Characterization of human tissue carnosinase.

Human tissue carnosinase (EC 3.4.13.3) had optimum activity at pH9.5 and was a cysteine peptidase, being activated by dithiothreitol and inhibited by p-hydroxymercuribenzoate. By optimizing assay conditions, the activity per g of tissue was increased 10-fold compared with values in the literature. The enzyme was present in every human tissue assayed and was entirely different from serum carnosinase. Highly purified tissue carnosinase had a broader specificity than hog kidney carnosinase. Although tissue carnosinase was very strongly inhibited by bestatin, it did not hydrolyse tripeptides, and thus appears to be a dipeptidase rather than an aminopeptidase. It had a relative molecular mass of 90 000, an isoelectric point of 5.6, and a Km value of 10 mM-carnosine. Two forms of kidney and brain carnosinase were separated by high-resolution anion-exchange chromatography, although only one form was detected by various electrophoretic methods. Homocarnosinase and Mn2+-independent carnosinase were not detected in human tissues, although these enzymes are present in rat and hog kidney.     

Homocarnosinase: a hog kidney dipeptidase with a broader specificity than carnosinase.

A dipeptidase was isolated from hog kidney; it is the first enzyme described that has the capacity to cleave homocarnosine. It was purified to apparent homogeneity and split carnosine, anserine, and several other dipeptides in addition to homocarnosine. Homocarnosinase had a molecular weight of 57,000 as determined by sodium dodecyl sulfate-gel electrophoresis; it appeared to consist of a single polypeptide chain and did not contain sulfhydryl groups or serine residues essential to its activity. The enzyme was activated by Co²⁺ and by Mn²⁺, cobaltous ions being much more effective than manganous ions. Its isoelectric point was 5.6 and no evidence of isozymes was seen during isoelectric focusing. Homocarnosinase had a broader specificity, higher solubility, lower stability, and different metal ion sensitivity than hog kidney carnosinase (EC 3.4.13.3). Carnosinase was present in most tissues of the rat, whereas homocarnosinase was detected only in kidney, uterus, lung, and liver.     

Human cytosolic carnosinase: evidence of identity with prolinase, a non-specific dipeptidase

In separate papers published in 1985, human cytosolic carnosinase and prolinase were purified and characterized for the first time. Prolinase had activity against many dipeptides not containing proline; carnosinase also had broad specificity. The present paper reports that carnosinase and prolinase activities were not separated from one another during chromatography on columns of DEAE-cellulose, AGMP-1, gel filtration media, hydroxylapatite or butyl-agarose. Both activities had identical pH-stability curves at 50 degrees C, being stabilized by manganese ions and dithiothreitol. Prolinase substrates competitively inhibited carnosinase activity and carnosinase substrates inhibited prolinase activity. Bestatin was a potent inhibitor of both activities, while cilastatin inhibited neither. It was concluded that prolinase and carnosinase activities reside in the same enzyme. High performance anion-exchange chromatography of extracts from kidney, liver or brain separated the enzyme into two forms having isoelectric points of 5.6 and 5.1. Because of the broad specificity of this dipeptidase, it is recommended that it be termed "human cytosolic non-specific dipeptidase".     

Specificity and distribution of mammalian carnosinase.

Hog kidney carnosinase (EC 3.4.13.3) was found to have a narrow specificity; it hydrolyzed carnosine, anserine and glycyl-L-histidine, but did not split L-alanyl-L-histidine or homocarnosine. The isoelectric point of this enzyme was 5.8 and its molecular weight was about 84 000. Carnosinase was found to be widely distributed in various tissues of the rat. Uterus, kidney, liver and lung contained high levels of carnosinase, whereas moderate concentrations were found in spleen, heart and brain, with low levels in small intestine, skeletal muscle and stomach, and none in blood.     

Bestatin inhibition of human tissue carnosinase, a non-specific cytosolic dipeptidase

Bestatin is a dipeptide containing a unique beta-amino acid. It is usually referred to as an aminopeptidase inhibitor. Current interest has focused on the immunostimulating activity of bestatin and several clinical trials have demonstrated that it is an effective adjunct to radiation or chemotherapy in the treatment of certain types of cancer. We found that bestatin was much more effective against human tissue carnosinase than against aminopeptidases. Inhibition was competitive, with a Ki of 0.5nM. Carnosinase did not hydrolyse bestatin and the enzyme-inhibitor complex formed rapidly. A hog kidney dipeptidase similar to human tissue carnosinase was equally sensitive to this inhibitor. Bestatin has a backbone structure identical to that of carnosine; however, our results indicate that the inhibitory activity of this compound is primarily attributable to the side chains of the beta-amino-acid moiety. Human tissue carnosinase is a non-specific dipeptidase, actively hydrolysing many dipeptides, including prolinase substrates. Inhibition of this cytosolic enzyme is probably at least partially responsible for the intracellular accumulation of dipeptides which occurs following the in vivo administration of bestatin.     

Salmonella typhimurium peptidase active on carnosine.

Wild-type Salmonella typhimurium can use carnosine (beta-alanyl-L-histidine) as a source of histidine, but carnosine utilization is blocked in particular mutants defective in the constitutive enzyme peptidase D, the product of the pepD gene. Biochemical evidence for assigning carnosinase activity to peptidase D (a broad-specificity dipeptidase) includes: (i) coelution of carnosinase and dipeptidase activity from diethylaminoethyl-cellulose and Bio-Gel P-300 columns; (ii) coelectrophoresis of carnosinase and dipeptidase on polyacrylamide gels; and (iii) inactivation of carnosinase and dipeptidase activities at identical rates at both 4 and 42 degrees C. Genetic evidence indicates that mutations leading to loss of carnosinase activity map at pepD. Several independent pepD mutants have been isolated by different selection procedures, and the patterns of peptide utilization of strains carrying various pepD alleles have been studied. Many pepD mutations lead to the production of partially active peptidase D enzymes with substrate specificities that differ strikingly from those of the wild-type enzyme. The growth yields of carnosinase-deficient strains growing in Difco nutrient broth indicate that carnosine is the major utilizable source of histidine in this medium.     

Similarity of tuna N-acetylhistidine deacetylase and cod fish anserinase.

1. The brain and ocular fluid of skipjack tuna (Katsuwonus pelamis) contained high levels of N-acetylhistidine deacetylase. 2. This enzyme had a molecular weight of about 120,000 and was activated by zinc or cobaltous ions. 3. Cod (Gadus callarias) brain, ocular fluid and muscle contained a similar metal-activated thiol hydrolase, the muscle enzyme being known as anserinase. 4. The purified enzymes hydrolyzed N-acetylhistidine, carnosine, homocarnosine, anserine and certain other dipeptides. 5. Their specificity resembled that of hog kidney homocarnosinase. 6. In both fish, brain and ocular fluid were rich sources of this hydrolase, whereas muscle contained only trace amounts.     

Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-aminobutyric acid-L-histidine) are two naturally occurring dipeptides with potential neuroprotective and neurotransmitter functions in the brain. Peptidase activities degrading both carnosine and homocarnosine have been described previously, but the genes linked to these activities were unknown. Here we present the identification of two novel cDNAs named CN1 and CN2 coding for two proteins of 56.8 and 52.7 kDa and their classification as members of the M20 metalloprotease family. Whereas human CN1 mRNA and protein are brain-specific, CN2 codes for a ubiquitous protein. In contrast, expression of the mouse and rat CN1 orthologues was detectable only in kidney. The recombinant CN1 and CN2 proteins were expressed in Chinese hamster ovary cells and purified to homogeneity. CN1 was identified as a homodimeric dipeptidase with a narrow substrate specificity for Xaa-His dipeptides including those with Xaa = beta Ala (carnosine, K(m) 1.2 mM), N-methyl beta Ala, Ala, Gly, and gamma-aminobutyric acid (homocarnosine, K(m) 200 microM), an isoelectric point of pH 4.5, and maximal activity at pH 8.5. CN2 protein is a dipeptidase not limited to Xaa-His dipeptides, requires Mn(2+) for full activity, and is sensitive to inhibition by bestatin (IC(50) 7 nM). This enzyme does not degrade homocarnosine and hydrolyzes carnosine only at alkaline pH with an optimum at pH 9.5. Based on their substrate specificity and biophysical and biochemical properties CN1 was identified as human carnosinase (EC ), whereas CN2 corresponds to the cytosolic nonspecific dipeptidase (EC ).     

Cilastatin-sensitive dehydropeptidase I enzymes from three sources all catalyze carbapenem hydrolysis and conversion of leukotriene D4 to leukotriene E4

The dipeptidase, dehydropeptidase I (EC 3.4.13.11), was purified to homogeneity from rat lung, rat kidney, and hog kidney. Analysis of physical parameters (subunit molecular weights, Km values for glycyldehydrophenylalanine, Ki values for dehydropeptidase I inhibitors, and immunoreactivity) showed the rat dipeptidases to be similar to each other but different from the hog dipeptidase. However, all three enzymes hydrolyzed imipenem and converted leukotriene D4 to leukotriene E4, and these activities were inhibited by cilastatin.     

Characterization of two carnosine-degrading enzymes from rat brain. Partial purification and characterization of a carnosinase and a beta-alanyl-arginine hydrolase

From rat brain extracts, two carnosine-degrading enzymes have been identified and partially purified by ion-exchange chromatography, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B and gel filtration. These enzymes exhibit distinct differences in their chemical characteristics and substrate specificities. One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low Km value (0.02 mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine-degrading enzyme hydrolyzes beta Ala-Arg considerably faster than carnosine and, therefore, has been tentatively designated beta Ala-Arg hydrolase. This enzyme exhibits high Km values with carnosine (Km = 25 mM) and beta Ala-Arg (Km = 2 mM). Homocarnosine and gamma-aminobutyryl-arginine are not degraded by beta Ala-Arg hydrolase. Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carnosinase is also not inhibited by bestatin but beta Ala-Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50 = 50 nM). While carnosinase is strongly inhibited by thiol-reducing agents such as dithioerythritol and 2-mercaptoethanol, beta Ala-Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal-chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by Mn2+ as well as by heavy metal ions. In contrast, beta Ala-Arg hydrolase requires Mn2+ ions for full enzymatic activity. Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles, but considerable differences in their relative distribution among different tissues are also observed.     

Purification and identification of a novel primitive secretory enzyme catalyzing the hydrolysis of imidazole-related dipeptides in the jawless vertebrate Lethenteron reissneri

Imidazole-related dipeptides, such as carnosine and anserine, occur widely in skeletal muscles of jawed vertebrates. All of the known enzymes that catalyze the hydrolysis of these dipeptides belong to the M20A metallopeptidase subfamily; two secretory enzymes, serum carnosinase (EC 3.4.13.20) and anserinase (EC 3.4.13.5), and one non-secretory enzyme, cytosolic nonspecific dipeptidase (EC 3.4.13.18). Here we report the enzymatic characterization and molecular identification of an unidentified enzyme, which catalyzes the hydrolysis of these dipeptides, from the skeletal muscle of Far Eastern brook lamprey (Lethenteron reissneri). A 60-kDa subunit protein of the enzyme was purified to near homogeneity. We cloned two M20A genes from the skeletal muscle of Far Eastern brook lamprey; one was a secretory-type gene encoding for the 60-kD protein, and another was a non-secretory-type gene presumably encoding for cytosolic nonspecific dipeptidase. Our findings indicate that the purified enzyme is a N-glycosylated secretory M20A dipeptidase distributed specifically in the jawless vertebrate group, and may be derived from a common ancestor gene between serum carnosinase and anserinase. We propose that this dipeptidase is a novel secretory M20A enzyme and is classified as neither serum carnosinase nor anserinase.     

New glycoside derivatives of carnosine and analogs resistant to carnosinase hydrolysis: synthesis and characterization of their copper(II) complexes

Carnosine (β-alanyl-L-histidine) is an endogenous dipeptide widely and abundantly distributed in muscle and nervous tissues of several animal species. Many functions have been proposed for this compound, such as antioxidant and metal ion-chelator properties. However, the main limitation on therapeutic use of carnosine on pathologies related to increased oxidative stress and/or metal ion dishomeostasis is associated with the hydrolysis by the specific dipeptidase carnosinase. Several attempts have been made to overcome this limitation. On this basis, we functionalized carnosine and its amide derivative with small sugars such as glucose and lactose. The resistance of these derivatives to the carnosinase hydrolysis was tested and compared with that of carnosine. We found that the glycoconjugation protects the dipeptide moiety from carnosinase hydrolysis, thus potentially improving the availability of carnosine. The copper(II) binding properties of all the new synthesized compounds were investigated by spectroscopic (UV-Visible and circular dichroism) and ESI-MS studies. Particularly, the new family of amide derivatives that are not significantly hydrolyzed by carnosinase is a very promising class of carnosine derivatives. The sugar moiety can act as a recognition element. These new derivatives are potentially able to act as chelating agents in the development of clinical approaches for the regulation of metal homeostasis in the field of medicinal inorganic chemistry.     

Purification and identification of two carnosine-cleaving enzymes, carnosine dipeptidase I and Xaa-methyl-His dipeptidase, from Japanese eel (Anguilla japonica)

Three enzymes, carnosine dipeptidase I (EC 3.4.13.20, CNDP1), carnosine dipeptidase II (EC 3.4.13.18, CNDP2), and Xaa-methyl-His dipeptidase (or anserinase: EC 3.4.13.5, ANSN), are known to be capable of catalyzing the hydrolysis of carnosine (β-alanyl-l-histidine), in vertebrates. Here we report the purification and identification of two unidentified carnosine-cleaving enzymes from Japanese eel (Anguilla japonica). Two different dipeptidases were successfully purified to homogeneity from the skeletal muscle; one exhibited a broad substrate specificity, while the other a narrow specificity. N-terminal amino-acid sequencing, deglycosylation analysis, and genetic analysis clearly revealed that the former is a homodimer of glycosylated subunits, encoded by ANSN, and the latter is another homodimer of glycosylated subunits, encoded by CNDP1; that is, Xaa-methyl-His dipeptidase, and carnosine dipeptidase I respectively. This is the first report on the identification of carnosine dipeptidase I from a non-mammal. Database search revealed presence of a CNDP1 ortholog only from salmonid fishes, including Atlantic salmon and rainbow trout, but not from other ray-finned fish species, such as zebrafish, fugu, and medaka whose genomes have been completely sequenced. The mRNAs of CNDP1 and ANSN are strongly expressed in the liver of Japanese eel, compared with other tissues, while that of CNDP2 is widely distributed in all tissues tested.     

Kidney and liver carnosinase activity and carnosine level in goose.

Activity of kidney and liver carnosinase and concentration of carnosine in leg muscles were determined for 8 weeks in old geese of three races: Italian white, Bilgoraj and Lublin. significant differences were noted between the three races with respect to all parameters under study. the following correlations were found: 1. Between live goose weight and carnosine concentration in muscles (r= 0.5276). 2. Between weight of leg muscles and carnosine level in these muscles (r=0.4912). 3. Between liver weight and carnosine level in muscles (r= 0.3292). 4. Between kidney carnosinase activity and liver carnosinase activity (r= .2104). 5. Between liver carnosinase activity and carnosine level (r= 0.2280). 6. Between kidney carnosinase activity and carnosine level (r= -0.1675). 7. Between the ratio of kidney:liver carnosinase activity and carnosine level in muscles (r =0.1816).     

Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick disease

Summary Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%.Sphingomyelinase activity was separated into three peaks (I–III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced.Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.     

Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain

High performance liquid chromatography studies documented the presence of an enzyme activity, N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase), in rat brain membranes that cleaves the endogenous brain dipeptide, N-acetyl-L-aspartyl-L-glutamate to N-acetyl-aspartate and glutamate. With ion exchange chromatography, which quantitatively separated [3,4-3H]glutamate from N-acetyl-L-aspartyl-L-[3,4-3H]glutamate, we found that NAALA dipeptidase activity was essentially restricted to nervous tissue and kidney. We characterized NAALA dipeptidase activity in lysed synaptosomal membranes obtained from rat forebrain. Membrane-bound NAALA dipeptidase activity was optimal between pH 6.0 and 7.4 at 37 degrees C. Eadie-Hofstee analysis of kinetic data revealed a rather high apparent affinity for N-acetyl-L-aspartyl-L-glutamate with a Km = 540 nM and a Vmax = 180 nM/mg of protein/min. While NAALA dipeptidase showed a requirement for monovalent anions such as Cl-, the polyvalent anions phosphate and sulfate inhibited enzyme activity 50% at 100 microM and 1 mM, respectively. The divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which was partially restored by manganese. Treatment of membranes with 1 mM dithiothreitol abolished NAALA dipeptidase activity. NAALA dipeptidase activity was also sensitive to the aminopeptidase inhibitors bestatin and puromycin, although not to the selective aminopeptidase A inhibitor amastatin. Structure-activity relationships inferred from inhibitor studies suggest that this enzyme shows specificity for N-acetylated alpha-linked acidic dipeptides. NAALA dipeptidase was also potently inhibited by the excitatory amino acid agonist L-quisqualate. Comparison of the properties of NAALA dipeptidase to those of previously characterized enzymes suggests that this is a novel peptidase which may be involved in the synaptic degradation of N-acetyl-L-aspartyl-L-glutamate.     

Multiple forms of the cobalt(II)-carnosine complex.

Cobalt(II) ion and L-carnosine produce two different complexes when mixed in aqueous solution at pH 7.2. One complex has coordination of N-3 of the imidazole ring to the cobalt(II) and is produced when the concentration of peptide exceeds that of cobalt(II). The second complex has chelation of three nitrogen atoms of a single carnosine. This second complex produces a reversible oxygen carrier by making stable mixed chelates with additional carnosine, histidine or cysteine. These results indicate that cobalt complexes with mixed ligands should be of more importance $\text{in}\text{vivo}$ than those with carnosine as the only ligand. They provide an explanation for the high activity and substrate specificity of carnosinase in kidney.     

Low plasma carnosinase activity promotes carnosinemia after carnosine ingestion in humans

A polymorphism in the carnosine dipeptidase-1 gene (CNDP1), resulting in decreased plasma carnosinase activity, is associated with a reduced risk for diabetic nephropathy. Because carnosine, a natural scavenger/suppressor of ROS, advanced glycation end products, and reactive aldehydes, is readily degraded in blood by the highly active carnosinase enzyme, it has been postulated that low serum carnosinase activity might be advantageous to reduce diabetic complications. The aim of this study was to examine whether low carnosinase activity promotes circulating carnosine levels after carnosine supplementation in humans. Blood and urine were sampled in 25 healthy subjects after acute supplementation with 60 mg/kg body wt carnosine. Precooled EDTA-containing tubes were used for blood withdrawal, and plasma samples were immediately deproteinized and analyzed for carnosine and β-alanine by HPLC. CNDP1 genotype, baseline plasma carnosinase activity, and protein content were assessed. Upon carnosine ingestion, 8 of the 25 subjects (responders) displayed a measurable increase in plasma carnosine up to 1 h after supplementation. Subjects with no measurable increment in plasma carnosine (nonresponders) had ～2-fold higher plasma carnosinase protein content and ～1.5-fold higher activity compared with responders. Urinary carnosine recovery was 2.6-fold higher in responders versus nonresponders and was negatively dependent on both the activity and protein content of the plasma carnosinase enzyme. In conclusion, low plasma carnosinase activity promotes the presence of circulating carnosine upon an oral challenge. These data may further clarify the link among CNDP1 genotype, carnosinase, and diabetic nephropathy.     

Cyclic nucleotide phosphodiesterase activities from pig coronary arteries. Lack of interconvertibility of major forms

DEAE-cellulose chromatography, with or without dithiothreitol and over a pH range of 6.0 to 8.5, resolved two phosphodiesterase activities (peaks I and II) from the soluble fraction of pig coronary arteries. The activity of peak I was increased by calmodulin (3-7-fold), whereas that of peak II was not. Chromatography of peak I on Biol-Gel A-0.5 m columns resolved two peaks of phosphodiesterase activity (peaks Ia and Ib). Peak Ia was eluted in the presence or absence of 0.1 M KCl and was relatively insensitive to calmodulin. Peak Ib was eluted only in the presence of KCl and was sensitive to calmodulin. The substrate specificity and kinetic behavior were the same for peaks I, Ia, and Ib. Repeated gel chromatography of either peak Ia or Ib, under appropriate conditions, yielded a mixture of peaks Ia and Ib. Peak Ia appears to be a reversible aggregate of peak Ib. Gel chromatography of peak II resolved only one phosphodiesterase activity, which was eluted without KCl, was highly specific for cyclic AMP, was not sensitive to calmodulin and migrated differently on the gel column than either peak Ia or Ib. Sucrose density gradient centrifugation of the soluble fraction from pig coronary arteries in the presence or absence of dithiothreitol resolved two peaks of phosphodiesterase activity (6.6 S and 3.6 S) which were similar to peaks I and II separated by DEAE-cellulose chromatography with regard to their substrate specificity and their sensitivity to calmodulin. Upon recentrifugation, each of the two peaks of phosphodiesterase activity gave a single peak of activity which migrated with the same S value as did its parent. These results indicate that the two major forms of phosphodiesterase of pig coronary arteries, which are representative of those found in many tissues, are not interconvertible in cell-free systems.     

Reversible phosphorylation control of skeletal muscle pyruvate kinase and phosphofructokinase during estivation in the spadefoot toad,Scaphiopus couchii

Both pyruvate kinase (PK) and phosphofructokinase (PFK) occur in two different forms, separable by isoelectric focusing (IEF), in skeletal muscle of the spadefoot toad Scaphiopus couchii. During estivation (aerobic dormancy) the proportions of the two forms changed compared with controls; in both cases the amount of enzyme in Peak I (pI = 5.3-5.4) decreased whereas activity in Peak II (isoelectric point = 6.2-6.4) increased. In vitro incubation of crude muscle extracts with 32P-ATP under conditions that promoted the activity of cAMP-dependent protein kinase led to strong radiolabeling associated with Peak I, but not Peak II, and reverse phase HPLC confirmed that 32P was associated with the subunits of both PK and PFK found in Peak I. Specific radiolabeling of Peak I PK and PFK by protein kinase A was further confirmed using immunoprecipitation. In total, this information allowed identification of the Peaks I and II enzymes as the phosphorylated and dephosphorylated forms, respectively, and the effect of estivation was to increase the proportion of dephosphorylated PK and PFK in muscle. Analysis of the kinetic properties of partially purified PK and PFK revealed significant kinetic differences between the two forms of each enzyme. For PK, the Peak II (low phosphate) enzyme showed a 1.6-fold higher Km for phosphoenolpyruvate and a 2.4-fold higher Ka for fructose-1,6-bisphosphate than did the Peak I (high phosphate) form. These kinetic properties suggest that Peak II PK is the less active form, and coupled with the shift to predominantly the Peak II form during estivation (87% Peak II vs. 13% Peak I), are consistent with a suppression of PK activity in estivating muscle, as part of the overall metabolic rate depression of the estivating state. A similar shift to predominantly the Peak II, low phosphate, form of PFK (75% Peak II, 25% Peak I) in muscle of estivating animals is also consistent with metabolic suppression since phosphorylation of vertebrate skeletal muscle PFK is typically stimulated during exercise to enhance enzyme binding to myofibrils in active muscle. Peak II PFK also showed reduced sensitivity to inhibition by Mg:ATP (I50 50% higher) compared with the Peak I form suggesting that the enzyme in estivating muscle is less tightly regulated by cellular adenylate status than in awake toads. The data indicate that reversible phosphorylation control over the activity states of enzymes of intermediary metabolism is an important mechanism for regulating transitions between dormant and active states in estivating species.