Alterations in AMP deaminase activity and kinetics in skeletal muscle of creatine kinase-deficient mice

Peter C. Tullson1, James W. E. Rush1, Bé Wieringa2, and R. L. Terjung1

1 Department of Physiology, State University of New York, Health Science Center at Syracuse, Syracuse, New York 13210; and 2 Department of Cell Biology and Histology, University of Nijmegen, 6500 HB Nijmegen, The Netherlands

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Alterations in the competency of the creatine kinase system elicit numerous structural and metabolic compensations, including changes in purine nucleotide metabolism. We evaluated molecular and kinetic changes in AMP deaminase from skeletal muscles of mice deficient in either cytosolic creatine kinase alone (M-CK-/-) or also deficient in mitochondrial creatine kinase (CK-/-) compared with wild type. We found that predominantly fast-twitch muscle, but not slow-twitch muscle, from both M-CK-/- and CK-/- mice had much lower AMP deaminase; the quantity of AMP deaminase detected by Western blot was correspondingly lower, whereas AMP deaminase-1 (AMPD1) gene expression was unchanged. Kinetic analysis of AMP deaminase from mixed muscle revealed negative cooperativity that was significantly greater in creatine kinase deficiencies. Treatment of AMP deaminase with acid phosphatase abolished negative cooperative behavior, indicating that a phosphorylation-dephosphorylation cycle may be important in the regulation of AMP deaminase.

muscle energetics; adenine nucleotides; inosine monophosphate; enzyme kinetics; enzyme phosphorylation

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE ONSET OF CONTRACTION by skeletal muscle can rapidly increase the rate of ATP hydrolysis ~100-fold within seconds. Skeletal muscle sustains these transitions via a high-energy phosphate network that facilitates the maintenance of the free energy of ATP hydrolysis (Delta GATP). This network attenuates changes in muscle adenylates via disequilibrium of key energy buffering reactions catalyzed by creatine and adenylate kinase (8, 28).

Creatine kinase (CK) effectively buffers both temporal and spatial changes in ATP concentration occurring during exercise by mediating the near-equilibrium phosphotransferase reaction of phosphocreatine (PCr) and ADP, yielding creatine (Cr) and ATP
PCr + MgADP<SUP>−</SUP> + H<SUP>+</SUP> ⇔ Cr + MgATP<SUP>2−</SUP>
Accumulation of ADP is also lessened by the near-equilibrium transphosphorylation of free ADP to ATP and AMP through the action of adenylate kinase
MgADP<SUP>−</SUP> + ADP<SUP>3−</SUP> ⇔ MgATP<SUP>2−</SUP> + AMP<SUP>−</SUP>
In muscle with an intact CK system, free ADP is held at low concentrations (~5-40 µM) until phosphocreatine is nearly depleted (2). Increases in free ADP lead to net forward flux through adenylate kinase giving rise to elevated free AMP concentrations. The reduction in Delta GATP may be further forestalled by deamination of the adenylate pool to IMP via the nonequilibrium reaction catalyzed by AMP deaminase
AMP<SUP>−</SUP> ⇒ IMP<SUP>−</SUP> + NH<SUB>3</SUB>
Thus deamination occurs when ATP utilization exceeds ADP rephosphorylation and probably serves to facilitate net forward flux through the adenylate kinase reaction by consuming AMP. The decline in the ATP/ADP ratio is thereby limited.

AMP deaminase is a tetrameric enzyme composed of identical 80-kDa subunits controlled, at least in part, by AMP concentration, allosteric modulators such as ADP and inorganic phosphate (20), and ATP turnover associated with muscle contraction (13). During intense contractions, AMP deamination can occur at an extremely high rate in fast-twitch muscle, sufficient to deplete the ATP concentration by ~50% (20). Once formed, IMP is retained within the myocyte until muscle energy balance is restored sufficiently for reamination back to AMP via the purine nucleotide cycle (18).

Feeding the creatine analog beta -guanidinopropionic acid (beta -GPA) to deplete the muscle creatine-phosphocreatine pool produces a functional CK system deficiency that results in several compensatory metabolic and morphological changes (9, 14, 23). In particular, the activity of muscle AMP deaminase is decreased as much as ~85% in fast-twitch muscle (10, 19). Interestingly, this occurs with no loss of ability to form IMP during exercise. It is presently unclear whether these changes are a result of the loss in the CK energy system per se or secondary to specific changes in phosphocreatine concentration and/or beta -GPA itself.

The purpose of this study was to determine the nature and extent of changes induced in AMP deaminase in the muscles of transgenic mice deficient in CK (16, 25, 29). Thus we have performed studies on AMP deaminase from skeletal muscles of mice possessing both cytosolic and mitochondrial isoforms of CK (wild type), mice deficient only in the cytosolic CK isoform (M-CK-/-), and mice with neither cytosolic nor mitochondrial CK isoforms (CK-/-). Specifically, AMP deaminase gene expression, activity, and kinetics have been investigated. Further in vitro experiments have established the likelihood that phosphorylation may play a key role in regulating AMP deaminase kinetic properties.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

CK-deficient strains. Transgenic mice with a homozygous null mutation for the gene encoding the M-subunit of CK (M-CK-/-) have no cytosolic CK (23, 24) but do express the mitochondrial isoform. Transgenic mice deficient in both the cytosolic and mitochondrial isoforms (CK-/-) were generated by crossing M-CK-/- and ScCKmit-/- mice (16). Each group was composed of five female mice.

Muscle sampling. Mice were anesthetized with 3-bromoethanol; the soleus, superficial vastus lateralis, and the mixed-fiber muscle of the upper and lower hindlimb were removed and frozen in liquid nitrogen and stored at -80°C until use. The mouse soleus is composed primarily of slow-twitch red fibers (4); the superficial vastus lateralis is expected to be primarily fast-twitch white fibers as is the superficial portion of the mouse gastrocnemius muscle (4) because there is generally excellent correspondence in fiber-type composition between the superficial quadriceps and gastrocnemius muscle in other rodents (1) and these knockout mice do not appear to exhibit significant changes in muscle fiber composition (cf. Refs. 16, 22, 23).

Enzyme assays. Frozen skeletal muscles/sections were extracted and assayed for AMP deaminase activity by direct assay of IMP formation via HPLC analyses, as described in detail previously (21). Reaction conditions were linear over the time of measurement and proportional to the amount of muscle extract used. Total AMP deaminase activity was determined on whole muscle homogenates, and kinetic analyses were performed on 13,000 g supernatants using an extensive range of AMP concentrations from 0.04 to 15 mM, as described previously (11). To minimize the potential for enzyme modification/inactivation, we did not further process the supernatants (dialyze to eliminate effectors); however, muscle extracts were diluted ~2,000-fold for the assay procedure. This would likely eliminate possible confounding influences of soluble effectors present in the muscle. There were no changes in the 80-kDa signal and no accumulation of any lower-molecular-weight immunoreactive AMP deaminase species in the wild-type or CK-/- samples over the time for kinetic determination. Maximal velocities and Michaelis-Menten (Km) constants were calculated by double-reciprocal plots.

Citrate synthase was assayed in duplicate using the same whole muscle homogenates as above after freeze-thawing three times (15).

Treatment of muscle extracts with acid phosphatase. The potential role of phosphorylation in governing AMP deaminase muscle kinetics was tested by treating muscle extracts from creatine-deficient mice with acid phosphatase. Acid phosphatase prepared from wheat germ (Sigma) was added to aliquots of the soluble cell fractions of M-CK-/- and CK-/- mixed muscle at a dose of 10 U/ml. These mixtures were incubated at room temperature for 30 min, after which they were used for kinetic analysis of AMP deaminase as described above. Preliminary experiments demonstrated that these conditions of dose and time were sufficient to elicit the maximal acid phosphatase-induced response in AMP deaminase kinetics. There were no accumulations of lower-molecular-weight immunoreactive AMP deaminase species with acid phosphatase treatment; however, the 80-kDa AMP deaminase signal was slightly reduced in both wild-type and CK-/- mice (~15%).

RNA analyses. Frozen mixed-muscle samples were weighed and pulverized in a mortar chilled with liquid nitrogen. Total cellular RNA was isolated using a kit (Ultraspec RNA; Biotecx, Houston, TX). Total RNA was quantitated by absorbance at 260 nm, and mRNA was determined by slot blot analysis of 0.25 and 0.5 µg of total RNA using end-labeled oligo(dT)12-18 (6).

Plasmid Bluescript containing the rat AMP deaminase-1 (AMPD1) cDNA insert was linearized using the restriction enzyme Stu I (New England Biolabs). A riboprobe was generated using T3 RNA polymerase, following the kit instructions (Riboprobe Combination System-T3/T7; Promega). To confirm mouse AMPD1 transcript size, we performed Northern blots with this riboprobe after agarose gel electrophoresis of 5 and 10 µg of total RNA. Protocols described in detail previously (12) were followed except that RNA was transferred to nylon membranes (MSI MagnaCharge; Fisher). AMPD1 transcript abundance was determined by probing slot blots loaded with 5 and 10 µg of total RNA. Radioactive bands identified on exposed X-ray film were digitized to an eight-bit gray scale image at 600 dots per inch (dpi) using a flatbed scanner (Hewlett Packard), with the darkest area of the region of interest set to 255 and the lightest to 0. Analyses were performed on a Macintosh computer using the gel-plotting macro of the public domain National Institutes of Health (NIH) Image software (version 1.60).

Western blot. Western blot analysis was performed using muscle samples extracted exactly as described previously (19) using the same polyclonal antiserum raised against purified rat skeletal muscle AMP deaminase (7). Developed nitrocellulose membranes were digitized and analyzed using NIH Image as described above.

Statistics. One- or two-way analyses of variance were used to evaluate significant differences among means. Tukey's omega -procedure was used to calculate least significant difference at P < 0.05.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

AMP deaminase activity. Total muscle AMP deaminase activity is altered by CK deficiencies in a fiber-type-specific fashion. As shown in Table 1, AMP deaminase activity was markedly reduced (by 70-75%) in the fast-twitch superficial vastus lateralis muscle from both single and double CK-deficient mice, whereas no change was detected in the soleus muscle containing predominantly slow-twitch red fibers. Citrate synthase activity, a marker of oxidative capacity, was increased in fast-twitch (200%) and slow-twitch (80%) CK-/- muscle (Table 1).

                              
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Table 1.   Total tissue AMP deaminase and citrate synthase activity in superficial vastus lateralis and soleus muscles of wild-type, M-CK-/-, and CK-/- mice

Distal hindlimb muscle composed of a mixture of fast- and slow-twitch fibers had lower AMP deaminase activities and smaller, although still highly significant, declines in AMP deaminase activity associated with CK deficiency than did the superficial vastus lateralis (cf. Table 2). Increases in citrate synthase activity in lower hindlimb muscles from CK-deficient animals were more modest than in the fast-twitch vastus lateralis, with significantly higher levels only in the M-CK-/- animals (Table 2).

                              
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Table 2.   Total tissue AMP deaminase and citrate synthase activity in lower hindlimb mixed-fiber muscle of wild-type, M-CK-/-, and CK-/- mice

AMP deaminase Western blot data. Figure 1A shows a representative Western blot for AMP deaminase from muscle of M-CK-/- and wild-type mice. The apparent molecular mass of AMP deaminase in mice of 80 kDa (cf. Fig. 1A) was similar to that found in rat skeletal muscle (19). The AMP deaminase protein in M-CK-/- mice was less abundant than in wild-type mice; increased amounts of smaller immunoreactive species having apparent molecular masses of ~60 and ~56 kDa were often observed in CK-deficient muscle (cf. Fig. 1B). Densitometric analysis of Western blots of mixed-fiber muscle, illustrated in Table 3, showed that the 80-kDa AMP deaminase protein was significantly decreased in M-CK-/- (by 50%) and CK-/- (by 80%) animals. Blot signals were proportional to protein load applied to the gel. The decreases were similar or greater in the vastus lateralis muscle section (cf. Table 3). In contrast and in agreement with the activity data (Table 1), the AMP deaminase protein of the soleus muscle was not significantly different in CK-/- mice.


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Fig. 1.   A: representative Western blot of lower hindlimb mixed-fiber muscle AMP deaminase from wild-type mice and mice deficient in cytosolic creatine kinase (M-CK-/-) (n = 3 each); 12 µg of protein from each extract were analyzed. For comparison of apparent molecular mass and to confirm proportionality, 4, 8, and 16 µg of protein from rat mixed-fiber plantaris muscle were included. After 10% SDS-PAGE, gels were blotted and probed using polyclonal antiserum raised against purified rat AMP deaminase isoform A (7). B: representative density profiles after Western blot analysis of 12 µg of protein from lower hindlimb mixed muscle of wild-type and M-CK-/- mice. Note the decrease in ~80-kDa protein and accumulation of smaller-sized immunoreactive proteins (~60- and ~56-kDa bands).

                              
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Table 3.   Relative levels of AMP deaminase 80-kDa protein in mixed, vastus lateralis, and soleus muscles of M-CK-/- and CK-/- mice relative to wild type

AMP deaminase gene expression. Data regarding AMPD1 gene expression are somewhat more complex (Table 4). Results of Northern blots showed expression to be marginally lower with CK deficiency, when expressed relative to the constant amount of total RNA applied to the blot. However, if AMPD1 gene expression is corrected for the relative amount of mRNA, as assessed by oligo(dT)12-18 hybridization, the decline in AMPD1 signal was significant in the CK-/- mice. Yet, the yield of total RNA was progressively higher from the M-CK-/- and CK-/- than from wild-type muscle. If AMPD1 gene expression is corrected for this increased amount of total RNA per gram, there was no change in AMPD1 mRNA abundance per gram of muscle. Thus it appears that an unchanged amount of AMPD1 mRNA was diluted by an increase in total muscle RNA found in the CK-deficient muscle.

                              
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Table 4.   Relative levels of total RNA, mRNA content, and AMPD1 gene expression in mixed-fiber muscle of wild-type, M-CK-/-, and CK-/- mice

AMP deaminase kinetics. Complex kinetic changes in AMP deaminase were observed in the mixed-fiber lower hindlimb muscle of CK-deficient mice. Figure 2 is a double-reciprocal plot of data from wild-type and M-CK-/- mice (results for CK-/- mice were similar to M-CK-/-; data not shown). The pronounced concave nature of the double-reciprocal plots for all groups reflects negative cooperativity. The kinetic changes were analyzed by independently evaluating the biphasic quasilinear portions of the double-reciprocal plot, representing two ranges of AMP concentration from 0.04 to 0.15 mM and from 0.2 to 15 mM, as illustrated in Fig. 2. The Km thus derived are presented in Table 5. The Km of AMP deaminase over the low AMP concentration range is significantly lower in muscle of single and double CK-deficient mice than in muscle of wild-type mice. It is noteworthy that, unlike normal rat mixed-fiber muscle (19), muscle from all three groups of mice had AMP deaminase that exhibited a higher substrate affinity over the lower range of AMP concentrations.


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Fig. 2.   Double-reciprocal plot of relative AMP deaminase activity from lower hindlimb mixed-fiber muscle of wild-type (WT; bullet ) and M-CK-/- (open circle ) mice (n = 5). Velocity (V) was normalized as a percentage of the activity measured at saturation (15 mM) to correct for differences in the amount of enzyme present in wild-type and M-CK-/- muscle. AMP deaminase from wild-type and M-CK-/- muscle displays negative cooperativity, evidenced by two linear phases representing a low-affinity component (solid line) and a high-affinity [low Michaelis-Menten constant (Km)] component (dashed line). Negative cooperativity is much greater in the M-CK-/- muscle.

                              
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Table 5.   Km of AMP deaminase from lower hindlimb mixed-fiber muscle of wild-type, M-CK-/-, and CK-/- mice

Effect of phosphatase treatment. Incubation of muscle extracts with purified acid phosphatase induced a remarkable change in enzyme kinetics. Figure 3 shows double-reciprocal plots of AMP deaminase from CK-/- mice after incubation in buffer with or without acid phosphatase. Incubation without acid phosphatase showed the expected biphasic kinetic pattern (cf. Figs. 2 and 3). Acid phosphatase treatment abolished the negative cooperativity, yielding a single, essentially linear, double-reciprocal plot. Similarly, essentially linear kinetic double-reciprocal plots were induced by acid phosphatase treatment in M-CK-/- and wild-type (data not shown) mice. Interestingly, the five- to eightfold increases in the phosphoserine-, phosphothreonine-, and phosphotyrosine-specific antibody signal in the 80-kDa AMP deaminase gel position for the CK-/- mouse muscle (compared with the wild-type mouse using equivalent AMP deaminase loads on the gel) were eliminated (88-93%) by acid phosphatase treatment (unpublished observations). Although confirmation is necessary, it appears that the AMP deaminase kinetic change coincides with specific amino acid phosphorylation-dephosphorylation.


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Fig. 3.   Double-reciprocal plot of relative AMP deaminase activity from lower hindlimb mixed-fiber muscle of mice with double CK deficiency (CK-/-) with (bullet ) and without (open circle ) treatment with acid phosphatase (n = 5). Velocity was normalized as in Fig. 2. Treatment with acid phosphatase abolishes negative cooperativity as shown by a single essentially linear phase representing a single low-affinity (high Km) component.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

CK deficiencies in skeletal muscle profoundly alter the morphological and metabolic properties of muscle tissue in ways that likely serve to compensate for the loss of ATP buffering capacity. Transformations occurring in the phenotype of CK-deficient muscle include alterations in contractile function (3, 22, 26), energy-transducing metabolic pathways (16, 22), and Ca2+ management (16, 22). The present work describes adaptive changes in AMP deaminase evident in CK-deficient muscle that we believe reveal specific regulatory mechanisms of fundamental importance for the control of adenine nucleotide metabolism in muscle.

Fast-twitch CK-deficient muscle exhibits markedly lower AMP deaminase activity that can be attributed to a loss of native tetrameric enzyme on the basis of the decreased quantity of the full-size (80 kDa) monomeric subunit as quantified by Western blot following SDS-PAGE. These results parallel the pattern established in rat skeletal muscle during creatine depletion using the creatine analog beta -GPA (19). In contrast, AMP deaminase activity in the predominantly slow-twitch red soleus muscle was similar for M-CK-/-, CK-/-, and wild-type mice; this finding was corroborated by the similar abundance in Western blot signal between wildtype and CK-/- mice (Table 3). These results expand the report (26) of the unique fiber-specific responses to CK deficiency in showing the distinctions between fast-twitch and slow-twitch skeletal muscle for AMP deaminase.

The extensive expansion of mitochondrial content in deficient mice, indicated by increases in citrate synthase activity, was nonuniform in different muscles and not strictly related to changes in AMP deaminase (Tables 1 and 2). Citrate synthase activity was elevated in CK-deficient hindlimb mixed-fiber muscle, as found by others (16, 22). The greatest differences in citrate synthase activity were found in the superficial vastus muscle, where CK-/- > M-CK-/- > wild type; AMP deaminase, however, was decreased similarly in the single and double CK-deficient animals. Changes evident in citrate synthase and AMP deaminase seem to be related to fiber composition. In predominantly fast-twitch superficial vastus muscle, differences in the two enzymes are reciprocal, whereas, in the predominantly slow-twitch soleus muscle, higher citrate synthase activity exists without diminished AMP deaminase. Thus we interpret the more modest changes in AMP deaminase of the mixed-fiber muscle of the hindlimb as an averaged response based on fiber composition. There have been no remarkable changes in fiber type, size, or distribution reported for muscle of M-CK-/- mice (27), although there is precedence for such changes induced by creatine analog feeding in normal mice (9) and in M-CK-/- mice (23). Whether gross alterations in muscle-fiber type or more subtle differences in myosin expression exist in the superficial vastus or soleus muscles used in this study is not presently known.

Our results argue that the decreased activity of AMP deaminase is not attributed to altered gene expression, based on the similar AMP deaminase mRNA abundance across groups (cf. Table 4). It is possible that translation efficiency of the AMP deaminase mRNA is diminished and/or that degradation, and consequently turnover, of AMP deaminase protein is increased. The frequent observation of smaller size immunoreactive species (~56 and 60 kDa) in Western blots (Fig. 1) suggests elevated proteolysis of the enzyme; if true, an increased turnover of the protein would be expected. These smaller, presumably inactive, species are even observed at low levels in blots of normal muscle (Fig. 1). In rats fed a diet containing beta -GPA, the lower apparent molecular weight species accumulated to a significant degree during the course of AMP deaminase enzyme loss (11, 19).

Kinetic experiments revealed that AMP deaminase from mixed muscle of wild-type and deficient mice displayed negative cooperativity (Fig. 2); furthermore, AMP deaminase from M-CK-/- and CK-/- exhibited significantly lower Km values (increased affinity) than the enzyme from wild-type muscle when evaluated at low-AMP concentrations (<0.2 mM) (Table 5). In both M-CK-/- and CK-/- mice, the absence of CK is associated with a loss of total AMP deaminase activity that is coupled to increased substrate affinity at low-substrate concentrations. A similar situation is established with creatine depletion in rats fed beta -GPA (11). Calculated flux through AMP deaminase at low physiologically relevant AMP concentrations, using the measured kinetic constants (Table 5) and maximal activities (Table 2), shows that the higher substrate affinity partially compensates for the lower quantity of enzyme. Thus it may be expected that AMP deaminase would function to effectively produce IMP during intense contraction conditions in vivo despite its lower enzyme protein abundance, similar to that observed for beta -GPA-treated muscle (10, 19).

Several years ago, Tovmasian et al. (17) showed that rat skeletal muscle AMP deaminase could be phosphorylated by the rat brain protein kinase C. Thus it was important to evaluate whether phosphorylation of AMP deaminase could be the basis for the kinetic alteration seen in enzyme from M-CK-/- and CK-/- mice. The remarkable effect that treatment with acid phosphatase had in abolishing the negative cooperativity kinetic effect, similar to that observed in muscle from beta -GPA-fed rats (11), is consistent with a critical role for a phosphorylation-dephosphorylation cycle in the control of AMP deaminase activity in vivo. If further experiments support this hypothesis, AMP deaminase phosphorylation-dephosphorylation may prove to be of critical importance in the management of the muscle adenine nucleotide pool during exercise.

The specific mechanism(s) linking functional changes in the CK system to altered AMP deaminase activity is not known. CK and AMP deaminase buffer decrease in the Delta GATP by facilitating the consumption of ADP during intense muscle contraction conditions. The synergistic roles of these enzymes suggest that a chronically altered energy balance resulting from CK system deficiencies might exert a regulatory effect on AMP deaminase. This may also be evident to a modest extent in resting muscle. Our working hypothesis is that a modified energy state (cf. Ref. 5) and/or Ca2+ management (cf. Ref. 16), established in resting muscle by loss of the CK energy buffer system, favors phosphorylation of AMP deaminase; this, in turn, induces negative cooperativity kinetic behavior (Fig. 3). The resulting elevated activity at physiological AMP concentrations (Km low at ~0.02 mM), in concert with previously identified noncovalent modulators of AMP deaminase, such as ADP, activates deamination. This serves to attenuate declines in Delta GATP during periods of high energy demand. Similarly, the loss in ATP buffering capacity and/or its effects on H+ and phosphate homeostasis could be involved. It is intriguing to speculate further that the altered conformation due to phosphorylation increases the susceptibility of AMP deaminase to proteolytic degradation. This could potentially account for the increased abundance of the 56- and 60-kDa AMP deaminase fragments identified in the Western blots.

In summary, metabolic changes brought about by CK deficiencies include changes in the quantity of AMP deaminase activity and the 80-kDa subunit enzyme. Kinetic behavior exhibiting negative cooperativity is greater in the enzyme from deficient mice. If muscle metabolism of the transgenically altered mice is similar to that observed in creatine-depleted rats (11, 19), we would predict that, despite the smaller amounts of enzyme, the ability to deaminate AMP during intense exercise may be maintained by increases in the enzyme's affinity for AMP.

    ACKNOWLEDGEMENTS

The expert technical assistance of Judy Freshour and Mary Graziano (SUNY Health Science Center) and Mr. Frank Oerlemans (University of Nijmegen) is gratefully recognized. The polyclonal antibody and cDNA probe to AMPD1 were the kind gift of Dr. Richard L. Sabina (Medical College of Wisconsin, Milwaukee, WI).

    FOOTNOTES

This study was supported by National Institutes of Arthritis and Musculoskeletal and Skin Diseases Grant AR-21617 and by a program grant from the Dutch organization for Scientific Research (Medical Sciences). J. W. E. Rush was the recipient of a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship B predoctoral fellowship.

Address for reprint requests: R. L. Terjung, Biomedical Sciences, College of Veterinary Medicine, E 102 Vet. Med. Bldg., Univ. of Missouri, Columbia, MO 65211.

Received 10 November 1997; accepted in final form 5 February 1998.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

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AJP Cell Physiol 274(5):C1411-C1416
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society