THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed m U. S. A. Vol. 255. No. 18, Issue of September 25, pp. 8458-8464. 1980

Purification, Structure, and Catalytic Properties of L-myo-Inositol-1- phosphate Synthase from Rat Testis*

(Received for publication, June 18, 1979, and in revised form, May 2, 1980)

Toshio Maeda and Frank Eisenberg, Jr. From the Laboratory of Biochemistry and Metabolism, National Institute of Arthritis, Metabolism and D%estive Diseases, National Institutes of Health, Bethesda, Maryland 20205

L-myo-Inositol-1-phosphate synthase (EC 5.5.1.4) has been purified to homogeneity for the first time and its properties were investigated. By means of ammonium sulfate precipitation from rat testis supernatant, fol- lowed by chromatography on DEAE-cellulose, Ultro- gel, glucose 6-phosphate-Sepharose, and hydroxylapa- tite, the synthase was purified 460-fold to a specific activity of 250 milliunits/mg of protein (4.2 mkat/kg). The enzyme requires NAD+ and is optimally active at pH 7.7. The molecular weight determined by chroma- tography, electrophoresis, and sedimentation equilib- rium is 210,000; the subunit weight is 68,000, a result which suggests a rare trimeric structure. Monovalent cations, among which N€L’ is the most effective, de- creased the K,,, for both glucose 6-phosphate and NAD’ from the basal values of 3.8 l l l ~ and 17.9 p ~ , respec- tively. With the exception of Li’, which is inhibitory, monovalent cations increased the activity up to 400%, suggesting a possible regulatory role for these ions. Divalent cations had no effect and heavy metals in- hibited strongly. EDTA had no effect, suggesting that although metals can alter the activity, metal is not required for basal activity. 2-Deoxyglucose Q-phos- phate and 2-deoxyglucitol 6-phosphate inhibited strongly. Antibody against the homogeneous enzyme, prepared in rabbits, cross-reacted with testis, epididy- mis, and brain synthases showing identity of the three enzymes. The immunoprecipitated enzyme complex from either testis or brain is about 60% as active as the corresponding soluble enzyme. No binding of labeled substrate through a Schiff base could be detected, nor was the enzyme inactivated by borohydride reduction. Recent studies showing that epididymis is a richer source of synthase than testis could not be confirmed. In testis of diabetic animals, although free inositol lev- els were 2 to 3 times higher than normal, there was no change in the level of inositol-1-phosphate synthase.

L-myo-Inositol-1-phosphate synthase (EC 5.5.1.4) catalyzes the irreversible isomerization of D-glucose 6-phosphate to L- myo-inositol 1-phosphate (3). The enzyme is essential to the organism as the sole supplier of the 6-carbon myo-inositol ring, the precursor of the inositol phospholipids which are important structural components of all membranes. Since NAD’ is required for the reaction, intermediate oxidoreduc-

‘This work was presented in part a t the 69th and 71st annual meetings of the American Society of Biological Chemists, June 4-8, 1978, Atlanta, Georgia (1) and June 1-5, 1980, New Orleans, La. (2), respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

tion is probably involved in the catalytic mechanism. Al- though intermediates have not been isolated, indirect evidence supports a mechanism in which substrate is oxidized to 5- ketoglucose 6-phosphate which in turn is cyclized to inosose- 21-phosphate in an intramolecular aldol condensation (4-9). Reduction by NADH, the putative nucleotide intermediate, then leads to the product.

Owing to the lack of purified synthase, it has not been possible to ascertain whether the isomerization is catalyzed by a single enzyme, how subunits are implicated, to which class (I or 11) the postulated aldolase function conforms, and what effect cations have on the activity.

With the realization of homogeneous enzyme for the first time most of these questions have been answered. Moreover, the purification of the enzyme has enabled the preparation of antibody, with the result that synthases from various sources under normal and pathological conditions can be compared, isolation of the enzyme is simplified, and sensitivity of both detection and assay is enhanced.

With purified enzyme, furthermore, it has been possible to resolve a conflict in interpretation of the mechanism of attack of the enzyme on the substrate. Our data are consistent with the conclusion of Sherman et al. (10) who could find no Schiff base involvement in the synthase mechanism, contrary to Hoffmann-Ostenhof et al. ( l l ) , who argue for a Schiff base intermediate.

RESULTS’

Purification-Table I summarizes the purification of s p - thase from rat testis. Owing to interfering enzymes, it was impossible in early experiments to determine specific activity in crude fractions, precluding an overall estimate of purifica- tion. With the later development of antibody specific to the pure enzyme (see below), it became feasible to assay crude fractions as shown in the first part of the table. The second part of Table I thus deals with relatively pure fractions only. From the two sets of data, overall purification was estimated at 450-fold. The agreement between the specific activities in the two ammonium sulfate fractions is within experimental error.

Fig. 1, a to e shows electrophoretic patterns of the second fractions of Table I. With successive purification, the number of protein bands decreased to a single band at the hydroxyl-

’ Portions of this paper (including “Experimental Procedures,” Figs. 1 to 11 and Tables I to VI) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014. Request Document No. 79M-1209, cite authors, and include a check or money order for $3.15 per set of photocopies. Full sized photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

8458

I. -myo-Inositol-1-phosphate Synthase 8459

apatite stage, interpreted a t this point to be homogeneous enzyme. Although there was little effect on the electrophoretic pattern, the specific activity nearly doubled as a result of affinity chromatography (Table I). Fig. 2 identifies the main protein band with the active enzyme at the affiiity stage. Fig. 3 illustrates the sharp elution of synthase from the affinity adsorbent with the KC1 gradient.

In a further test of homogeneity, the purified enzyme was examined for several other activities: glucose-6-phosphatase, inositol-1-phosphatase (12), glucose-6-phosphate dehydrogen- ase (13), glucose-6-phosphate isomerase (14), fructose-1,6-bis- phosphate aldolase (15), lactate dehydrogenase (15), glycerol- 3-phosphate dehydrogenase (15), and the inosose reductase system (16). None of these activities was found, although all were demonstrable in crude testis, and all have some feature in common with synthase, substrate, cofactor, or probable mechanism.

NAD+ Requirement-To elicit maximal activity at all stages of purification, the addition of NAD' was required as shown in Table I, last column. With increasing purity the relative activity declined to about 2% of maximal activity with NAD'. We conclude that homogeneous enzyme has an abso- lute requirement for NAD'.

pH Optimum-Maximal activity was found at pH 7.7. At pH 8 the activity was halved and completely abolished at pH 6.

Stability-Enzyme at the ammonium sulfate stage was stable for several months at -20°C. With further purification, stability decreased but no significant loss of activity occurred during the 10 to 14 days required for purification. Purified enzyme rapidly lost activity on freezing and thawing.

Molecular Weight-Three methods were used to determine the molecular weight: polyacrylamide gel electrophoresis (Fig. 4), ultracentrifugal sedimentation equilibrium (Fig. 5), and gel fitration (Fig. 6). All methods gave the same value of 210,000 in agreement with earlier reports (4, 17).

Subunit Weight-Although continuous sodium dodecyl sul- fate-polyacrylamide gel electrophoresis of the enzyme showed a single major subunit band (Fig. If), discontinuous electro- phoresis revealed a doublet (Fig. lg) suggesting that the subunits are not identical. Electrophoretic comparison with standard proteins established the subunit weight at approxi- mately 68,000 (Fig. 7).

Effect of Inhibitors-In agreement with Barnett et al. (18) the activity was inhibited by 2-deoxyglucose 6-phosphate and 2-deoxyglucitol6-phosphate to the extent of about 50% at 0.1 mM and completely at 1 m. Analogous to the observed failure of N-acetylglucosamine 6-phosphate to inhibit (M), neither glucosamine 6-phosphate nor glucosaminitol 6-phos- phate inhibited at 1 mM.

Effect of Cations and EDTA-With 5 m~ glucose 6-phos- phate and 1 m NAD' in 50 m~ Tris buffer, the enzyme exhibited basal activity linear with enzyme concentration up to 15 pg/O.5 ml and time for at least 2 h. Table I1 shows the effect of cations at 1 m~ concentration. These ranged from strongly inhibitory (Cu2+, H e , Zn", and Cd") to strongly stimulatory (K' and NH4'). EDTA up to 100 KIM had no effect on basal activity. The lack of metal requirement implied by this result eliminates the enzyme from Class 11 (metal- requiring) aldolases. At 10 m, EDTA added first protected the enzyme from heavy metal inhibition; addition of EDTA after the metal reversed Cd2' and Zn2+ inhibition but not CU*+. Fig. 8 shows the effect of monovalent cations as a function of concentration. NH,' and K+ are powerful stimu- lants, significantly effective at as low as 0.02 m~ and 0.2 mM, respectively. Li' varied from slightly stimulatory to slightly inhibitory between 10 and 100 m ~ . Although the effect of Na'

was also small, it interfered strongly with NH,' and K', reducing the stimulatory effect of each cation by a factor of 10 (not shown). Li' had a similar but smaller effect. Ca2' and M e depressed the basal activity 50% at 40 mM with similar effect on NH4' and K' stimulation. Table I11 shows the effect of cations on K,,, for glucose 6-phosphate; although Li' and NH,' had vastly different effects on activity (Fig. 8), both ions caused a sharp decrease in K,,, from the basal value of 3.89 m ~ . These cationic effects may be important in the regulation of synthase activity and will be described in greater detail in a later publication.

Mechanism of Action-Since synthase had already been excluded as a Class I1 (metal-requiring) aldolase by experi- ments just described, it was of interest to determine if the enzyme fitted the Class I description (Schiff base-forming). Conflicting results from two laboratories (10, 11) led us to attempt to answer this question through borohydride reduc- tion experiments essentially identical to those of Pittner and Hoffmann-Ostenhof (19). Contrary to these authors, as shown in Table IV, we found no specific inactivation (i.e. inactivation in excess of control without substrate) of the enzyme as a result of borohydride reduction in the presence of substrate at either pH 6.0 or 7.4. In Fig. 9 (solid circles), in agreement with Pittner and Hoffmann-Ostenhof (20), we found no stable binding of I4C-labeled substrate to the 68,000-dalton subunit after borohydride reduction. Both results are thus consistent with the conclusion of Sherman et al. (lo), based on the failure of either substrate or medium '*O to be incorporated into product, that a Schiff base is not involved. But Pittner and Hoffmann-Ostenhof's argument for a Schiff base mecha- nism is based mainly on observed binding of labeled substrate to a 35,000-dalton subunit (20), a finding which we could not confirm, since homogeneous enzyme is devoid of such a sub- unit. We could, however, mimic the result if we used impure enzyme at the DEAE-cellulose stage (Fig. 9, open circles). The implication of this finding is that synthase purified by NAD'-Sepharose (17) is not homogeneous.

Antisynthase Antibody-With pure enzyme now available, it became possible to prepare specific anti-synthase antibody, which cross-reacted with crude testis supernatant, brain DEAE-cellulose fraction, and crude epididymal supernatant (Fig. lo), showing identity among these synthases. Preimmune serum showed no precipitin lines in the same test, nor could precipitin lines be demonstrated with pituitary, pancreas, or uterus. A faint line was seen with ovary (not shown).

Fig. 11 illustrates a test of the enzymic activity of the immune complex of both testis and brain synthase. No dimi- nution in soluble synthase activity was seen on addition of preimmune serum. With specific antiserum there was com- plete removal of synthase activity from the supernatants. The activity resided solely in the pellets to the extent of about 60%, indicating partial (40%) masking of the active site by the antibody. The possibility that loss of activity was the result of washing of the immunoprecipitate was excluded by incubation of testis enzyme with excess antiserum and assay of activity without separation of the immune complex. Recovery of ac- tivity was 64%, showing no significant loss due to washing.

Recently, Robinson and Fritz (21) reported that whole epididymis is about 15 times as rich a source of synthase as whole testis. By means of immunoprecipitation with antibody, we examined epididymis of mature rats and found, on the contrary, the specific activity, even after partial purifcation with ammonium sulfate (Table V), only one-eighth that of testis (Table I, third line). Ouchterlony double diffusion of antibody with epididymis and testis extracts from young rats also failed to confirm the observation of Robinson and Fritz (not shown).

8460 L -myo-lnositol-l-phosphate Synthase

Inositol Metabolism in Diabetic Rats-In a further appli- cation of the immunochemical assay, synthase activity was determined in rat testis 3 weeks after induction of diabetes by streptozotocin (Table VI). With neither crude supernatant nor partially purified enzyme was there any difference be- tween diabetic and control animals. The large decrease (130%) in testicular synthase reported by Whiting et al. (22) might reflect the longer duration of diabetes (12 weeks) in those experiments. With respect to free inositol levels in various tissues (Table VI) of the diabetic rat, there was no difference in serum, about 25% increase in brain, and 170% increase in testis compared with normals. These results are in substantial agreement with those of Palmano et al. (23) who showed no change in serum and brain levels of free inositol 2 weeks after streptozotocin (35 mg/kg) induction of diabetes. The slight increase in brain inositol reported in Table VI could be the result of the larger dose of streptozotocin (75 mg/kg), and the longer duration of diabetes in our study. The biosynthetic pathway from glucose 6-phosphate to free inositol requires, in addition to synthase, specific inositol-1-phosphatase which is present in excess (3). Since the synthase level remained con- stant during the diabetic period, the formation of free inositol could not have increased. Decreased utilization must therefore account for the accumulation.

DISCUSSION

Affinity Chromatography-After several unsuccessful at- tempts to retard the enzyme on glucose 6-phosphate immo- bilized on Sepharose through various types of amino spacer (24,25), it became apparent that a net negative charge on the adsorbent was essential to the binding of the enzyme. For this reason, glucose 6-phosphate bound to epoxy-activated Seph- arose was selected for afti-mity chromatography. Consistent with these observations is the fact that inhibitors of the enzyme are all negatively charged and aminosugar phosphates (net zero charge) are without effect (18). Although three additional chromatographic steps were necessary to bring the enzyme to homogeneity, the use of affinity chromatography alone following ammonium sulfate fractionation effected a 15- fold purification (data not shown). That the column was specific for glucose 6-phosphate-dependent enzymes was shown by the failure of glycerol phosphate dehydrogenase to be retarded and the identical elution pattern of both synthase and glucose-6-phosphate dehydrogenase.

Subunit Weight-Although the molecular weight (210,OOO) determined in this study agrees with previous estimates for the rat testis enzyme, subunit weights differ markedly from those reported by Pittner et al. (17) for synthase purified on NAD'Sepharose. Our preparation contained no 35,000-dal- ton subunit as reported by these authors. In the present study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed heterogeneous subunits of about 68,OOO daltons, re- sults that suggest that the intact enzyme is a trimer composed of at least two different subunits (26). Duckweed (Lemna gibba) synthase is also thought to be a trimer (27). The role subunits play in the synthase reaction remains to be eluci- dated.

Mechanism of Action-Postulation of an intramolecular aldol condensation in the synthase catalytic mechanism has raised the question of the type of aldolase the enzyme repre- sents, Class I (Schiff base-forming) or Class I1 (metal-requir- ing). Our results, showing lack of an EDTA effect, confirm earlier observations (17, 18,28) that animal synthases are not of Class 11. Does that necessarily place them in Class I? On the basis of l 8 0 exchange studies, Sherman et al. (lo), using enzyme purified similarly to the Ultrogel stage, found no incorporation of isotope into product from either substrate or

medium, an approach classically used to determine Schiff base involvement in enzymatic reactions (29). Synthase was thus eliminated from Class I, prompting Sherman et al. (10) to propose an alternative mechanism for ring closure.

Contrary to Sherman et al., Pittner and Hoffmann-Ostenhof (19) found incorporation of medium l80 into product, pointing to the involvement of a Schiff base. Clearly the results of the two laboratories are in conflict. In view of our inability to find a subunit smaller than 68,000 daltons, we suspected that the discrepancy between the two schools lay in the purity of the NAD'-Sepharose enzyme (17). The difference in specific ac- tivity between that preparation (154 milliunits/mg) and the present glucose 6-phosphate-Sepharose preparation (250 milliunits/mg) alone suggested inhomogeneity. Recently, these authors reported crystallization of a homogeneous pro- tein from rat testis (30); evidence sufficient to identify it as synthase was not given.

If the results of Pittner and Hoffmann-Ostenhof can be accounted for by an impurity, it should be possible to repeat their observation of irreversible binding with impure enzyme. Fig. 9 confirms this prediction by showing binding of substrate to a M, = 35,000 species associated with synthase at the DEAE-cellulose stage of purification. Since this material ul- timately disappears with further purification, the observation in no way supports the Schiff base mechanism. On the con- trary, our evidence, like that of Sherman et al., fails to categorize synthase as either a Class I or Class I1 aldolase. It is interesting in connection with the crystallization reported by Pittner and Hoffmann-Ostenhof (30) that we likewise succeeded in crystallizing a protein at the affiiity chromatog- raphy stage; it was completely devoid of synthase activity.

In attempting to adhere as closely as possible to the condi- tions of Pittner and Hoffmann-Ostenhof (20), we also tested for specific inactivation of the enzyme after reduction at pH 6.0 (Table IV) and found none, although the enzyme was largely inactivated nonspecifically. The selection of pH 6.0 by Pittner and Hoffmann-Ostenhof for their binding study has been explained by Morse and Horecker (31) and Schellenberg (32), who showed that the optimal condition for reduction of Schiff bases is pH 6.0, even though the pH optimum for catalytic activity may be considerably different, as with syn- thase.

Antibody-This is the first preparation of antibody specitic to synthase, a development following from the purification that should facilitate both the elucidation of the reaction mechanism and the localization of the enzyme within organs and subcellular organelles. By means of antibody it was pos- sible to determine overall purification of the enzyme from crude extracts under conditions which precluded the use of conventional assay methods (Table I). Furthermore, we were able to show that synthases from all organs of the rat studied are identical (Fig. 10). On this basis we examined epididymal synthase and, contrary to the report by Robinson and Fritz (21), confmed our earlier findhgs, based on a radiochemical assay (33, that epididymis exhibits only a fraction of the synthase activity of testis. A more puzzling result of this recent work (21) is the exceedingly high specific activity of whole testis synthase observed (0.311 nmol/min/pg), making this preparation far and away the most active synthase yet dem- onstrated from any source, some 650 times as active as high speed supernatant in our hands ( O . o o o 4 s nmol/min/pg, Table I). Whether such high activity is realistic is open to question.

Acknowledgment-The authors thank Dr. Edward Steers, Jr., National Institute of Arthritis, Metabolism and Digestive Diseases, for the determination of molecular weight by sedimentation equilib- rium.

L -myo-Inositol-1-phosphate Synthase 8461

Note Added in Proof-The effects of streptozotocin-induced dia- betes on testicular levels of synthase and free inositol (Table VI) are in remarkably close agreement with the recent results of Rancour and Wells (34). Bovine testis synthase has recently been purified to homogeneity (35).

REFERENCES

1. Maeda, T., and Eisenberg, F., Jr. (1978) Fed. Proc. 37, 1525 2. Maeda, T., and Eisenberg, F., Jr. (1980) Fed. Proc. 39, 1854 3. Eisenberg, F., Jr. (1967) J. Biol. Chem. 242, 1375-1382 4. Barnett, J. E. G., Rasheed, A., and Corina, D. L. (1973) Biochem.

5. Eisenberg, F., Jr. (1978) in Cyclitols and Phosphoinositides (Wells, W. W., and Eisenberg, F., Jr., eds) pp. 269-278, Aca- demic Press, New York

6. Chen, I.-W., and Charalampous, F. C. (1967) Biochim. Biophys. Acta 136,568-570

7. Chen, C. H.-J., and Eisenberg, F., Jr. (1975) J. Biol. Chem. 250,

8. Sherman, W. R., Stewart, M. A., and Zinbo, M. (1969) J. Biol. Chem. 244,5703-5708

9. Hauska, G., and Hoffmann-Ostenhof, 0. (1967) Hoppe-Seyler’s 2. Physiol. Chem. 348, 1558-1559

10. Sherman, W. R., Hipps, P. P., Mauck, L. A., and Rasheed, A. (1978) in Cyclitols and Phosphoinositides (Wells, W. W., and Eisenberg, F., Jr., eds) pp. 279-295, Academic Press, New York

11. Hoffmann-Ostenhof, O., Pittner, F. and Koller, F. (1978) in Cycli- tols and Phosphoinositides (Wells, W. W., and Eisenberg, F., Jr., eds) pp. 233-247, Academic Press, New York

12. Eisenberg, F., Jr. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed) Vol. 3, 2nd English Ed., pp. 1337-1341, Academic Press, New York

13. Ghr, G. W., and Waller, H. D. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed) Vol. 2, 2nd English Ed., pp. 636-643, Academic Press, New York

14. King, J. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed) Vol. 2, 2nd English Ed., pp. 1113-1117, Academic Press, New York

15. Bergmeyer, H. U., Gawehn, K., and Grassl, M. (1974) in Methods

J. 131,21-30

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of Enzymatic Analysis (Bergmeyer, H. U., ed) Vol. 1, 2nd English Ed., pp. 425-522, Academic Press, New York

16. Hipps, P. H., Sehgal, R. K., Holland, W. H., and Sherman, W. R. (1973) Biochemistry 12,4705-4712

17. Pittner, F., Fried, W., and Hoffmann-Ostenhof, 0. (1974) Hoppe- Seyler’s 2. Physiol. Chem. 355,222-224

18. Barnett, J. E. G., Rasheed, A., and Corina, D. L. (1973) Biochem. SOC. Trans. 1,1267-1269

19. Pittner, F., and Hoffmann-Ostenhof, 0. (1976) Hoppe-Seyler’s 2. Physiol. Chem. 357, 1667-1671

20. Pittner, F., and Hoffmann-Ostenhof, 0. (1978) Hoppe-Seyler’s 2. Physiol. Chem. 359,1395-1400

21. Robinson, R., and Fritz, I. B. (1979) Can. J . Biochem. 57, 962- 967

22. Whiting, P. H., Palmano, K. P., and Hawthome, J . N. (1979) Bwchem. J. 179,549-553

23. Palmano, K. P., Whiting, P. H., and Hawthorne, J . N. (1977) Biochem. J. 167,229-235

24. Funkhouser, E. A., and Loewus, F. A. (1975) Plant Physiol. 56,

25. Jeffrey, A. M., Zopf, D. A., and Ginsburg, V. (1973) Biochem.

26. Wood, W. A. (1977) Trends Biochem. Sci. 2,223-226 27. Ogunyemi, E. O., Pittner, F., and Hoffmann-Ostenhof, 0. (1978)

Hoppe-Seyler’s 2. Physwl. Chem. 359,613-616 28. Naccarato, W. F., Ray, R. E., and Wells, W. W. (1974) Arch.

Biochem. Biophys. 164, 194-201 29. Horecker, B. L., Tsolas, O., and Lai, C. Y. (1972) in The Enzymes

(Boyer, P., ed) Vol. 7, pp. 213-258, Academic Press, New York 30. Pittner, F., and Hoffmann-Ostenhof, 0. (1979) Mol. Cell. Bio-

chem. 28,23-26 31. Morse, D. E., and Horecker, B. L. (1968) Adu. Enzymol. 31, 125-

181 32. Schellenberg, K. (1963) J. Org. Chem. 28,3259 33. Eisenberg, F., Jr., and Bolden, A. H. (1964) Nature 202,599-600 34. Rancour, T. P., and Wells, W. W. (1980) Arch. Biochem. Biophys.

35. Mauck, L. A., Wong, Y.-H., and Sherman, W. R. (1980) Biochem-

Additional references are found on p. 8464.

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istry 19, 3623-3629

8462 L -myo-Inositol-1-phosphate Synthase

C h d c r l r epoxy-actlwtcd SCphamse 6 8 ) 1.1 washed wlth d i s t i l l e d water for 1 h on a glass fllter and then incubated f o r 20 h a t 37' i n 20 nl of 0.1 M b i c a h a t e - c a d m a t e b u f h r (pH 9.1) containing 50 nu glycose 6-pkmphata. The binding of substrate m s mnitond by 9 s - l i q u i d chromatography of the supernatant; In the absence o f support them was no da- c m s c I n the aaunt o f so lub le glucose 6-phmphatc. The incubation was mntlnued an addit-

ing -4 groups. Flnal ly the d twi .1 was Mashed r l t h water. 0.1 M b r a t * buffer (pH 0.0). ional M h a t 37' i n 0.1 M HaHCO containing 1 M Cthanolamlne a t pH 9 . 5 to Inac t iva te -in-

0.1 M acetate buffer (pH 4.5). and 50 dl TPis-eCetdte buf fer (pH 7.1).

Vrep.r&tion of AfflniW Adsorbent - 10 g of bisox lnne- l inked s . h m s e (Ph.mcla Fine

- Mala rabbi ts (2.5 kg) were inunind by U l t i p l e i n t r a d c m l .1 *.ch Of an ~ l S i O " -ad O f m yg hwg.Mous r a t t n t i s

Jurant (Calbiocha). Four mlu l a t e r a s l m i l w i n j e c t i o n was g l r m f o l l a e d OIU w m k l a t a r synthase i n 1 m1 O f 0.1 M hC1. M dl Tris-acetat. m1x.d With 1 11 o f Freund's c o q l e t a ad-

by bleeding of the mlml from an tap artery. Twenty-five a1 o f s e w decanted fmn the Clot after spontaneous co Ulation overnight i n the cold 1.1 di lu ted w i t h an equal v o l u of phMphate-buffered ralineqGrBC0) and mixed Wlth 50 a1 Of I l t U I a t a d m n i m Sulfate. Tha p 'cc lp l ta te a f tw cen t r i fugat ion was dlssolvad i n phosphate-buffered saline. the v o l u ad- justed to 20 ml. and bmught t o 201 r s t v n t i o n with addltion Of 5 nl of saturated Mi- sulfate. The supernatant a f te r cen t r i fugat ion was bmught to 331 saturation by the addi t ion of another 5 m1 of saturated a n a n l m sulfate. me resu l t i ng pNCip i t s ta was washed thm

of i r is-acetate buf fer ( 5 0 M , pH 7.7) containing 0.1 M KC1. The IOlUtlon was dldlyzed tilnel " 1 0 33S saturated m n l m SUlfdte i n phosphate buf fered ra l lne and dissolved I n 10 m1

agalnrt the < m e buffer overnight. F m 25 nl o f a n t i r e r u ZW mg o f p m t e i n was obtalncd. A control w l t h an uninnunlzed rabbi t ( p r e l m n e s e n d produced 150 mg o f protein.

Nbaka+of Inos i to l 1- rphate 5 thase - S t k s e or synthase-antibody clxlplex was i n -

(pH 7.4) a t 37'. Inos i to l 1-phosphate p m d m d during 1 h o w of incubatton was measured i n wo w a y : i n general. for m u t i n e assay the periodate method of Barnett e t a l . ( 1 1 *as used

M e r conditions &re I hlgh inorganlc phosphate background w u l d Interfere, gas- l iqu id In which inorganic phosphate I s l iberated specifically fm Inos i to l 1-phoTpiiite and assayed.

chmnawraphy Of the t r i m e t h y l s i l y l ethen was used d l described by Wajundcr and Elsenberg

depmtelnization. (2 ) . m d l f i e d to include r m v a l of Cat ims rlth Reryn 101(H) (F i rher Sc lent i f ic Co.) a f t e r

1 K g l u c o s e 6"ph%hate (5 b lUo'(1 e) i n 0.5 111 of 50 dl Trls-HCl buffer

chmatogra and the apperMnce. on subseqwnt gas-liquid chmatography, of an equivalent The i d m t l t y o f inositol 1-phaph.ta tias nrificd by i t s c o q l e t a d i s a p p M r w e fm the

m t of Inos i to l and inorganic pkmp?ak as t r lmethy ls i l y l der iva t ives a f te r t reahent of

0.1 to 0.3 w g l c o f I n o s i t o l 1 - W r p h a t e was produced for assay. the synthase reaction mixture with P ~ J -&pendolt specific i m r l t o l 1-phosphatase (3). F m

~- P u r i f i c a t i o n o f - a l l s t e p were carried o u t a t 4' except as now. H-izatim. C M t r i f W a t i m . M d & a t TRlt"lt - W5-S UZighing 120 g fmn 50 Orborn-&n&l ra ts IZW-ZM g each1 were decapsulated and h-nired i n 6 batches. 2 mi" each. i n a Potter-Elvehjm h m - genlzcr In a t o t a l v o l m of 240 m1 Of 0.154 M KC1 cmta in ing 0.2 M d l t h i o t h r e i t o l . The hnogenate MI Centrifuged a t 10.000 x 9 for 1 h w r and the resul t ing supernatant (3.9 9 of proteln) was heated a t M)' f o r 2 nin. Centri fugation at 40.W x p gave 240 ml of supernatant.

m i m Sulfate Fractlonation - the p e l l e t Dbtained between 30 and Un saturation was dissolved I n 76.1 o f standard buffer (50 Iw Tris-acetate. pH 7.1. Contdlnlng 0.2 nll d l t h i o t h r e i t o l ) and dlalyzed against the Sam? buffer overnight.

C h m t o q r a p h y on 0E"CellulOSe - the dialyzed en- Was appl ied to

wi th s tanddk buf fer . Af ter a 1 liter wash with the fam buffer. the a c e l m ( 2 4 x 30 on1 Of DL r ( H l a t n a n 1 r h l c h had been equi l ibrated

e n z p was eluted rlth 600 ml of standad buffer cmtalning 1 l lnear a n d l e n t o f KC1 f m 0 to 0.5 M. Fractions Of 5 "1 *ere col lected a t kJ.1 pr h w r .

6rl F i l t m t i a coltm (2.5 x 97 a) Of U l t m g e l AcA 34 ( L B ) was

fraCtiOM wen apQ1i.d and Slutad With the S U SOlutlOn. F r s c t l o M wen collected as described above. A f f l n i t y LhmwtWrav - a c o l u l (1.5 x 15 a) o f glucose 6-phosp)ute bund to e rmxy-ac t ivak fsphemsa vas equi l ibrated w i t h standard buffer. P w l e d f m c t l o m were dlalyzed against stanbrd buffer and appl ied to the colvm followed by Xa nl o f standard buf fer and then another 180 nl O f the s d m buffer containing a l i n e a r g r l d i m t o f KC1 fmln 0 t o 0.3 M. F m c t i o m Of 3 a1 wen Col lected a t M ml par h. Chmutoqra h on d m l a t i r e - a c o l u n (1.0 x 15 a) of Blo-6el

containing 0.1 M KC1 and 0.2 nll dl th lo thre i to l . Pwled f ract ions wem RT (Blo-Rad? e%ll%ts w V h Sodlm phMphate buffer (5 M, pH 7.4)

appl ied to the calm followed by 40 m l of the s a buffer. Ylth rCl and dlthlothrei to l concentmtlons hold constant. the concentration of phosphate was raised s t e p l s e (25. 50, 100. and 330 m), 60 m1 a t each step. Fractions of 1.5 ml rere col lected a t 5 m1 wr h and assayed f o l protein. Peak fractions we" pooled and assayed f o r enzymic act-

Up t o 1W mM phosphate. only that eluted with 25 .)I phosphate was MZ- i v i t y by gas-liquid chmllatography. Although p m t e l n was eluted with

GFilma%- th s t a h r d buffar mteining 0.1 U El. Pwled act ive

YniCdllY actire.

Tabla I

Pur i f icat ion of L ~ i m i t o l 1-phOrph.ta Smthasa

Synthu. fm r a t t a s t i s haopmta *u pUr4fi.d as d n c r t b a d i n Expwiat.1 Pmudum. Fractions abow the b m h lln w n ass& altar iunopmclp- i tat ion wi th ant ibody and sp.clflc a c t i d t i n warn cowactad for Un l m s o f a c t i v l t y i n the i u n O p n c i p i t a t . ( s a a F i g 11). Onr.11 purification was 21610.48 or l i o - f o l d . h l a t l w e c t l r i t y I s p-t a c t l v l t y I n the ab%- of adhd rw* n l a t l n t o t h a t with addad W* (1 *). Ore dJ of m y r pm- dwsd 1 m a l of i n o l i t o l 1-phorphrt. wr min fm 5 dl IYCM. 6-phorphat.. Vmtc in was datanimd by the mthod of L m y .r a. (17.

561. 155 30

xx)..

68 10

" """.

5.7 2.6

0.48 1.2 5.3

8.1

104 36

1% 216-

8.5 0.9 1.2 1.9 1.8

a b C d e f 9

SLICE NUMBER BPB

(-1 (+:

L -myo-Inositol-

FRACTION NUMBER

Figure3 - 4 f f i n i t y c h r m t a p r a p h y O f l n o s l t o l 1-phosphate synthase. Eight hund- red Ru o f e n z w eluted frm Ul t rogel vas chrwtographed on glucose 6-phosphate Dmnd t o epoxy-activated Sepharare es described i n Exprlmental Pwcedure.

I

15 t I-1-P SYNTHASE

LBSA (Dimer)

20 25 MOLECULAR WEIGHT IX lodl

Twenty t o M YB each Of i n o s i t o l 1-phosphate lynthase hydroxylapatite f ract ion FIQUIP 4 - Molecular WelJht of l n o r l t o l I-phosphate synthase by electrophoresis

(I-1-P), owlbumin IOV), bovine serum albumin (BSA) , and alcohol dehydrogmare

38:l O f acrylamide t o N,N'-mthylene blrdcrylmide. Negat lve slope^, CalCUldted ( A w l ) were electrophoresed on 3.9% polyacrylamide gels at a constant rat lo of

w t h molecular weight according to the method of Hednck and Smth (8). frm plots o f r e l a t l v e l n o b i l i t l e r versus gel COnCentrat lmI, varied l inearly

14,000 RPM 3.0

MOLECULAR WEIGHT:

1.5 209,800 49 50

c m 2

F79ut-e 5 - lb lecular weight of i n o s i t o l I-phosphate synthase by r e d i m n t - a t ian equlllbrium. P r t a n d a r d buffer solut ion of p u r i f i e d synthase

el E a n a l y t i c a l Y l t r ~ e n t r i f u g e equipped with Rayleigh interference (about 3 mg/rnlTrar centr i fuged a t 14.000 rm at 25- i n the Spina, m d c

w t i c r . m l e r u l a r weight was calculated fm the slope of the curve E l a t i n g f r i n g e d i r p l a c m n t 1") to ce l l rad ius lm) by the mthod o f Yphantir (9).

I

Y 2 0 .~1

0' 6 8 10 I

15 20 25 MOLECULAR WEIGHT (x 104t

1 -phosphate Synthase I I

0.9 - MAJOR BAND

0.7

0 -

0.5 - a MINOR BAND

0.3 - 19) - 4 6 8 10 15 202530

MOLECULAR WEIGHT (X lo4)

Table I1

E f f e c t Of Cations on Synthase A c t i v i t y

PUrifled synthase Y11 assayed 15 described I n Experimental Procedure with cations replaced by T r i l f o r basal a c t i u t y (100%). Values were corrected ln sepwate controls fo r

after incubation. Added sa l ts (1 rrH) were a11 chlorides the effect On the periodate al lay method O f cations added

except Zn++ vhich was the acetate.

Cation % A c t l v i t y

Fe+++ 82

C"++

Hg++

10- 2

CDt' 66

M"++ 90

hi+ 94

Cat+ 96

h + + 101

Ll+ 97

Nd+ 120

K t 275

nn4+ 4m

0

0

Cd" 2

[sal t l I(n nu nll

8463

Basal 3.89

NaCl 50 1.35

KC 1 50 0.31

NHnCl 20 0.26

LiCl 50 0.28

8464 L -myo-Inositol-1-phosphate Synthase

Table I V

E f f e c t O f Borohydride Reduction on Syntkare k t i v i t y

TwV 0.5 np q u a n t i t i p Of p u r i f i e d e n m (hydmxylapatite fraction. 250 nlllnp protein) were pre-incubated a t 37 and pH 7.4 for 30 s 4 t h Substrates indicated i n 1 nl O f 50 nll lr is-HCl buffer containing 2 nll NWCl and 50 p p I n t i f o s n C Emlsion (Signa). One sample was then ac id i f ied to pH 6.0 with 2 I4 acetic acid. treated with 5 q HaBH4. and i m d i a t e l y c h i l l e d On

with borohydride a t pH 7 . 4 and chi l led; 15 mi " l a t e r another a l i q w t O f borohydride was added ice; three additional a l i q w t s Of borohydride were added over 1 h. The other was treated

foll-d 45 m l n l a t e r by 2 N acetic acid to destroy m i n i n g bomhydride. 80th $ampler were dialyzed against standad buffer at 4' and assayed f o r synthase a c t l v i t y w i t h HA0 ( I rill) and glucose 6-phosphate I5 rill) a t pH 7.4.

Pre-incubation pH 7.4 (37')

Substrate added t: A c t i v i t y

66. 100

21 82

24 93

22 82

24 86

4 m 2 0.0 02 0.4 0.6 0.8 1.0 12 BFWN

GAMMA GLOBWN lmgl

0.0 0.4 08 12 1.6 20

Fiuure 11 - Smthase a c t i v i w of t e s t i s and bra in i l lunoorec ip i ta tes. Pure t e s t I synt are nll an ra n -ce Y ore Fact on .43 mg 2 .22r lJ ) re: in!%% & z ~ a d l y ttbd f%l "1: !it, :ncmisin?-tl O f g a m g lobu l in f rdc t im O f r a b b i t t n t i s e m or pmimme s c m i n 0.5 nl Trls-acetate buffer (50 nll. pH 7.7) COntaining 0.1 II KC1. then kept a t 4' overnight. After centrifugation at 3ooo rpn for 20 min the i m w p e c i p i t a t e s rere washed 3 times and suspended i n 0.3 ml Of the same buffer. Supernatants and rurpenr imr were assayed for synthase act iv i ty . Act iv i t ies are presented as percent o f t h a t i n supernatant incubated without g- globulin. (A) ter- t i s 01 bra in synthase plus p r e - l m n c serw; (0-0) t e r t i r supernatant plus antiservni (0-4) bra in supernatant plus a n t i s e m i (LO t e s t i s i l u n o - precipitate; (0---81 bra in iWnoPreC ip i t l t e .

E x p r v r e Of the en- t o pH 6.0 buf fer for 1 h a t 4' resulted i n i r r e v e r s i b l e l o s s o f 34% Of the x t i y i t y melSYred a t pH 7.4.

70,000 %,OOO BPB

4 4 4 Table V

Pur i f i ca t ion o f Rat Epidid-1 Synthase

E p i d i m i d e r (3.93 g) f m rats weighing 250 g were pmcessed as described i n p u r i f l c a t i m o f t e s t i s synthase. except t h a t 0-IOI saturated amiu sul fa te was used. k t i v i t y of me i m m p p e c i p i t a t e was &ternid as dercribed i n F i g 11.

Pmtein

lulq protein - f o l d

mted supematant

miu sulfate 0.67

Slice Number

F i w R 9 - Bindinq Of substrate to e n w by bomkrdride reduction. Hydmxyl-

l . W i q :rotein) !&?.c8b:::d separat:ll atd3;'A:01 ilm:n Mi ih 10 uCi lU-1Gl- glucose 6-phosphate. 5 nll glucose 6-phosphate. and 1 nll HAD' i n 1 nl iris-HCl buf fer (pH 7.4 ) containing 2 nll NHaCl and 50 pp" Ant i fom C h l s i o n (Signal. A t the end of incubdt im and 15 n i n l a t e r 0.1 ml Of 1 U HaBH4 was added. After 1 hour reduction m ice excell borohydrldc "dl destroyed by addition Of 2 N acet lc ac id . lo td l prote in was prec ip i ta ted and washed with alcohol to m v c nOrt of labeled substrate. and c q l e t e l y dissolved i n 0.2 m1 of phosphate buf fer (10 nll. pH 7.0) containing 1% mrcaptoethanel. 1% sodium doddecyl Sulfate. dnd 0.5 N urea. F i f t y y l O f the so lut ion was heated 3 min a t 100' and elect ro- phoresed m 7 .51 acrylmide gel as described i n F i g 19. The 'pl was s l iced in -

aqyews Protosol (Nol England Nuclear) and Counted i n 10 m l of 3a20 (RPl Corp.) t o 1 nm sect ims and each section was dl lso lved overn ight in 0.8 m l Of 0.05 n

f r d c t l m . l i q u i d s c i n t i l l a t i o n solvent. (0) Hydmxylapat i te f ract im; (0) D M - c e l l u l o s e

&pat te r a c t on q p m t e n an -ce Y ore ract ion (36 nll F i w R 9 - Bindinq Of substrate to e n w by bomkrdride reduction. Hydmxyl- &pat te r a c t on l.Wina : m d n l zL?.c:b% reoaratelv at 37' for 2 min Mith 10 uCi t u - 1 6 -

q prote in) and OEAE-cellulose f ract ion (36 nll l a b l o V I

L N ~ S o f synthase and F m m i t m r i t o l in Diabetic Rats g1;;or; & - & & p h a ~ . ~ 5 nll glucose 6-phosphate. and 1 nll HAD' i n 1 nl l r i i - H C l . buf fer (pH 7.4 ) containing 2 nll NHaCl and 50 pp" Ant i fom C h l s i o n (Signal. A t the end of incubdt im and 15 n i n l a t e r 0.1 ml Of 1 U HaBH4 was added. After 1 hour reduction m ice excell borohydrldc "dl destroyed by addition Of 2 N acet lc ac id . lo td l prote in was prec ip i ta ted and washed with alcohol to m v c nOrt of labeled substrate. and c q l e t e l y dissolved i n 0.2 m1 of phosphate buf fer (10 nll. pH 7.0) containing 1% mrcaptoethanel. 1% sodium doddecyl Sulfate. dnd 0.5 N urea. F i f t y y l O f the so lut ion was heated 3 min a t 100' and elect ro- phoresed m 7 .51 acrylmide gel as described i n F i g 19. The 'pl was s l iced in -

aqyews Protosol (Nol England Nuclear) and Counted i n 10 m l of 3a20 (RPl Corp.) t o 1 nm sect ims and each section was dl lso lved overn ight in 0.8 m l Of 0.05 n

f r d c t l m . l i q u i d s c i n t i l l a t i o n solvent. (0) Hydmxylapat i te f ract im; (0) D M - c e l l u l o s e

Diabetes was Induced by a single intraper l tq lC.1 in ject ion of s t n p m m m c i n (75 q l k d in male rats weighing m0 g. I n i m l s with blood sugav leve ls above 3w q l d l wen wed a f t c r 3 web. Synthase a c t i v i t y was measured l n the iumppecipitate as dercrlbed i n Fig 11.

or butanebomnate esters (Baa) (13) with o-methylglucoride or g luc i to l . mpec t i ve l y . as ln- F m iwritol was measured by gas-liquid chraatography Of the t r i m e t h y l l i l y l eth.n (nS) (2)

ternal standards. Values represent the man f S . E . for 6-8 aniM1s each.

o iabet ic ! ION1

Synthase (testis) I Wq pmteln

Supernatant

(m4)2so, f ract lon

0.290 f 0.0152 0.2% f 0.0123

3.15 t 0.131 3.19 t 0.200

F n e = i w s i t o l unol tg t issue or m1 r e m

Testis (ma) (BBA)

Bra in (lK) (BBA)

3.79 t 0.123 3.73 t 0.117

1.40 t 0.066 1 . a t 0.037

7.19 t 0.212 5.63 f 0.055 7.11 t 0.193 5.71 t 0.105

S e m (WA) 0.068 t 0.001 0.070 t 0.001

REFEREKES

2. Wlmder. A. L.. M d E i m b e q . F.. Jr. (1974) Uiochn. U t a . b s . CM. @. 1. Barnett. J . E. 6.. Brice. 17. E . , and Corina. 0. L. (1970) Biocha. J. 119. 183-186.

3. Eisenbeq. F.. Jr. (1974) i n lkthods o f En-tic I n a l s i s (8emyer. H. U.. ed.)

4. Lmry. 0. H.. Rorcbmugh. N. J . . Farr. A. L.. and Randall. R. J . (1951) J. Uial. UU. Vo1. 3. Second English Edition. w. 1337-1341, Academic Press. kr Yo*.

5 . Omstein. 1. (1964) Inn. 11.1. Aud. Sci. lZJ, 321-349. 6 . Davis. 8. J . (1964) Inn. N.I. Acad. Sci. 121. 404-427. 7. L m m l i . V. K. (1970) Nature 227. 680-685. 8. Hedrick. J . L.. and Smith. A. J . (1968) Arch. U i o c h . EioDhvr. 126. 155-16).

10. A n d m r . P. (1964) Blahem. J . 91. 222-233. 9. Iphantis. 0. A. (1964) E i r a h m i s t w 3. 297-317.

133-139.

- 193. 265-275.

11. Yeber. K., M d Orborn. N. (1969) J. 0101. ckn. 244. UM-4412. 12. L i m r a v e r . H.. M d nu*. 0. (1934) J . k. Cha. Sa. s, 650466. 13. E ismbeq, F.. Jr. (1972) i n lkthods i n En-lopy. (6insbun). V.. ad.) Val. 28.

pp. 168-178, Academic Pmss. New Yo*.