ARCHIVES

OF HIOCHEMISTRY

AND

RIOPHYSICS

Vol. 278, No. 1, April, pp. 99-105, 1990

Separate Effects of Mg*+, MgATP, and ATP4- on the Kinetic Mechanism for Insulin Receptor Tyrosine Kinase Pasquale

P. Vicario’

and Alfred

Bennun*

Department of Biochemical Endocrinology, Merck Sharp & Dohme Research Laboratories, Rahway, New Jersey 07065, and *Department of Biological Sciences, Rutgers State University, Newark, New Jersey 07102

Received June 30, 1989; and in revised form December 7,1989

The separate effects of the equilibrium species Mg2+, MgATP substrate, and ATP4- on the reaction catalyzed by insulin receptor tyrosine kinase were examined. The separated kinetic constants show that the Ko.5 value for from 23 to 0.43 mM and the Hill coMg2+ decreased efficient for Mg2+ (hMg2+) decreased from 1.43 to 0.668 when the concentration of ATPT (MgATP + ATP4-) was increased from 50 to 1000 PM. The apparent Ki for ATP4- increased from 0.20 to 136 ELM and the Hill coefficient for ATP4- (h *rp4-) decreased from 1.41 to 0.82 as the concentration of total ATP (ATPr) increased. These findings suggest that the [ATP4-]/[Mg2’] ratio modulates the shift from positive to negative cooperativity. It was also shown that the apparent affinity of the kinase for MgATP increased as the concentration of free Mg2+ increased and that the apparent affinity of the kinase for free Mg2+ increased as the concentration of MgATP substrate increased. Thus, Mg2+ and MgATP interact with the kinase in a mutually inclusive manner which leads to an increase in the ratio of the enzyme (E) rate-limiting species, [Mg-E-MgATP]/[E-MgATP]. Free ATP4- not only acts as a competitive inhibitor of the substrate but also decreases the relative concentration of Mg-E-MgATP. ATP,-dependent activation of the kinase is, therefore, a result of MgATP’s increasing the affinity of the kinase for Mg2+, thereby leading to saturation of the enzyme with Mg2’ at lower concentrations of the divalent metal. This results in an increase in the [Mg-E-MgATP]/[E-MgATP] ratio, and therefore decreases saturation of the kinase with ATP4- inhibitor, not only at the active site but also at a kinetically distinct regulatory site. This kinetic relationship allows not only for the mutually inclusive interaction between Mg2+ and MgATP, but also for the mutually exclusive interaction toward ATP4-, hence indicating that the effect of Mg2+ will be to form an enzyme com-

1 To whom correspondence

should be addressed.

0003.9861/90 $3.00 Copyright C 1990 by Academic Press, All rights of reproduction in any form

Inc. reserved.

plex (Mg-E) which will have a higher affinity for MgATP substrate and a lower affinity for ATP4- than E alone. The role of the equilibrium concentrations of Mg-E, E, and ATP-E on the activation of insulin receptor tyrosine kinase is discussed which may account, at least in part, for modulation of cooperativity and the metal-dependent increase in turnover (V,). cc 1990 Academic

Press,

Inc.

The insulin receptor is a transmembrane glycoprotein composed of two (Y (135 kDa) and two p (95 kDa) subunits (1). Interaction of insulin with the extracellular cy subunit activates a tyrosine kinase activity associated with the intracellular p subunit (2). Once activated, this receptor-associated enzymatic activity is rendered insulin independent (3) and is capable of phosphorylating endogenous proteins in the cell (4-7), as well as exogenous peptides and proteins (8,9). Results of site-directed mutagenesis, as well as studies with an inhibitor of insulin receptor tyrosine kinase (lo), have strongly supported a role for the kinase in insulin action. Substitution of tyrosine residues 1150 and 1151 on the p subunit with phenylalanine (11) or replacement of lysine at the ATP binding site (12) with arginine, alanine, or methionine not only decreases insulin receptor tyrosine kinase (IRTK)2 activity but also reduces the ability of insulin to stimulate glucose uptake into Chinese hamster ovary (CHO) cells. Thus, regulation of IRTK takes on an important role in mediating the biological effects associated with insulin. The relative concentrations of divalent metals (Mn2+ and/or Mgzt) or metal-ATP (MnATP and/or MgATP) ’ Abbreviations used: IRTK, insulin receptor tyrosine kinase; CHO, Chinese hamster ovary; pp, polypeptide; E, enzyme; Me, metal; RARE, rapid random equilibrium; ATPT, total ATP; Mg,, total Mg; Hepes, 4-(2-hydroxyethylj-l-piperazineethanesulfonic acid.

99

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substrate influence the catalytic activity of IRTK (1315). Kinase activity is enhanced by Mn2+ or Mg2+ when present in excess of that required for metal-ATP substrate formation, suggesting the presence of a metal-dependent regulatory site on the enzyme. Mn2+ is more potent than Mg2+ in this regard (13-15). In the presence of a near-saturating concentration of MnClz, insulin-dependent IRTK activity increases as a function of increasing MgCl, (13). Also, increasing concentrations of MgClz enhance only insulin-dependent kinase activity, whereas basal activity (absence of insulin) remains relatively unaffected. This latter result was also observed using the endogenous IRTK substrate pp”’ (6). These data suggest that divalent metals interact with IRTK at several sites: a metal-ATP (active) site (MnATP = MgATP), a Me’+-dependent regu latory site (Mn2+ > Mg’+) which activates IRTK activity independent of insulin, and a Mg2+-dependent site which confers insulin dependence to the kinase (13). In this paper we discuss the effects of free ionic magnesium, MgATP substrate, and free ATP4- on IRTK activity and suggest a mechanism which accounts for the modulation of cooperativity and activation of the kinase by divalent metals. EXPERIMENTAL

PROCEDURES

Materials. Male CD rats (ZOO-250 g) were purchased from Charles River (Boston, MA). [y-“2P]ATP (3000 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Val’-angiotensin II was obtained from Vega Biotechnologies (Burlingham, CA). Phosphocellulose (P81) paper was purchased from Ace Scientific (East Brunswick, NJ). Soluble insulin receptor was preInsulin receptor preparation. pared from rat liver membranes as previously described (16) and partially purified by wheat germ affinity chromatography (17). Protein was determined by the method of Lowry et al. (18) using bovine serum albumin as a standard. Peptide phosphorylation assay. Insulin receptor (l-5 fig protein) was incubated for 15 min at 23°C in a final reaction volume of 0.025 ml containing (at final concentrations) 20 mM Hepes (pH 7.5), 1.0 pM insulin, 5% glycerol, 30 mM NaCl, 100 pM sodium orthovanadate, and 12 mM p-nitrophenyl phosphate. The concentrations of MgClz used are indicated in Table I and in the figures. [Y-~‘P]ATP (10 cpm/fmol at 50 FM; 1 cpm/fmol at 1000 pM ATPr) was added and the incubations continued for 5 min at 23°C. Val’-angiotensin II (2.0 mM) was added and the incubations continued for an additional 5 min at 23°C a time at which the rate of peptide phosphorylation by IRTK is linear. Isolation of the phosphorylated peptide was achieved using phosphocellulose (P81) paper as previously described (19). Other methods. The concentrations of the ionic forms of Mg”‘, its chelated species MgATP, and uncomplexed ATP4- were determined using an iterative FORTRAN IV program (20) and the calculations based on their association constants (21,22). The equilibrium concentrations of Mg’+, MgATP, and ATP4- at fixed concentrations (50 and 1000 pM) of ATPr and varying concentrations of MgClz are shown in Table I. Linear and nonlinear regression analyses were performed using GraphPad (IS1 Software, Philadelphia, PA).

RESULTS

The mass action effect of an increase from 1.0 to 75 MgC12 in the presence of 50 pM ATPr shifts the equi-

mM

BENNUN TABLE

Equilibrium

I

Concentrations of M$+, MgATP, at 50 and 1000 pM ATPT Concentration (mM) equilibrium

M&l, bM) (A)

1.0 10 20 35 50

(B) ;?O 0.25 0.50 1.0 2.5 5.0 10

and ATP4-

at

ATP, (mM)

0.05 0.05 0.05 0.05 0.05 0.05 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Mg” 0.646 9.950 19.950 34.950 49.950 74.950 0.008 0.021 0.042 0.084 0.577 3.835 8.936

MgATP 0.0438 0.0495 0.0497 0.0498 0.0499 0.0499 0.084 0.186 0.314 0.478 0.864 0.977 0.990

ATP40.00618 0.00045 0.00025 0.00013 0.00009 0.00006 0.91605 0.81360 0.68577 0.52181 0.13646 0.02323 0.01010

[ATP*-~]/[Mg’+] 0.009567 0.000045 0.000013 0.000004 0.000002 0.000001 109 38 16 6 0.240 0.010 0.001

Note. The equilibrium concentrations of Mg”+, MgATP, and ATP4were determined using an iterative FORTRAN IV program (20) and were based on the association constants described by Taqui-Khan and Martall (21,22).

librium concentrations and increases the concentration of MgATP by only 1.14-fold (Table I). This effect is amplified by a corresponding 116-fold increase in free ionic Mg2+ and a IOl-fold decrease in free ATP4-. Under these experimental conditions, insulin-stimulated IRTK activity increased from 0 to 111 pmol/min/mg, with halfmaximal activation occurring at approximately 17 mM MgC12. In contrast, the mass action effect of an increase from 0.10 to 10 mM MgClz in the presence of 1000 pM ATPr shifts the equilibrium concentrations and increases the concentration of MgATP 11.8fold. This effect is amplified by a corresponding 1063-fold increase in free ionic Mg2+ and a go-fold decrease in free ATP4-. In the presence of a higher concentration of ATPr (1000 PM), increasing concentrations of MgC12 (0.10-10 mM) increase IRTK activity from 3.5 to 500 pmol/min/mg, with half-maximal activation at about 1.28 mM MgC12. Figure 1 illustrates Hill (23) plots obtained as a function of a change in the concentration of total magnesium uncomplexed magnesium (Mg ‘+), and free (M&J, ATP4-. The observed responses for Mgr represent a summation of the individual responses of the kinase to Mg2+, MgATP, and free ATP4-. The apparent K0.5 for Mgr (Fig. 1A) decreased from 23 mM (r = 0.994) to 1.91 mM (r = 0.992) and the Hill coefficient (h,,) decreased slightly from 1.43 to 1.29 (Table II) as the concentration of ATPr increased from 50 to 1000 PM. The apparent & for Mg2+ (Fig. 1B) decreased from 23 mM (r = 0.994) to 0.43 mM (r = 0.989) and the Hill coefficient decreased from 1.43 to 0.668 (Table II) as the concentration of

EFFECTS

OF DIVALENT

METALS

ON INSULIN

RECEPTOR

I

I

0.10 MS+4

O.‘O L 0.10

1.0

10 ATP4-bM)

100

1000

0.01

KINASE

I

1.0 Mg2+(mM)

0.10

101

KINETICS

I

I

10

100

1.0

MgATP(mM)

FIG. 1. Effect of MgCl,, Mg’+, ATP’-, and MgATP on insulin-stimulated IRTK activity as a function of ATPr MgCI,-saturation curves were plotted according to Hill (23) for the individual components of the reaction mixtures at 50 FM (0) and 1000 pM (0) ATPT. V,, maximal velocity; u, initial velocity. Note that only a single data point is shown for 50 pM ATPr in (D) since MgATP increased only 1.14-fold over the range of MgC& concentrations used.

ATPr increased. Figure 1C shows that the apparent K, for ATP4- increased from 0.20 PM (1. = 0.991) to 136 PM (r = 0.973) and that the Hill coefficient decreased from 1.41 to 0.82 (Table II) as the concentration of ATPr increased from 50 to 1000 PM. Figure 1D (r = 0.979) shows that at high (1000 PM) ATPr (high ATP4-, low Mg2+) the apparent KM of the kinase for MgATP is approximately 700 PM and that the Hill coefficient is 3.23 (Table

TABLE Cooperativity

II

Values of IRTK for MgT, ATP4-, and MgATP

Mg’+,

ATPr Species

50 FM

h Me h MgZ+ h AT@ h M&TP

1.43

1.29

1.43 1.41 -

0.668 0.820 3.23

1000 JLM

II), suggesting that MgATP interacts with the kinase at distinct sites, one or more of which may play a regulatory rather than active site role. A comparison to low (50 PM) ATPr is not reliable since the MgATP concentration increased only 1.14-fold (Table I) over the range of MgCl, used in the assay. Thus, only a single point for low ATPr (at 50 mM MgC12) is shown for comparison. Although the MgATP-saturation data at 50 PM ATPr did not lend themselves to analysis, it is apparent that under conditions of low ATPr (low ATP4-, high Mg2’) the curve is shifted to the left, thus yielding a lower apparent KM of the kinase for MgATP substrate which was estimated to be about 100 PM. When the concentrations of MgATP are maintained at a 540-fold excess over ATP4- and the concentration of free Mg2+ is kept constant at about 30 mM (13), the apparent KM of the kinase for MgATP is about 100 PM (13,24). Computation appears to indicate that the change in the apparent KM would be minimized by correcting for the effect of excess ATP4- over that necessary for substrate formation. The data in Fig. 2 show the relationship between IRTK activity and [ATP”-]/[Mg2’] ratio at low (50 pM)

102

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BENNUN

mation of Mg-E-MgATP complex is considerably less when MgATP concentrations are high, thus facilitating the mutual inclusion of Mg2+ (activator) and MgATP (substrate). Kinetically, this may be related to EMgATP having a higher affinity for Mg2’ and E-Mg” having a higher affinity for MgATP than E alone. In contrast, the curves for ATP4- at both low (50 /IM) (Fig. 3C) and high (1000 PM) ATPr (Fig. 3F) are not linear over the range of ATP4- plotted, suggesting that ATP4interacts with more than one site on the kinase. Table III summarizes the kinetic and thermodynamic constants for the various enzyme complexes.

1

I

I

I

I

I

I

-6

-4

-2

0

2

4

Log [ATP4J [Mg *+I FIG. 2. Regulation of IRTK activity by [ATP4-]/[Mg”]. Data from Fig. 1 were replotted according to Hill (23) as a function of the ratio of [ATP4-]/[Mg*+] at low (0) and high (0) ATPT. V,, maximal velocity; 0, initial velocity.

and high (1000 PM) ATPr . The cooperativity (Hill coefficient) of the response of IRTK toward the [ATP4-]/ [Mg’+] ratio at low ATPr had a value of 0.706 which decreased to 0.572 when the concentration of ATPr increased. Thus, the [ATP4-]/[Mg’+] ratio appears to modulate the shift to a more negative cooperativity (Figs. 1B and C, Table II). The kinetic plot shown in Fig. 2 did not adequately illustrate the cause-effect relationship involved in the [ATP4-]/[Mg’+] ratio dependence for the transition toward negative cooperativity. Although Fig. 2 suggested that the ratio of [ATP4-]/[Mg’+] influenced cooperativity, we analyzed the data in terms of an arbitrary thermodynamic expression. Treatment of the data in this manner allowed us to compare the ligand-dependent activation for each individual reaction within the multiple equilibria of the kinase with Mg2+ and ATP4-. Values for energy of activation (E,) are typically obtained by plotting the log V, vs l/T (“K-l). In this study, however, we wanted to estimate the activation of IRTK by Mg2+ rather than temperature. Thus, by plotting log u vs [Mg2+]-l or [ATP4-I-‘, our values are not in standard units, but in arbitrary units which then allowed US to examine the relationships capable of explaining the transition from positive to negative cooperativity. As shown in Fig. 3, linear relationships were obtained for MgCl, and Mg2+ at both low (50 PM) ATPr (Figs. 3A and B) and high (1000 PM) (Figs. 3D and E) ATPr. Expressed in arbitrary units, the ligand-dependent activation of IRTK by Mg2+ decreased from 30 units (Fig. 3B) to 0.12 unit (Fig. 3E) when the concentration of ATPr was high. This suggests that the energy required for for-

DISCUSSION

Our goal in these studies was to describe the interactions of IRTK with Mg2+, MgATP, and free ATP4- and to elucidate a mechanism(s) whereby these reactants modulate IRTK activity. As shown in Fig. lB, increasing concentrations of ATP4- decrease the concentration of Mg2+ required for half-maximal activation of IRTK. Thus, it is possible that ATP4- interacts with IRTK at or near the metal-dependent regulatory site and alters its conformation in such a way as to modulate the affinity of the enzyme for Mg2+. Increasing concentrations of ATP4- not only decrease the affinity of IRTK for ATP4(Fig. 1C) but also decrease its cooperativity toward ATP4-. Accordingly, our data suggest that a high ( [ATP4-]/[MgATP]) ratio modulates both the affinity for and the cooperativity of IRTK toward Mg2+ and ATP4- (Figs. 1B and C). The data shown in Figs. 3C and F suggest that ATP4- interacts at more than one site on the enzyme. One site is the active (metal-ATP) site at which free ATP4- is likely to compete with metal-ATP substrate. A second site could be regulatory in nature since it was observed (Fig. 1C) that a high concentration of ATPr (low free Mg2+, high free ATP4-) decreases the affinity of the kinase for ATP4-; i.e., the Ki for ATP4increased from 0.20 to 136 ~.LM when the concentration of ATPr increased. When the reaction velocity increases as a function of MgATP substrate, formation of the rate-limiting species E-MgATP will allow for a cooperativity equal to about 1.0. This value could increase to greater than 1.0 if the increases also as a function of ratio of [Eactivell[~less active1 an increase in [substrate]. For this to be possible under experimental conditions in which [E] remains constant, it is required to postulate that the enzyme exists initially in a low KM and/or a low V, form. This equilibrium between T (low affinity) and R (high affinity) forms (25), however, has been classically considered as a function of a single equilibrium between substrate (reactants) and modulators. Their analysis appears to be insufficient to define the true kinetic parameters of proteins whose physical-chemical properties indicate a tendency to es-

EFFECTS

OF DIVALENT

METALS

ON INSULIN

RECEPTOR

KINASE

103

KINETICS

2.2

1.6

1.6

0

0.02

0.04

0.06

0.08

0.10

0

0.02

0.04

0.06

0.08

0

0.10

4000

8000 (ATP4-)-I

(Mg*+)-l(mM)-l

(MgCl2)-l(mM)-1

12000

16000

20000

80

100

(mM)-1

0

2.4 -I

2.0 1 161

2.0,

1.61 0

0.4

0.8

1.2

1.6

2.0

0

(MgC12)-'(mM)-'

5

10

15

(Mg*+)-'

(mM)-'

20

25

0

20

40 (ATP4-)-1

60 (mu)-'

FIG. 3. Ligand-dependent interactions of IRTK with Mg”+ and ATP4-. Data from Fig. 1 for MgCl,, Mg”+, and ATP4- were replotted as a function of log u at 50 FM (0) and 1000 HIM (0) ATP4 The ligand-dependent activation (LD,) was calculated from the slopes of each curve according to the expression slope = -LD,/2.3R.

tablish multiple equilibria. Hence, several saturation parameters capable of modulating enzyme activity may change when the data are examined using varying concentrations of a single reactant. As the data in Fig. 1 illustrate, a single reactant (MgCl,) is capable of modify-

TABLE

III

and Thermodynamic Constants for the Indicated E Complexes

Kinetic

Enzyme complex Mn-E Mg-E EmMnATP EmMgATP E-peptide Mn-E-MnATP MgmE-MgATP MnE-peptide

Kd (mM)

KM (mid

-A@ (kcal)

0.50 1 .oo 0.82 0.10 0.09 1.47

6.50 4.54 4.45 4.04 4.16 5.39 5.45 3.82

0.015 0.430

Note. The kinetic and thermodynamic constants were determined from data shown in Fig. 2 and previously reported (13,14). A@ values were determined from the expression AC?’ = -2.3RT log K,, as described by Hitzemann (31). Kd, dissociation constant; KM, MichaelisMenten constant; X”, standard free energy.

ing the saturation state of the kinase not only with Mg2+ but also with MgATP and ATP4-, states which also change when the concentration of MgClz changes. The data indicate that interaction of Mg2+ with the kinase increases its affinity for MgATP substrate (13) and that binding of MgATP to the enzyme increases its affinity for Mg2+ (13) (Fig. 1B). This effect could yield cooperativity values greater than 1.0 if E differs from Mg-E by their observed difference in affinity for substrate. Thus, E meets the criteria for a T form of an enzyme and MgE for those of an R form. At high concentrations (50 mM) of MgCl,, the cooperativity of the response of the kinase toward Mg2+ (hMs2+) and ATP4- (h *ri+) is positive because the concentration of Mg-E, the R form of the enzyme, is increasing, whereas the concentration of ATP-E, the T form of the enzyme, is simultaneously decreasing. In contrast, when the concentration of ATPr is high (1000 PM), an increase in MgC12 is largely utilized to form MgATP substrate. Thus, the MgCl,-mass action displacement of the equilibria from a T form to an R form is reduced. Therefore, a shift to negative cooperativity in the response of the kinase to Mg2+ (hr&+) is observed (Fig. lB, Table II). On the other hand, the values for h MgT (Fig. lA, Table II) reflect the summation of the

104

VICAR10

+

Sl .-

b

AND

E-S, + s 2 \ KA

E +

2’

-4

Products

E\S, s,,

E-S2

+ S,

KA

+ Me KEA

11 -

+ ‘lb

Me-E-Sl+S,

BKa

Me-EH:-Prod”cts

Me-E

‘S* + SPY

Me-E-S ~KA

2 +S I

FIG. 4. Multiple equilibria among IRTK, metal-ATP, and peptide substrate in the absence and presence of excess divalent metal. KA represents the dissociation constant for the reaction of enzyme (E) with S, (MnATP or MgATP) and KB represents the dissociation constant for the reaction of E with S, (peptide), in the absence of excess free divalent metal. aKA represents the dissociation constant for the reaction of Me-E with S, and /3KB represents the dissociation constant for the reaction of Me-E with S,. The factors by which the dissociation constants KA and KB are altered by the presence of Me’+ (in excess of that required for metal-ATP substrate formation) are represented by cy and 0. KEA represents the dissociation constant for the Me-E complex. Changes in the affinity of E for MeATP substrate are depicted as differences in the thickness of the arrows.

BENNUN

control of turnover of the E complexes of this nature may at least partially account for the Me2+-induced activation of IRTK. Thus, even if the reaction mechanism remains a RARE bi-bi type, there will be a mass action effect favoring the formation of Me-E-MeATP over that of Me-E-peptide. Hence, the initial formation of Me-E-MeATP will be thermodynamically favored and the kinetic sequence could approach that of an ordered bi-bi. The rate of product formation will be increased by the mass action effect of an increase in the concentration of Me-E-MeATP substrate. An increase in turnover of this nature may be reflected by a simultaneous increase in both maximal velocity ( VM) and affinity (KM). Thus, the prior interaction of IRTK with Me’+ will be preferred since its effect is to facilitate the inclusion of MeATP substrate since Me-E is more reactive with MeATP than E alone. ACKNOWLEDGMENTS The authors thank Dr. M. A. Cascieri, Dr. R. Saperstein, and Mr. H. V. Strout for their constructive criticism of the initial manuscript.

REFERENCES 1.

cooperativity induced by the joint mass action of Mg2+ and MgATP on the R/T ratio. Adenylate cyclase has been reported to react with MgATP in a rapid random equilibrium (RARE) bi-bi mechanism (26). In this instance, what is observed as cooperativity, but which could also be termed mutual interaction, could be due to the capability of the enzyme to exist in two forms, E and Mg-E, both of which could react with MgATP. Hence, the classical hypothesis appears incomplete because it fails to predict a variable strength of interaction between sites with different roles, but capable of affecting each other even if present in a single subunit (27). It could also be predicted that liver glucokinase, a monomeric enzyme (28), may display its cooperativity (29) because it interacts with Mg2+ and MgATP at two distinct sites. Although the kinetic mechanism for the phosphorylation of exogenous peptides is of the RARE type (14,30), divalent metals influence this kinetic mechanism in that they thermodynamically favor the initial formation of a Me-E-MeATP complex. In the absence of excess free divalent metal, the affinity of the kinase for MeATP is low (13, 14). When divalent metals are present in excess of that required for MeATP substrate formation, the affinity of the enzyme for MeATP substrate increases at least an order of magnitude (13,14). Figure 4 illustrates that the interrelationship between the enzyme complexes allows for a Me ‘+-induced change in the affinity of IRTK for MeATP substrate, which is then capable of modifying the mass action ratios among E complexes. A

Czech, M. P. (1977) Annu. Rev. B&hem. 22,1059-1073. F. A., and Kahn, C. R. (1982) Science 215, 185-187. Rosen, 0. M., Herrera, R., Olowe, Y., Petruzzelli, L. M., and Cobb, M. H. (1983) Proc. N&l. Acad. Sci. USA 80,3237-3240. Graves, C. R., Gale, R. D., Laurino, J. R., and McDonald, J. M. (1986) J. Biol. Chem. 261,10429-10438. Kadowaki, T., Nishida, E., Kasuga, M., Ariyama, T., Tabaku, F., Ishikawa, M., Sakai, H., Kathuria, S., and Fujita-Yamaguchi, Y. (1985) Biochem. Biophys. Res. Commun. 12’7,493-500. Rees-Jones, R. W., and Taylor, S. I. (1985) J. B&l. Chem. 60,

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P. P., Saperstein, R., and Bennun, A. (1988) Arch. Biothem. Biophys. 26 1,336-345. 15. White, M. F., Haring, H. U., Kasuga, M., and Khan, C. R. (1984) J. Biol. Chem. 259,255-264. 16. Klein, H. H., Freidenberg, G. R., Cordera, R., and Olefsky, J. (1985) Biochem. Biophys. Res. Commun. 127,254-263.

EFFECTS 17. Hedo, J. A., Harrison, 3285-3293.

OF DIVALENT

METALS

L. C., and Roth, J. (1981) Biochemistry

18. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, (1951) J. Biol. Chem. 193,265-275.

ON INSULIN 20, R. J.

19. Casnelli, J. E., Harrison, M. L., Pike, L. J., Hellstrom, K. E., and Krebs, E. G. (1982) Proc. N&l. Acad. Sci. USA 79,282-286. 20. O’Sullivan, W. J., and Smithers, G. W. (1979) in Methods in Enzymology (Purich, D. L., Ed.), Vol. 63, pp. 294-336, Academic Press, New York. 21. Taqui-Khan, 10-15.

M. M., and Martell,

22. Taqui-Khan, M. M., and Martell, Sot. S&668-671.

A. E. (1962) J. Phys. Chem. 66, A. E. (1966) J. Amer. Chem.

23. Hill, A. V. (1922) Biochem. J. 7,471-480.

RECEPTOR

KINASE

KINETICS

105

24. Kwok, Y. A., Nemenoff, R. A., Powers, A. C., and Avruch, J. (1986) ’ Arch. Biochem. Biophys. 244,102-113. 25. Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Biol. Chem. 12,888118. 26. Ohanian, H., Borhanian, K., DeFrias, S., and Bennun, A. (1981) J. Bioenerg. Biomemb. 13,312-355. 27. Bennun, A. (1987) Biomed. Biochim. Acta 46,314-319. 28. Holroyde, M. J., Allen, M. B., Storer, A. C., Warsy, A. S., Chesher, J. M. E., Trayer, I. P., Cornish-Bowden, A., and Walker, D. G. (1976) Biochem. J. 153,363-373. 29. Cardenas, M. L., Rabajille, E., and Niemeyer, H. (1978) Arch. Biothem. Biophys. 190,142-148. 30. Walker, D. H., Kuppuswamy, D., Visvanathan, A., and Pike, L. J. (1987) Biochemistry 26,1428-1433. 31. Hitzemann, R. (1988) Trends Pharm. Sci. 9,408-411.