A calpain-like proteolytic activity produces the limited cleavage at the N-terminal regulatory domain of rabbit skeletal muscle AMP deaminase: evidence of a protective molecular mechanism

On storage at 4 degrees C, rabbit skeletal muscle AMP deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.     

Effect of pH and KCl on aggregation state and sulphydryl groups reactivity of rat skeletal muscle AMP deaminase

The effects of pH and KCl on sedimentation properties and SH groups reactivity of rat skeletal muscle AMP deaminase have been investigated. The values obtained for apparent molecular weight are consistent with an association of AMP deaminase subunits in response to increasing KCl concentration. Increasing pH value from 6.0 to 8.0 causes a reduction in the apparent molecular weight of the enzyme at high KCl concentration, which can be interpreted as due to a deprotonation-induced isomerization process. Removal of Zn2+ from AMP deaminase has effect similar to alkalinization in modifying the sedimentation properties of the enzyme. In the native enzyme at high K+ concentration about 7, 9 and 12 SH groups can be titrated with Nbs2, approximately 1, 2 and 4 SH groups reacting as fast sets, at pH 6.0, 7.0 and 8.0, respectively. Substitution of the 12 SH groups reactive with Nbs2 at pH 8.0 has no effect on the pH-dependent allosteric behaviour of the enzyme. Removal of K+ causes considerable changes in the reactivity of AMP deaminase towards Nbs2, unmasking a class of additional SH groups, so that the total number of titratable SH groups approaches that of 30 determined in denaturing conditions. In the enzyme previously treated with N-ethylmaleimide to alkylate the fast reacting class of SH groups, the class of additional SH groups are substituted by Nbs2 at basic pH, but not at acidic pH, with a concomitant reduction of the enzyme activity.     

Activation of trout gill AMP deaminase by an endogenous proteinase--I. Effects on the regulatory properties of the enzyme

Some regulatory properties of trout gill AMP deaminase were determined in crude extracts, before or after modification of the enzyme by the endogenous proteinase. After proteolysis, the optimal concentrations for activation by sodium and potassium were shifted from 10 to 75 mM, resulting in a large increase of enzyme activity near the physiological potassium concentration. This activation was shown to be the consequence of a much lower sensitivity of AMP deaminase to inhibition by increasing ionic strength. The modified enzyme was also less sensitive to modifications of pH and to inhibition by physiological concentrations of inorganic phosphate. When all these modifications were considered, limited proteolysis of gill AMP deaminase resulted in a 40 times increase of enzyme activity under in vivo conditions.     

Activation of trout gill AMP deaminase by an endogenous proteinase--II. Modification of the properties of the enzyme during starvation, pollution and salinity changes

The relative amount of modified AMP deaminase has been determined by taking advantage of the different effects of monovalent cations on the two enzymatic forms. When trout were subjected to different environmental perturbations (starvation, pollution of the water by a pesticide, transfer to sea water or reverse transfer to fresh water), modified AMP deaminase could be detected in the gill extracts. Depending on the nature of the stress and the period of experimentation, 8 to 100% of the enzyme had been modified by limited proteolysis. As a consequence of the much higher activity of the proteolyzed AMP deaminase form, a 2 to 12 times increase of the intracellular AMP deaminase activity could be expected. At the same time, limited proteolysis will modify the regulatory properties of the enzyme, since it can be estimated that 50 to 100% of the enzyme activity expressed in the cell will be an AMP deaminase form less sensitive to inhibition by inorganic phosphate and ionic strength, and to variations of the intracellular pH. Limited proteolysis will result in increased AMP deaminase activity under conditions of increased energy demand, where the concentration of inorganic phosphate is dramatically increased. The consequence should be stabilization of the adenylate energy charge.     

The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis

The key regulatory enzyme in the biosynthetic pathway of the plant hormone ethylene is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.14). We have partially purified ACC synthase 6,000-fold from Cucurbita fruit tissue treated with indoleacetic acid + benzyladenine + aminooxyacetic acid + LiCl. The enzyme has a specific activity of 35,000 nmol/h/mg protein, a pH optimum of 9.5, an isoelectric point of 5.0, a Km of 17 microM with respect to S-adenosylmethionine, and is a dimer of two identical subunits of approximately 46,000 Da each. The subunit exists in vivo as a 55,000-Da species similar in size to the primary in vitro translation product. DNA sequence analysis of the cDNA clone pACC1 revealed that the coding region of the ACC synthase mRNA spans 493 amino acids corresponding to a 55,779-Da polypeptide; and expression of the coding sequence (pACC1) in Escherichia coli as a COOH terminus hybrid of beta-galactosidase or as a nonhybrid polypeptide catalyzed the conversion of S-adenosylmethionine to ACC (Sato, T., and Theologis, A. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6621-6625). Immunoblotting experiments herein show that the molecular mass of the beta-galactosidase hybrid polypeptide is 170,000 Da, and the size of the largest nonhybrid polypeptide is 53,000 Da. The data suggest that the enzyme is post-translationally processed during protein purification.     

Effects of storage on activity and subunit structure of rabbit skeletal-muscle AMP deaminase.

On storage, AMP deaminase is converted into a form exhibiting hyperbolic kinetics even at low KCl concentration. This effect results from cleavage of the enzyme subunit (mol.wt. 79 000) to a product of similar size to the component of approx. mol.wt. 70 000 present in trace amounts in AMP deaminase just prepared from fresh muscle.     

Purification and characterization of the DNA-dependent RNA polymerase from Clostridium acetobutylicum.

The DNA-dependent RNA polymerase (EC 2.7.7.6) from Clostridium acetobutylicum DSM 1731 has been purified to homogeneity and characterized. The purified enzyme was composed of four subunits and had a molecular mass of 370,000 Da. Western immunoblot analysis with polyclonal antibodies against the sigma 70 subunit of Escherichia coli RNA polymerase identified the 46,000-Da subunit as an immunologically and probably functionally related protein. The other three subunits of 128,000, 117,000, and 42,000 Da are tentatively analogous to the beta, beta', and alpha subunits, respectively, of other eubacterial RNA polymerases. The RNA polymerase activity was completely dependent on Mg2+, nucleoside triphosphates, and a DNA template. The presence of Mg2+ or Mn2+ in buffers used for purification or storage caused irreversible inactivation of the RNA polymerase.     

Monoclonal antibodies recognizing nitrite oxidoreductase of Nitrobacter hamburgensis, N. winogradskyi, and N. vulgaris.

Three monoclonal antibodies (MAbs) against nitrite oxidoreductase (NOR) of Nitrobacter hamburgensis were produced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of the purified enzyme showed that the MAbs named Hyb 153.1 and Hyb 153.3 both recognized a protein with a molecular mass of 64,000 Da, while Hyb 153.2 recognized a protein with a molecular mass of 115,000 Da. The molecular masses of these proteins are in the same range as those of the proteins of the alpha (115,000-Da) or beta (65,000-Da) subunit of the NOR. By using the antibodies, the amount of NOR was shown to be dependent on the growth conditions. The highest level of NOR was observed in N. hamburgensis when cells were growing mixotrophically. Analysis of whole-cell extracts of N. hamburgensis, N. winogradskyi, and N. vulgaris indicated serological homology of the NORs from these species of the genus Nitrobacter. The immunological analysis enables detection of the key enzyme of the genus Nitrobacter.     

Modulation of arginine decarboxylase activity from Mycobacterium smegmatis. Evidence for pyridoxal-5'-phosphate-mediated conformational changes in the enzyme

Arginine decarboxylase (arginine carboxy-lyase, EC 4.1.1.19) from Mycobacterium smegmatis, TMC 1546 has been purified to homogeneity. The enzyme has a molecular mass of 232 kDa and a subunit mass of 58.9 kDa. The enzyme from mycobacteria is totally dependent on pyridoxal 5'-phosphate for its activity at its optimal pH and, unlike that from Escherichia coli, Mg2+ does not play an active role in the enzyme conformation. The enzyme is specific for arginine (Km = 1.6 mM). The holoenzyme is completely resolved in dialysis against hydroxylamine. Reconstitution of the apoenzyme with pyridoxal 5'-phosphate shows sigmoidal binding characteristics at pH 8.4 with a Hill coefficient of 2.77, whereas at pH 6.2 the binding is hyperbolic in nature. The kinetics of reconstitution at pH 8.4 are apparently sigmoidal, indicating the occurrence of two binding types of differing strengths. A low-affinity (Kd = 22.5 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations and a high-affinity (Kd = 3.0 microM) binding to apoenzyme at high pyridoxal 5'-phosphate concentrations. The restoration of full activity occurred in parallel with the tight binding (high affinity) of pyridoxal 5'-phosphate to the apoenzyme. Along with these characteristics, spectral analyses of holoenzyme and apoenzyme at pH 8.4 and pH 6.2 indicate a pH-dependent modulation of coenzyme function. Based on the pH-dependent changes in the polarity of the active-site environment, pyridoxal 5'-phosphate forms different Schiff-base tautomers at pH 8.4 and pH 6.2 with absorption maxima at 415 nm and 333 nm, respectively. These separate forms of Schiff-base confer different catalytic efficiencies to the enzyme.     

The (Na,K)-ATPase of Friend erythroleukemia cells is phosphorylated near the ATP hydrolysis by an endogenous membrane-bound kinase

Friend murine erythroleukemia cells (MEL cells) contain a cAMP-independent protein kinase which phosphorylates the 100,000-Da catalytic subunit of the (Na,K)-ATPase both in living cells and in the purified plasma membrane (Yeh, L.-A., Ling, L., English, L., and Cantley, L. (1983) J. Biol. Chem. 258, 6567-6574). We have taken advantage of the selective phosphorylation of the 100,000-Da subunit in purified plasma membranes and the similarity between the proteolysis patterns of the MEL cell and dog kidney (Na,K)-ATPase to map the site of kinase phosphorylation on the MEL cell enzyme. The chymotryptic and tryptic cleavage sites of the dog kidney (Na,K)-ATPase have previously been located (Castro, J., and Farley, R. A. (1979) J. Biol. Chem. 254, 2221-2228). The 100,000-Da catalytic subunits of the dog kidney and MEL cell enzymes were specifically labeled at the active site aspartate residue by incubation with (32P)orthophosphate in the presence of Mg2+ and ouabain. Digestion of these two enzymes with chymotrypsin or trypsin revealed similar active site aspartate containing proteolytic fragments indicating a similar structure for the two enzymes. Chymotryptic digestions of MEL cell (Na,K)-ATPase labeled in vitro with [gamma-32P]ATP localize the region of kinase phosphorylation to within a 35,000-Da peptide derived from the middle of the 100,000-Da subunit. Tryptic digestion of the MEL cell plasma membranes degraded the 100,000-Da subunit to an NH2-terminal 43,000-Da peptide which contained the active site aspartate but which did not contain the kinase-labeled region. These results further locate the region of kinase phosphorylation to the COOH-terminal half of the 35,000-Da chymotryptic peptide. This location places the site of phosphorylation between the active site aspartate residue which accepts the phosphate of ATP during turnover and an ATP-binding site which has previously been located by labeling with fluorescein 5'-isothiocyanate (Carilli, C. T., Farley, R. A., Perlman, D. M., and Cantley, L. C. (1982) J. Biol. Chem. 257, 5601-5606). Phosphorylation of the (Na,K)-ATPase in this region may serve to regulate the activity of this enzyme.     

Isolation and regulation of piglet cardiac AMP deaminase

The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a K_m of 2 mM for 5-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the K_m of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD⁺, whereas, the K_m was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in V_max with no change in K_m. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.     

Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli

Feedback inhibition of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli. Cloning ilvA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl 1-thio-beta-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts. High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity 3-fold greater than previous preparations. The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/56,000-Da chain. Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation. The enzymic dehydration of threonine to alpha-ketobutyrate measured by steady-state kinetics, performed at 20 degrees C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a K0.5 of 8.0 mM. The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and K0.5 of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a K0.5 of 5.7 mM. Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme.     

Molecular forms of pigeon skeletal muscle AMP deaminase

Phosphocellulose chromatography of pigeon leg muscle extract revealed the existence of two well-separated forms of AMP deaminase. This was in contrast to the pigeon breast muscle extract, which yielded only one form. The two leg muscle enzyme isoforms manifested similar kinetic and regulatory properties. They were activated by very low concentration of potassium ions and demonstrated similar patterns of pH and effector dependence. At pH 6.5, as well as at other pH values tested. ADP and ATP slightly stimulated, whereas GTP and orthophosphate inhibited the two molecular forms of pigeons leg muscle enzyme. Surprisingly, the molecular form of AMP deaminase present in pigeon breast muscle was inhibited by ATP at all pH values tested. The kinetic and regulatory properties of the three molecular forms of pigeon skeletal muscle AMP deaminase examined do not resemble those which have been described for pigeon heart muscle enzyme.     

Structure of enoate reductase from a Clostridium tyrobutyricum (C. spec. La1)

Enoate reductase from Clostridium tyrobutyricum was purified by a rapid novel procedure. Chromatography on DEAE-Sepharose and on hydroxyapatite resulted in a high yield of about 90% pure enzyme in less than 10 h. A purity greater than 98% could be obtained by additional chromatography on Sephacryl S-300. The enzyme sediments in the analytical ultracentrifuge as a single, symmetrical boundary with a velocity of S(0)20,w = 24.9 S. Equilibrium ultracentrifugation yielded a molecular mass of 940 000 +/- 20 000 Da. The enzyme contains one type of subunit as shown by dodecyl sulfate electrophoresis and partial sequence determination. A subunit molecular mass of about 73 000 Da was established by dodecyl sulfate electrophoresis and by sedimentation equilibrium analysis in guanidine hydrochloride. In addition to FAD, iron and labile sulfur, the enzyme purified by the new method showed approximately 0.7 mol of FMN per mol of subunit. A dissociation product sedimenting at a velocity of S(0)20,w = 9.8 S can be obtained by various experimental protocols. The fragment was obtained in pure form by gel permeation chromatography. The molecular mass was 230 000 +/- 10 000 Da as shown by sedimentation equilibrium analysis. Thus it appears that the dissociation product is a trimer of the 73 000-Da subunit. The formation of the 10-S fragment by dissociation of the native enzyme is accompanied by the loss of most of the FMN, whereas the FAD content is not changed. The fragment catalysed the reduction of acetylpyridine adenine dinucleotide by NADH. However, enoate reductase activity with NADH or methylviologen as cosubstrate was low. Electron micrographs of negatively stained enoate reductase show trigonal symmetry. The data suggest that enoate reductase is a dodecamer (tetramer of trimers) with tetrahedral symmetry.     

Nuclear-membrane-associated protein kinase and substrates. The effect of growth state on activity and specificity.

Reaction of rat muscle AMP deaminase with low molar excess of tetranitromethane results in a rapid loss of free thiol groups and a concomitant decrease in enzyme activity at high, but not at low, AMP concentration. This modification appears to be limited to the same non-essential thiol groups reactive towards specific reagents in non-denaturing conditions. On incubation with higher molar excess of tetranitromethane, a loss of enzyme activity is observed, which correlates with nitration of tyrosine residues. By amino acid analysis, approximately there tyrosine residues per subunit are estimated to be nitrated in the completely inactivated enzyme. The kinetic properties of the partially inactivated AMP deaminase reveal a negative co-operatively behaviour at approximately half saturation. This suggests that modification of tyrosine residues is also responsible for alteration of the binding properties of the hypothesized activating site of AMP deaminase.     

AMP deaminases of rat small intestine

Phosphocellulose column chromatography revealed the existence of two forms of AMP deaminase both in whole tissue and in the intestinal epithelium. AMP deaminase I, which eluted from the column as a first activity peak, exhibited hyperbolic, nonregulatory kinetics. The substrate half-saturation constants were determined to be 0.3 and 0.7 mM at pH 6.5 and 7.2, respectively, and did not change in the presence of ATP, GTP and Pi. AMP deaminase II, which eluted from the column as a second activity peak, was strongly activated by ATP and inhibited by GTP and Pi. The S0.5 constants were 3.5 and 7.1 at pH 6.5 and 7.2, respectively. At pH 7.2 ATP (1 mM) S0.5 decreased to 2.5 mM and caused the sigmoidicity to shift to hyperbolic. The ATP half-activation constant was increased 9-fold in the presence of GTP and was not affected by Pi. Mg2+ significantly altered the effects exerted by nucleotides. The S0.5 value was lowered 10-fold in the presence of MgATP and 5-fold in the presence of MgATP, MgGTP and Pi. When MgATP was present, AMP deaminase II from rat small intestine was less susceptible to inhibition by GTP and Pi. A comparison of the kinetic properties of the enzyme, in particular the greater than 100% increase in Vmax observed in the presence of MgCl2 at low (1 mM) substrate concentration, indicates that MgATP is the true physiological activator. GuoPP[NH]P at low concentrations, in contrast to GTP, did not affect the enzyme and even activated it at concentrations above 0.2 mM. We postulate that AMP deaminase II may have a function similar to that of the rat liver enzyme. The significance of the existence of an additional, non-regulatory form of AMP deaminase in rat small intestine is discussed.     

Negative homotropic cooperativity in rat muscle AMP deaminase. A kinetic study on the inhibition of the enzyme by ATP

1. Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) at optimal KCl concentrations shows a biphasic response to increasing levels of the allosteric inhibitor ATP. 2. Up to 10 micrometer, ATP appears to convert the enzyme to a form exhibiting sigmoidal kinetics while at higher concentrations its inhibitory effect is manifested by an alteration of AMP binding to AMP deaminase indicative of negative homotropic cooperativity at about 50% saturation. 3. AMP deaminase is inactivated by incubation with the periodate oxidation product of ATP. The (oxidized ATP)--AMP deaminase complex stabilized by NaBH4 reduction shows kinetic properties similar to those of the native enzyme in the presence of high ATP concentrations. 4. A plausible explanation of the observed cooperativity is that ATP induces different conformational state of AMP deaminase subunits, causing the substrate to follow a sequential mechanism of binding to enzyme. 5. Binding of the radioactive oxidized ATP shows that 3.2 mol of this reagent bind per mol AMP deaminase.     

The correlation between Na+ channel subunits and scorpion toxin-binding sites. A study in rat brain synaptosomes and in brain neurons developing in vitro

Photoreactive derivatives of alpha- and beta-scorpion toxins have been used to analyze the subunit composition of Na+ channels in rat brain. In synaptosomes, both types of toxins preferentially labeled (greater than 85%) a component of 34,000 Da and, at a lower level, another component of 300,000 Da. Reduction of disulfide bridges shifted this latter band from 300,000 Da to 272,000 Da but did not modify the migration of the 34,000-Da component. Similarly, two bands were labeled in cultured brain neurons, one at 259,000 Da by alpha-scorpion toxins and the other at 34,000 Da by both alpha- and beta-scorpion toxins. Contrary to what was observed in synaptosomes, in cultured brain neurons reduction of disulfide bridges had no effect on the migration of the labeled high molecular weight component. Labeling of the smaller polypeptide was obtained even when cells were solubilized with sodium dodecyl sulfate immediately after cross-linking which proves that the 34,000-Da component is not a product of proteolysis. Binding sites for alpha- and beta-scorpion toxins, respectively, did not develop in parallel during neuronal maturation in culture: the increase in beta-scorpion toxin-binding site density was lower and later than that for alpha-scorpion toxin. When related to morphological development, the increase in alpha-scorpion toxin-binding sites was correlated to neurite growth, whereas the increase in beta-scorpion toxin-binding sites was associated with the development of chemical synapses. Finally, in cultured neurons, but not in synaptosomes, both the binding of beta-scorpion toxin and the labeling of the 34,000-Da component by beta-scorpion toxin were enhanced by depolarization of the cell membrane.     

Modification by liposomes of the adenosine triphosphate-activating effect on adenylate deaminase from pig heart.

Adenylate deaminase (AMP deaminase, EC 3.5.4.6) of a high substrate specificity was purified from pig heart by chromatography on cellulose phosphate. The enzyme shows a co-operative binding of AMP [h (Hill coefficient) 2.35, with SO.5 (half-saturating substrate concentration) 5mM]. ATP and ADP act as positive effectors, lowering h to 1.55 and SO.5 to 1 mM. The addition of liposomes (phospholipid bilayers) to ATP-activated or ADP-activated enzyme causes a further shift of the h value to 1.04 and SO.5 to 0.5 mM. For ATP-activated enzyme the addition of liposomes increases Vmax. by about 100%, and for ADP-activated enzyme by 50%. Liposomes have no effect on the kinetics of AMP deaminase in the absence of ATP and ADP, and neither do they influence the inhibitory effect of orthophosphate on heart muscle AMP deaminase. Metabolic implications of these findings are discussed.     

Characterization of the 1,4-dihydropyridine receptor using subunit-specific polyclonal antibodies. Evidence for a 32,000-Da subunit

The purified receptor for the 1,4-dihydropyridine Ca2+ channel blockers from rabbit skeletal muscle contains protein components of 170,000 Da (alpha 1), 175,000 Da (alpha 2), 52,000 Da (beta), and 32,000 Da (gamma) when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Subunit-specific polyclonal antibodies have now been prepared and used to characterize the association of the 32,000-Da polypeptide (gamma subunit) with other subunits of the dihydropyridine receptor. Immunoblot analysis of fractions collected during purification of the dihydropyridine receptor shows that the 32,000-Da polypeptide copurified with alpha 1 and alpha 2 subunits at each step of the purification. In addition, monoclonal antibodies against the alpha 1 and beta subunits immunoprecipitate the digitonin-solubilized dihydropyridine receptor as a multisubunit complex which includes the 32,000-Da polypeptide. Polyclonal antibodies generated against both the nonreduced and reduced forms of the alpha 2 subunit and the gamma subunit have been used to show that the 32,000-Da polypeptide is not a proteolytic fragment of a larger component of the dihydropyridine receptor and not disulfide linked to the alpha 2 subunit. In addition, polyclonal antibodies against the rabbit skeletal muscle 32,000-Da polypeptide specifically react with similar proteins in skeletal muscle of other species including avian and amphibian species. Thus, our results demonstrate that the 32,000-Da polypeptide (gamma subunit) is an integral and distinct component of the dihydropyridine receptor.