Non-michaelian kinetics of rabbit muscle uridine diphosphoglucose pyrophosphorylase

ARCHIVES

OF BIOCHEMISTRY

Vol. 227, No. 2, December,

AND BIOPHYSICS pp. 39’7-405, 1983

Non-Michaelian Kinetics of Rabbit Muscle Uridine Diphosphoglucose Pyrophosphorylase CARLO M. BERGAMINI,l AND

MARCO SIGNORINI, FRANC0 DALLOCCHIO

Istituto di Chin&a Biolo&a Received April

CARLO FERRARI,

d~ll’Univc?rsitci di Ferrara, 44 100 Fwrara, Italy

19, 1983, and in revised form July 26, 1983

The kinetic properties of rabbit muscle uridine diphosphoglucose (UDP-Glc) pyrophosphorylase have been studied, in both directions, with respect to substrate saturation, product inhibition, and cation requirement for activity. The results demonstrate that UDP-Glc pyrophosphorylase is a non-Michaelian enzyme: the synthetic reaction is characterized by a marked inhibition by glucose-l-phosphate (at concentrations higher than 0.3 mM) and by an hyperbolic saturation for UTP. In the reverse reaction, instead, the saturation function for UDP-Glc is hyperbolic and that for inorganic pyrophosphate is sigmoid, with a high Hill coefficient of (YQ) 2.5. The study of the metal requirement indicates a distinctive ability of cations to stimulate the reactions of synthesis and degradation of the sugar nucleotide and a different stoichiometry of the metal chelates involved. The reaction mechanism is of the ordered-sequential type and the data of product inhibition allowed the identification of glucose-l-phosphate as the first substrate bound and UDP-Glc as the last product released. The inhibition pattern by UDP-Glc gives evidence for cooperativity also in the binding of this molecule.

Uridine diphosphoglucose (UDP-Glc)’ plays a central role in metabolism, being involved in the biosynthesis of glycogen, glycoproteins, and complex carbohydrates, as well as in the reactions of sugar interconversion and of detoxification in the liver (l-5). The enzyme that catalyzes UDP-Glc synthesis according to the reaction: UTP + Glc-1-P + UDP-Glc + PPi, is UDP-Glc pyrophosphorylase (EC 2.7.7.9, UTP: a-Dglucose-l-phosphate uridilyltransferase); it has been detected in several tissues (6, ‘7) and isolated and fully characterized from liver (8,9), red blood cells (lo), amoebas (11,12), bacteria (13), and plants (14). i To whom correspondence should be addressed: Istituto di Chimica Biologica, Universiti di Ferrara, Via Borsari, 46, 44100 Ferrara, Italy. ’ Abbreviations used: UDP-Glc, uridine diphosphoglucose; Glc-l-P, glucose l-phosphate; PGM, phosphoglucomutose; GGP-DH, glucose-6-phosphate dehydrogenase; TEA, triethanolamine.

The enzyme has been described as following Michaelian kinetics with a compulsory mechanism of substrate addition and product release and to be dependent on divalent cations for activity (7). A lesser amount of information is available on the corresponding muscle enzyme, which was analyzed only in the reverse, unphysiological reaction (15) leading to Glc-1-P and UTP production from UDPGlc and inorganic pyrophosphate. In the present report we describe the kinetic properties of UDP-Glc pyrophosphorylase highly purified from rabbit skeletal muscle. Our most relevant findings are sigmoid saturation for pyrophosphate in the reverse reaction and substrate inhibition in the synthesis reaction. MATERIALS

AND

METHODS

Chemicals and enzymes. Phosphoglucomutase (PGM), glucose-&phosphate dehydrogenase (G6P-

397

0003-9861/83 $3.00 Copyright All rights

0 1983 hy Academic Press, Inc. of reproduction in any form reserved.

398

BERGAMINI

dH), alkaline phosphatase, Glc-l-P, UDP-Glc, NAD, NADP, and UTP were purchased from BoehringerMannheim. Radioactive [U-“C]Glc-1-P (295 mCi/ mmol) was obtained from New England Nuclear. All other reagents were of analytical grade and used without further purification. UDP-Glc dehydrogenase was purified 300-fold from calf liver acetone powder by a modification of the procedure of Zalidis and Feingold (16), involving ammonium sulfate preeipitation, acid heat treatment, polyethyleneglycol fractionation, and chromatography on CM-cellulose. The purified enzyme had a specific activity of 1.5-2.0 U/ mg and was free of detectable UDP-Glc pyrophosphorylase and phosphoglucomutase activity. Enzyme pur$cation UDP-Glc pyrophosphorylase was purified 3000-fold from rabbit muscle by an unpublished procedure; briefly, the homogenate was prepared and treated as described for the preparation of phosphorylase b (17), but with omission of the first dialysis. The fraction precipitated with 42-62% saturation ammonium sulfate was dialyzed and applied onto two consecutive columns of DEAE-cellulose in TEA buffer, pH ‘7.8; the enzyme was retained only on the second column from which it was eluted by a PPi gradient. The active fractions were concentrated by ammonium sulfate precipitation and chromatographed on a column of Sephadex G-200 in phosphate buffer, pH 7.0. The enzyme was then subjected to a final purification on aminohexyl-Sepharose. The recovery of activity was about 20% with a specific activity of 200-250 U/mg. Enzyme activity. This was measured by coupled spectrophotometric assays; for the reverse (pyrophosphorolytic) reaction, cuvettes contained, in a final volume of 0.8 ml, 40 mM Tris-acetate buffer, pH 7.8, indicated concentrations of UDP-Glc and MgPP, 0.2 mM NADP, 10 pg PGM, 1 gg GGP-dH, and l-5 munits of pyrophosphorylase. In the direct (synthetic) reaction, the cuvette contained, in a final volume of 0.65 buffer, pH 7.8, required conml, 40 mM Tris-acetate centrations of MgUTP and Glc-l-P, a 2 mM Mg acetate excess over UTP concentration, 1 mM NAD, loo-150 munits of UDP-Glc dehydrogenase, and 0.3-1.5 munits of UDP-Gle pyrophosphorylase. Under these conditions, in both assays, the reaction rate was proportional to the enzyme concentration and linear for at least 10 min. For the study of the UDP-Glc inhibition of the synthetic reaction, we employed a radiochemical assay with [U-r4C]Glc-1-P as substrate in the usual Trisacetate buffer (total volume 0.5 ml). At the required time, the reaction was stopped by acidification to pH 2.0 with 50 ~1 of 0.6 N HCl and the tubes were placed in ice; after 4 min the pH was raised to 8.3 with 100 pl of 1 M Tris base and the solution was stored frozen overnight. After thawing, each tube was treated with 50 pg of alkaline phosphatase and kept at 30°C for 60 min to allow a complete hydrolysis of residual Glc-

ET AL. 1-P. Labeled UDP-Glc was isolated by column chromatography of the reaction mixture on Dowex l- X 8, formate form. The resin was rinsed with water and UDP-Glc was eluted with 3 ml of 5% (v/v) HCl. Radioactivity was determined on 100-J aliquots of the eluate in 2 ml of Aquasol(New England Nuclear) using a liquid scintillation counter. other procedures. The concentration of UTP and UDP-Glc were determined by ultraviolet absorption (18), and those of Glc-1-P and PPi by enzymatic methods. The equilibrium constant of the pyrophosphorylase reaction, run in the pyrophosphorolytic direction at 37”C, was calculated by determining the concentration of UDP-Glc and Glc-1-P at different time intervals by standard spectrophotometric assays. Analysis of the kinetic data was performed according to the procedure of Cleland (19). RESULTS

Kinetic Properties of UDP-Glc Pyrophosphwyluse in the Synthetic Reaction When the reaction catalyzed by UDPGlc pyrophosphorylase is analyzed in the direction of synthesis of the sugar nucleotide, an increase of Glc-1-P concentration in the assay mixture (at different fixed levels of UTP) induces a marked substrate inhibition, easily appreciated in the Glc1-P saturation plot. The inhibitory effect of substrate appears as the concentration is raised over 0.3 mM and is so marked that at 8 to 10 mM Glc-1-P the velocity of the reaction is equal to only 20% of V,,,. As expected in these cases, the double-reciprocal plot does not give a straight line (Fig. 1); in any case it was possible to calculate an apparent Km of 40 PM for Glc-1-P. From the replot of the intercepts against the concentration of UTP, we determined a Km of 33 PM for this substrate. In perfect agreement with these results are those obtained in experiments devoted to the direct determination of the saturation plot for UTP at various fixed concentrations of Glc-1-P. The experimental curves show a clear hyperbolic saturation for UTP, although there are evident deviations from the Michaelian kinetics for a two-substrate enzyme in the double-reciprocal presentation (Fig. 2); in fact, the line corresponding to high concentrations of Glc-1-P intersects the ordinate at a value

KINETICS

OF MUSCLE

GLUCOSE

‘/[GIG-1-P

399

PYROPHOSPHORYLASE

I ImH-‘1

FIG. 1. Lineaweaver-Burk plot showing the dependence of enzyme activity on the concentration of Glc-1-P at different concentrations of UTP (synthetic reaction). Concentrations of UTP in the assay were (0) 30 and (0) 150 CM. In this, and in all other figures, activity is expressed as micromoles of product/minute/milligram of enzyme protein.

of El,, lower

than that obtained at lower concentrations of the fixed substrate. This effect is the direct consequence of the in-

hibition Glc-l-P, which has already been described, and prevents the calculation of a value of K, for Glc-1-P from the sec-

”

.2

‘/iUTPl

hntdl

FIG. 2. Lineaweaver-Burk plot showing the influence of UTP on the enzyme rate at different fixed concentrations of Glc-1-P. The concentration of Glc-1-P in the assay mixture was (A) 0.05, (V) 0.2, and (A) 2 mre. In the inset are reported the plots of slopes (0) and intercepts (0) as a function of the concentration of Glc-1-P.

400

BERGAMINI

ondary plot; indeed, the intercepts fit a curve, whereas the replot of the slopes gives rise to a straight line. These results suggest that the inhibition by Glc-1-P is of the uncompetitive type (20). From the region of the primary plot at low concentrations of Glc-1-P (up to 0.2 mM) it is possible to calculate a Km of 33 PM for UTP, in agreement with the results obtained from the replots of Fig. 1. As already described for the enzyme from other sources, UDP-Glc pyrophosphorylase from skeletal muscle also has an absolute requirement for divalent cations; the relevant data, presented in Table I, make clear that no activity is observed in TABLE

I

INFLUENCEOF~NORGANIC SALTS ONENZYMEACTIVITY Relative enzyme activity

Additions None M&b

CaCl, MnCI, ZnCl, CuCl, Na sulfate Na phosphate

Concentration Cm@

1 2 3 3 3 3 3 5 25 5 25

Direct reaction 0 35 100 140 45 185 0 0 82 38 42 11

Reverse reaction 0 65 100 100 0 0 0 0 75 40 47 10

Note. The reaction was run in Tris-acetate buffer, pH 7.8, with the indicated additions. For the direct reaction, the cuvette contained 0.2 mM Glc-l-P, and dehydrogenase0.4 mM UTP, and the NAD/UDP-Glc coupling system. For the reverse reaction, the enzyme was incubated with 0.4 mM UDP-Glc and 2 mM PP,, the reaction was stopped by heating in a boilingwater bath, and the Glc-1-P produced was measured by transferring aliquots of the content of the tubes to cuvettes containing NADP, PGM, GGP-dH, and 2 mM Mg acetate. In the case of the inhibition by sulfate and phosphate salts, cuvettes contained MgCla at 2 mM final concentration. Each value is presented relative to the activity recorded in the presence of 2 mM MgClZ.

ET AL.

the absence of cations and that the rate of synthesis of UDP-Glc is increased when the concentration of the metal is higher than that of the nucleotide. The published values of the dissociation constants of the Mg-UTP complex demonstrate that the nucleotide is always present in the chelate form and thus suggest the presence on the enzyme surface of some site(s) for binding free metals. It is noteworthy that magnesium, which is the physiological cofactor of the enzyme in the intracellular compartment, can be substituted for by manganese, which is an even better activator, and by calcium, but not by copper or zinc. Anions exert an inhibitory effect on enzyme activity, more apparent with phosphate than with sulfate salts. Kinetic Properties of UDP-Glc Pyrophosphmylase in the Pyrophosphm-o&tic Reaction Figure 3 shows the dependence of the rate of the pyrophosphorolytic reaction on the concentration of UDP-Glc at different fixed concentrations of MgPP, in the range l-6 mM: the hyperbolic curves are transformed in the double-reciprocal presentation into a set of straight lines convergent onto the x-axis at a value of Km for UDP-Glc of 48 PM. This result suggests that the enzyme follows a sequential-reaction mechanism. In contrast with this Michaelian response to UDP-Glc, a quite different behavior is observed when the activity is measured as a function of the MgPP concentration at different fixed levels of UDPGlc (Figs. 4A and B): in this case, we obtained sigmoid saturation curves which were not affected by the ratio of magnesium to pyrophosphate, by the addition of salts to increase the ionic strength, or by increasing the concentration of UDP-Glc up to 2 mM (data not shown). These anomalous kinetics preclude a direct determination of the K,,, for pyrophosphate because of upward bending of the doublereciprocal plot, a characteristic observed also in the secondary plot of intercepts against pyrophosphate concentration in experiments of the type presented in Fig. 3. An indicative value of the Km for pyro-

KINETICS

OF MUSCLE

GLUCOSE

PYROPHOSPHORYLASE

;5 “[UDP-GLcl

401

50 ImM-‘I

FIG. 3. Double-reciprocal plot describing the effect of the concentration of UDP-Glc on the rate of the pyrophosphorolytic reaction at different concentrations of Mg PPi added in stoichiometric amounts. Concentration of PPi was 1 (0), 2 (O), 4 (A), and 6 mM (A). In the inset, the replots of the slopes (@) and the intercepts (0) against the pyrophosphate concentration are shown.

V

A

B

2a I-

>; 1.0

lO( 1. z

2.5

3.2

1oglsl -1.0 0

2.0

Lo [PPII

ImMl

FIG. 4. Saturation plot for UDP-Glc pyrophosphorylase activity at variable concentrations of PPi and fixed concentrations of UDP-Glc. (A) PPi was added as the magnesium salt by addition of stoichiometric amounts of magnesium acetate and sodium pyrophosphate. Concentration of UDPGlc was (from top to bottom curves) 2’70,200,148, and 107 FM, respectively. In the inset is presented the correspondent Hill plot for the concentrations of UDP-Glc of 270 and 107 PM. (B) Results of a similar experiment of saturation for pyrophosphate, performed in the presence of 190 pM UDPGlc and variable PPi concentrations with fixed concentrations of magnesium acetate: (0) 0, (A) 1.25, (0) 2.5, and (0) 4 mM, respectively.

402

BERGAMINI

phosphate was derived from the secondary plot of Fig. 3 itself only because the data presented were obtained with concentrations of pyrophosphate (l-6 mM) which approach saturation, i.e., by fitting only the portion of the curve which approaches an hyperbolic function. When analyzed by the Hill plot, the data of Fig. 4 confirm the presence of high cooperativity in substrate binding, with Hill coefficients ranging from 2.0 to 2.6, depending on the concentration of UDP-Glc. According to this analysis, the half-saturation of the enzyme was obtained when pyrophosphate is about 1 mM. The requirement of cations for catalytic activity was also investigated in the pyrophosphorolytic reaction, at saturating concentrations of UDP-Glc and PPi, taking care to avoid artifacts due to the known dependence of PGM on magnesium ions (see legend to Table I). While in the absence of cations no activity at all is detectable, pyrophosphorolysis of the sugar nucleotide is observed only in the presence of magnesium; all the other cations being ineffective, in contrast to what is observed in the biosynthetic reaction. Furthermore, these data demonstrate the lack of effects of the free metal, since in the presence of 2 mM PPi, the same rate is obtained with 2 and with 3 mM Mg. This behavior is better explained by the experiments presented in Fig. 4B where the pyrophosphate saturation plot, in the presence of 0.25 mM UDPGlc, is presented in the absence of magnesium and in the presence of 1.25,2.5, and 4.0 mM cation. Clearly, the substrate-saturation curve is sigmoid in every instance and the rate of the reaction increases progressively until a maximal value, which corresponds to the presence of stoichiometric quantities of magnesium and pyrophosphate. Data of Table I also include the influence of anions on the rate of the pyrophosphorolytic reaction. Phosphate and sulfate inhibit both reactions to exactly the same extent (even at different pH values, results not shown) eliminating the possibility of artifacts due to the coupling systems and suggesting, therefore, some specific interaction between these anions and the enzyme. We also measured the equilibrium constant of the reaction at

ET AL.

37°C in the direction of pyrophosphorolysis of UDP Glc; the value obtained was 0.24, similar to that reported in the cases of the enzyme from liver (9) and red blood cells (10). Pattern of Product Inhibition of the UDP-Glc Pyrophosphwylase-Catalyzed Reaction

The results presented in the preceding section are consistent with a sequential reaction mechanism. To elucidate whether it is of the random or of the ordered type, we analyzed extensively the pattern of product inhibition. The results obtained from these experiments are presented in Fig. 5 in the double reciprocal form, together with the corresponding secondary plots of slopes and intercepts against the concentration of the individual inhibitor. The synthesis of UDP-Glc is inhibited, with great affinity, by both reaction products PPi and UDP-Glc. Inorganic pyrophosphate is a noncompetitive inhibitor against both UDP and Glc-1-P. On the contrary, UDP-Glc is a noncompetitive inhibitor against UTP, but a competitive inhibitor when tested against Glc-1-P; the corresponding replots of the slopes versus the concentration of UDP-Glc are parabolic, whereas the plots of the intercepts are linear. For partial confirmation of these data (i.e., competitive inhibition exclusively between Glc-1-P and UDP-Glc), we analyzed the pattern of product inhibition of the reverse reaction by UTP. The results of these experiments confirm that the inhibition between UDP-Glc and UTP is of the noncompetitive type (not shown). Taken together, these results and those of the steady-state kinetic experiments support the hypothesis of a compulsory mechanism of reaction for muscle UDPGlc pyrophosphorylase, in which Glc-1-P is the first substrate to bind to the enzyme surface, followed by UTP, while the order of product release is pyrophosphate first and UDP-Glc last. The values of the kinetic constants and the indication of the type of inhibition taking place for the reaction catalyzed by muscle UDP-Glc pyrophosphorylase are

KINETICS

OF MUSCLE

GLUCOSE

26

lb

“IS1

403

PYROPHOSPHORYLASE

10

r al

lmW1l)

FIG. 5. Patterns of product inhibition of the synthetic reaction catalyzed by UDP-Gle pyrophosphorylase determined by spectrophotometric (A, B) and radiochemical (C, D) assay. The inhibitory concentrations of pyrophosphate were 0.1 and 0.25 mM with substrate UTP at 200 jtM (A) and with Glc-1-P at 250 pM (B). In the bottom panels are presented the inhibitions obtained with 5 and 30 PM UDP-Glc with substrate UTP at 330 pM (C) and Glc-1-P at 225 pM (D). In the insets, the corresponding replots of slopes (0) and of intercepts (0) for every experiment.

summarized in Table II. It must be stressed that these results were obtained at low substrate concentration in the synthetic reaction (because of the inhibition by high concentrations of Glc-1-P) and at high substrate concentrations in the pyrophosphorolytic reaction, to avoid the complications deriving from the sigmoid saturation of PPi. DISCUSSION

In the present report we describe in detail the catalytic properties of purified rabbit muscle UDP-Glc pyrophosphorylase; this enzyme shares a number of properties with the corresponding protein from other tissues but is characterized also by features (cooperativity phenomena and substrate inhibition) that are presumably specific properties.

Our results clearly show that UDP-Glc pyrophosphorylase behaves like an allosteric enzyme: in the reserve reaction, the pyrophosphate saturation plot is sigmoid with high homotropic cooperativity in substrate binding (nH ranging from 2.0 to 2.6). A similar result was reported (15) by Villar-Palasi and Larner, who mentioned having encountered great difficulties in the determination of Km for pyrophosphate because of an upward curvature of the double-reciprocal plot. Evidence for cooperativity in PPi saturation is also found in the case of the liver enzyme (8) but not for that from red blood cells or heart (10). The significance of this effect is in every case difficult to interpret because the concentration of pyrophosphate is reported to be always very low in animal tissues (21). Possibly a more specific feature of the muscle enzyme is the inhibition of the di-

404

BERGAMINI TABLE KINETIC

Substrate Glc-1-P

Kl 0.040

PARAMETERS

ET AL. II

OF UDP-Glc

PYROPHOSPHOR~LASE

Kinetics N-M

Type of inhibition Competitive vs UDP-Glc Uncompetitive vs PPi (substrate

inhibition)

UTP

0.333

M

Uncompetitive

vs UDP-Glc

PPi

0.770

N-M

Uncompetitive

vs Glc-1-P and UTP

UDP-Glc

0.048

M

Competitive

and PPi

vs Glc-1-P and uncompetitive

vs UTP

Note. Reported Km values are millimolar concentrations calculated either from double-reciprocal plots or from replot of intercepts. The kinetics are symbolized by block letters N-M (non-Michaelian) and M (Michaelian).

rect reaction by Glc-1-P. It appears at substrate concentrations higher than 0.3-0.4 mM, is largely reduced at high concentrations of the second substrate UTP (data not shown); is of the uncompetitive type and cannot be ascribed to an ability of Glc1-P to bind magnesium ions. A possible explanation for this inhibition is the formation of an abortive complex. The structure of this complex is presently unknown, but we can exclude the type Enzyme/UDPGlc/Glc-1-P for two reasons: (a) UDP-Glc would occupy completely the active site, and (b) this type of complex would give a noncompetitive inhibition between Glc-lP and UDP-Glc. In fact, an increase of the concentration of Glc-1-P would also increase the proportion of the enzyme in the inactive form, and this possibility is in contrast with the reported competitive inhibition between Glc-1-P and UDP-Glc, at least unless the Kd for the dead-end complex is so small that it is missed during the kinetic measurements. An inherent difficulty in our experiments stems from the cooperative kinetics of the enzyme: this, and the low solubility of the pyrophosphate salts, necessarily restricted the range of substrate concentrations to narrow limits to get results amenable to analysis by the mathematical methods available. As a consequence, the kinetic parameters do not always fit the Haldane relationship perfectly. Furthermore, the agreement between the Ki values obtained by initial-rate and product-inhibition experiments is poor.

Nevertheless, even taking in account all these difficulties, some interesting conclusions can be drawn. The competitive inhibition between Glc-1-P and UDP-Glc and the noncompetitive inhibition for any other combinations of substrates and products are consistent with a sequential-ordered mechanism of reaction, Glc-1-P binding first with UDP-Glc being the last product released. These results can be summarized in the diagram: PPi UDP-Glc Glc-1-P UTP 1 1 f T The mechanism we propose differs substantially from that established on the basis of kinetic (7, 10) and binding data (22) for all other UDP-Glc pyrophosphorylases which bind UTP as the first substrate. We are beginning to confirm our present results by binding studies. Further careful examination of the product-inhibition experiments reveals some interesting characteristics, i.e., that the replots of slopes and intercepts are always linear for pyrophosphate, but those for the slopes are always parabolic in the case of UDP-Glc, independent of the other substrate. The situation is therefore reversed in comparison with the initial-rate measurements, in which pyrophosphate is the non-Michaelian substrate. Another important feature of the enzyme is the dependence of activity on divalent cations; in the reverse reaction, activity is observed only in the presence of magnesium, whereas both manganese and

KINETICS

OF MUSCLE

GLUCOSE

calcium can substitute in the direct reaction. This implies different mechanisms for the two reactions and this eventuality is supported by the stoichiometry of the metal-chelate complex involved; magnesium takes part in the reverse reaction as a one-to-one complex with pyrophosphate, whereas in the direct reaction there is evidence for the interaction of the free metal with specific sites on the enzyme surface. In this way, we can postulate either a metal-substrate-enzyme or a substratemetal-enzyme complex, and a Metal-enzyme-substrate complex as reaction intermediates in the reverse and in the direct reactions, respectively. Our results are thus in contrast with those reported, in a preliminary form, by Mildvan (23) for the crystalline liver enzyme. Further experiments by more sophisticated techniques like NMR are needed to clarify this point. Although we are unable at present to understand the physiological significance of the substrate-regulatory effect we have identified, our results suggest the existence of regulatory mechanisms in the synthesis of UDP-Glc in muscle, where most of the sugar nucleotide is involved in the synthesis of glycogen. It is appropriate to recall that the synthesis of the activated sugar is regarded as an important regulatory point in bacteria and in plants, where ADP-Glc is synthesized by the allosteric ADP-Glc pyrophosphorylase (24) but not in animal tissues, in which the reaction catalyzed by glycogen synthetase itself is the regulatory step (25). In any case, a detailed study of the determinants of enzyme activity in a reconstituted system, such as that performed by Roach et al. (18) for the liver enzyme, is still lacking in the case of muscle UDPGlc pyrophosphorylase. In conclusion, it is at present difficult to appreciate the physiological importance of the substrate inhibition by Glc-l-P, characteristic of the muscular enzyme because it is present in vitro only at concentrations of Glc-1-P much higher than those present in muscle (26). REFERENCES 1. LELOIR, L. F., AND GOLDBERG, S. H. (1960) J. BioL Chem 235, 919-923.

PYROPHOSPHORYLASE

405

2. LENNARZ, W. J. (1975) Science 188, 986-991. 3. BEHRENS, N. H., PARODI, A. J., LELOIR, L. F., AND KRISMAN, C. R. (1971) Arch. Biochem. Biophys. 143,375-383. 4. CHACKO, C. M., MCCRONE, L., AND NADLER, L. H. (1972) B&him. Biophys. Actu 286, 113-120. 5. STROMINGER, J. L., KALCKAR, H. M., AXELROD, J., AND MAXWELL, E. S. (1954) J. Amer. Chem Sot. 76, 6411-6413. 6. VILLAR-PALASI, C., AND LARNER, J. (1960) Arch Biochem Biophys. 86,270-273. 7. TURNQVIST, R. L. AND HANSEN, R. G. (1973) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 8, pp. 51-57, Academic Press, New York. 8. KNOP, J. K., AND HANSEN, R. G. (1970) J. Biol. Chem. 245, 2499-2504. 9. ALBRECHT, G. J., BASS, S. T., SEIFERT, L. L., AND HANSEN, R. G. (1966) J. Biol. Chem 241,29682975. 10. TSUBOI, K. K., FUKUNAGA, K., AND PETRICCIANI, J. C. (1969) J. Biol. Chem. 244, 1008-1015. 11. RUDICK, V. L., AND WEISMAN, P. W. (1974) J. Biol. Chem. 249, 7832-7840. 12. FRANKE, J., AND SUSSMAN, M. (1971) J. Biol Chem. 246, 6381-6388. 13. BERNSTEIN, R. L., AND ROBBINS, P. W. (1965) J. Biol. Chem. 240, 391-397. 14. GINSBURG, V. (1958) J. Biol. Chem. 232, 55-61. 15. VILLAR-PALASI, C., AND LARNER, J. (1960) AT&. B&hem. Biqphys. 86, 61-66. 16. ZALIDIS, J., AND FEINGOLD, D. S. (1969) Arch. Biochem Biophys. 132.457-465. 17. FISCHER, E. H., AND KREBS, E. G. (1958) J. Biol. Chem. 231, 65-71. 18. ROACH, P. J., WARREN, K. R., AND ATKINSON, D. E. (1975) Biochemistry 14, 5445-5450. 19. CLELAND, W. W. (1963) Biochim Biophys. Acta 67, 104-194. 20. CLELAND, W. W. (1979) in Methods in Enzymology (Purich, D. L., ed.), Vol. 63, pp. 500-513. Academic Press, New York. 21. GUYNN, R. W., VELOSO, V., LAWSON, J. W. R. AND VEECH, R. (1974) Biochem. J. 140, 369-375. 22. STEVENS, R. A. J., AND PHELPS, C. F. (1976) Biochem. J. 159, 65-70. 23. MILDVAN, A. S. (1970) in The Enzymes (Boyer, P. D. ed.), 3rd ed., Vol. 2, pp. 445-535, Academic Press, New York. 24. GHOSH, H. P., AND TREE, J. (1966) J. Biol. Chem. 241,4481-4504. 25. STALMANS, W., AND HERS, H. G. (1973) in The Enzymes (Boyer, P. D., ed.) 3rd ed., Vol. 9, pp. 309-361, Academic Press, New York. 26. RAHIN, Z. H. A., PERRETT, D., LUTAYA, G., AND GRIFFITHS, J. R. (1980) B&hem J. 186, 331341.