTHESIS OF GLUCOSAMINE 6-PHOSPHATE

in Table VI. G6P was included in the homogenization medium in these experiments. Although the aminotransferase has been consistently found in rat liver, only slight traces have been detected in other liver preparations. The reason for this is not yet known.

DISCUSSION

Gm6P appears now well established as an intermediate in mucopoly- saccharide biosynthesis. There are at present at least three pathways for its enzymatic formation: (1) by direct phosphorylation of n-glucosamine with hexokinase and adenosine triphosphate (21), (2) by reaction between F6P and NH,+ in the presence of appropriate enzyme and catalytic amounts of N-acetylglucosamine 6-phosphate (22), and (3) by the enzyme system described in this communication. The first pathway could be of signifi- cance only in mammalian mucopolysaccharide anabolism in which suffi- cient quantities of free glucosamine are present in the food intake. The marked stimulation produced by L-glutamine of radioactive sulfate and glucose incorporation into the chondroitin sulfate of cartilage slices points to the relative importance of the pathway from this nitrogen source in mammalian tissues (23, 24).

Although our experiments show G6P to be a more immediate precursor of Gm6P than F6P, Blumenthal, Horowitz, Hemerline, and Roseman (25) have reported the reverse to be true for the Neurospora enzyme. It may well be that a common intermediate is formed which is the immediate hex- ose precursor. The mechanism of the protection afforded by G6P to this enzyme is now under investigation. In any event, this substrate stabiliza- tion has aided us greatly in our studies of the aminotransferase and may be of more general usefulness in the study of other labile enzyme systems.

This amino transfer reaction represents another example of the role of the amide N of glutamine as an amino donor in biosynthetic reactions. Recently established enzymatic aminations requiring L-glutamine include the formation of 5-phosphoribosylamine from 5-phosphoribosylpyrophos- phate (26) and the synthesis of guanosine phosphate from xanthosine phos- phate (27). The biosynthesis of histidine also apparently involves this amide group (28).

Materials and Methods

Xubstrates-Crystalline barium G6P and sodium G6P were obtained from the Sigma Chemical Company. Barium F6P (Schwarz) was puri- fied by alcohol precipitation (29) after removal of any G6P by seeding a water solution with crystalline barium G6I’.7H&. The L-glutamine was chromatographically pure. n-Glucosamine hydrochloride (Pfanstiehl) was recrystallized from ethanol-water (30). n-Galactosamine hydro-

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B. M. POGELL AND R. M. GRYDER 711

chloride was obtained from the Nutritional Biochemicals Corporation. Synthetic crystalline Gm6P was a gift of Dr. Saul Roseman of the Uni- versity of Michigan.

Methods-The usual procedure for measurement of aminotransferase activity consisted of incubating 10 to 12.5 pmoles of sodium G6P, 15 pmoles of n-glutamine, enzyme, and 0.1 M sodium phosphate buffer of pH 7.4 in a final volume of 1 ml. for 2 hours at 38” in stoppered 15 ml. centrifuge tubes. 1 ml. of 0.4 N trichloroacetic acid was then added and a suitable aliquot of the supernatant fluid taken for hexosamine analysis. Control samples were stopped at zero time by addition of trichloroacetic acid.

Hexosamine was determined by the Blix-Elson-Morgan method (8) with slight modifications. After heating in sodium carbonate, 11 ml. of a 10: 1 mixture of ethanol and the p-dimethylaminobenzaldehyde reagent were directly added. In early experiments, the samples were hydrolyzed for 1 hour in 1 N HCl in a volume of 1 ml., and 1 ml. of 2.25 N sodium carbon- ate added before the second heating. This gave about a 40 per cent in- crease in color when supernatant liquids after precipitation with trichloro- acetic acid were directly analyzed, but no similar increase was found with synthetic Gm6P or the isolated reaction product. This preliminary hy- drolysis was not included in the later experiments.

Protein was determined by ultraviolet absorption measurements (9). The carbohydrate chromatograms were developed with aniline hydrogen phthalate reagent (31), and the amino acid chromatograms with ninhydrin reagent (32). The Dowex 50 cation exchange resin was 200 to 400 mesh with 12 per cent cross-linking. The enzyme purification steps were carried out at 4”.

SUMMARY

1. An enzyme has been found in rat liver extracts which catalyzes the following reaction: n-glucose 6-phosphate + n-glutamine -+ n-glucosa- mine-6-phosphate + L-glutamate. Properties of this system are de- scribed.

2. The presence of n-glucose B-phosphate was found to stabilize greatly this very labile enzyme.

We wish to express our deepest appreciation to the late Dr. Jonas S. Friedenwald, under whose guidance and stimulation the present investiga- tion commenced. Mr. Leslie A. Bard performed several of the chro- matographic analyses.

BIBLIOGRAPHY

1. Pogell, B. M., Biochim. et biophys. acta, 21, 205 (1956). 2. Leloir, L. F., and Cardini, C. IS., Biochim. et biophys. acta, 12, 15 (1953).

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712 SYNTHESIS OF GLIJCOSAMINE 6-PHOSPHATE

3. Lowther, D. A., and Rogers, H. J., Biochem. J., 62, 304 (1956). 4. Roseman, S., Moses, F. E., Ludowieg, J., and Dorfman, A. J. Biol. Chem., 203,

213 (1953). 5. Becker, C. E., and Day, H. G., J. Biol. Chem., 201, 795 (1953). 6. Topper, Y. J., and Lipton, M. M., J. Biol. Chem., 203, 135 (1953). 7. Roseman, S., Ludowieg, J., Moses, F. E., and Dorfman, A., J. Biol. Chem., 206,

665 (1954). 8. Blix, G., Acta them. Stand., 2, 467 (1948). 9. Pogell, B. M., and McGilvery, R. W., J. Biol. Chem., 208, 149 (1954).

10. LePage, G. A., in Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Manometric techniques and tissue metabolism, Minneapolis, 185 (1949).

11. Maley, F., and Lardy, H. A., J. Am. Chem. Sot., 78, 1393 (1956). 12. Paladini, A. C., and Leloir, L. F., Biochem. J., 61, 426 (1952). 13. Dische, Z., and Borenfreund, E., J. Biol. Chem., 184, 517 (1950). 14. Pontis, H. G., J. Biol. Chem., 216, 195 (1955). 15. Cabib, E., Leloir, L. F., and Cardini, C. E., J. Biol. Chem., 203, 1055 (1953). 16. Stoffyn, P. J., and Jeanloz, R. W., Arch. Biochem. and Biophys., 62, 373 (1954). 17. Kay, R. E.: Harris, D. C., and Entenman, C., Arch. Biochem. und Biophys., 63,

14 (1956). 18. Redfield, R. R., Biochim. et biophys. acta, 10, 344 (1953). 19. Consden, R., Gordon, A. H., and Martin, A. J. P., Biochem. J., 38, 224 (1944). 20. Buyske, D. A., Flowers, J. M., Jr., Wilder, P., and Hobbs, M. E., Science, 124,

1080 (1956). 21. Brown, D. H., Biochim. et biophys. acta, 7, 487 (1951). 22. Leloir, L. F., and Cardini, C. E., Biochim. et biophys. actu, 20, 33 (1956). 23. Bostrom, H., Roden, L., and Vestermark, A., Nature, 176, 601 (1955). 24. Roden, L., Ark. Kemi, 10, 333 (1956). 25. Blumenthal, H. J., Horowitz, S. T., Hemerline, A., and Roseman, S., Bact. Proc.,

137 (1955). 26. Goldthwait, D. A., J. Biol. Chem., 222, 1051 (1956). 27. Bentley, M., and Abrams, R., Federation Proc., 16, 218 (1956). 28. Neidle, A., and Waelsch, H., J. Am. Chem. Sot., 78, 1767 (1956). 29. Neuberg, C., Lustig, H., and Rothenberg, M. A., Arch. Biochem., 3,33 (1943-44). 30. Purchase, E. P., and Braun, C. E., Org. Syntheses, 26, 36 (1946). 31. Partridge, S. M., Nature, 164, 443 (1949). 32. Block, R. J., Durrum, E. I,., and Zweig, G., A manual of paper chromatography

and paper electrophoresis, New York, 88 (1955).

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Burton M. Pogell and Rosa M. GryderLIVER

GLUCOSAMINE 6-PHOSPHATE IN RAT ENZYMATIC SYNTHESIS OF

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