Effects of storage on activity and subunit structure of rabbit skeletal-muscle AMP deaminase.
Ontology highlight
ABSTRACT:
SUBMITTER: Ranieri-Raggi M
PROVIDER: S-EPMC1162007 | biostudies-other | 1980 Aug
REPOSITORIES: biostudies-other
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Project description:Mutation of the AMP deaminase 1 (AMPD1) gene, the predominate AMPD gene expressed in skeletal muscle, is one of the most common inherited defects in the Caucasian population; 2-3% of individuals in this ethnic group are homozygous for defects in the AMPD1 gene. Several studies of human subjects have reported variable results with some studies suggesting this gene defect may cause symptoms of a metabolic myopathy and/or easy fatigability while others indicate individuals with this inherited defect are completely asymptomatic. Because of confounding problems in assessing muscle symptoms and performance in human subjects with different genetic backgrounds and different environmental experiences such as prior exercise conditioning and diet, a strain of inbred mice with selective disruption of the AMPD1 was developed to study the consequences of muscle AMPD deficiency in isolation. Studies reported here demonstrate that these animals are a good metabolic phenocopy of human AMPD1 deficiency but they exhibit no abnormalities in muscle performance in three different exercise protocols.
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Project description:We examined the kinetic and regulatory properties of the two isoenzymes of red muscle AMP deaminase, forms A and B, corresponding respectively to the single isoenzymes present in the heart and white skeletal muscle. At the optimal pH value, 6.5, both enzymes show hyperbolic substrate-velocity curves and are inhibited by GTP, inducing sigmoid kinetics. An effect similar to that of GTP is exerted on form B by ATP, whereas form A is almost insensitive to this nucleotide. At pH 7.1 both enzymes follow sigmoid kinetics. ATP enhances the sigmoidicity of the substrate-velocity curve of form B, but it stimulates form A, reverting sigmoidal to hyperbolic kinetics shown by the enzyme at optimal pH. At pH 7.1, form A is also less sensitive to the inhibitory action of Pi and GTP. These results suggest that, owing to the presence of form A, AMP deamination occurs in red muscle also at moderate work intensity. A possible role of this process in counteracting the production of adenosine by 5'-nucleotidase is hypothesized.
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Project description:Reaction of rabbit skeletal-muscle AMP deaminase with a low molar excess of diethyl pyrocarbonate results in conversion of the enzyme into a species with one or two carbethoxylated histidine residues per subunit that retains sensitivity to ATP at pH 7.1 but, unlike the native enzyme, it is not sensitive to regulation by ATP at pH 6.5. This effect mimics that exerted on the enzyme by limited proteolysis with trypsin, which removes the 95-residue N-terminal region from the 80 kDa enzyme subunit. These observations suggest involvement of some histidine residues localized in the region HHEMQAHILH (residues 51-60) in the regulatory mechanism which stabilizes the binding of ATP to its inhibitory site at acidic pH. Carbethoxylation of two histidine residues per subunit abolishes the inhibition by ATP of the proteolysed enzyme at pH 7.1, suggesting the obligatory participation of a second class of histidine residues, localized in the 70 kDa subunit core, in the mechanism of the pH-dependent inhibition of the enzyme by ATP. At a slightly acidic pH, these histidine residues would be positively charged, resulting in a desensitized form of the enzyme similar to that obtained with the carbethoxylation reaction.
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Project description:AMP deaminase (AMPD) is characterized by a multigene family in rodents and man. Highly conserved rat and human AMPD1 and AMPD2 genes produce protein products that exhibit cross-species immunoreactivities (AMPD1, rat isoform A and human isoform M; AMPD2, rat isoform B and human isoform L). A third gene, AMPD3, has been described in humans, but antisera raised against its purified protein product (isoform E) reportedly does not cross-react with a third activity purified from rat tissues (isoform C). This study was designed to address this latter issue by cloning, sequencing and expressing rat AMPD3 cDNA species. Similarly to the human AMPD3 gene, the rat AMPD3 gene produces multiple transcripts that differ at or near their 5' ends. The boundary at which these alternative sequences diverge is precisely conserved in both species. Across the region that is common to all rat and human AMPD3 cDNA species, nucleotide and predicted amino acid sequences are 89% and 93% identical respectively, although the rat open reading frame is lacking two separate in-frame codons in the 5' end. Extreme 5' regions between the two species are entirely divergent, and one alternative rat sequence is predicted to confer at least 36 additional N-terminal residues to its encoded AMPD3 polypeptide. A comparison of 3' untranslated regions indicates that the rat sequence is 250 bp longer and contains multiple consensus polyadenylation signals. Examination of relative rat AMPD3 gene expression shows (1) variable patterns of alternative mRNA abundance across adult tissues, (2) developmental regulation in skeletal muscle and liver, and (3) greater mRNA abundance in adult red (soleus) than in mixed (plantaris) and white (outer gastrocnemius) skeletal muscle. Finally, baculoviral expression of rat and human AMPD3 proteins produces enzymes that are chromatographically and kinetically similar. Moreover, both recombinant activities immunoreact with anti-C and anti-E serum. These combined results demonstrate that rat isoform C and human isoform E are homologous cross-species AMPD3 proteins.
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Project description:Denaturation of rabbit skeletal-muscle AMP deaminase in acidic medium followed by chromatography on DEAE-cellulose in 8 M urea atpH 8.0 allows separation of two main peptide components of similar apparent molecular mass (75-80 kDa) that we tentatively assume correspond to two different enzyme subunits. Whereas the amino acid composition of one of the two peptides is in good agreement with that derived from the nucleotide sequence of the known rat and human AMPD1 cDNAs, the second component shows much higher contents of proline, glycine and histidine. N-Terminal sequence analysis of the fragments liberated by limited proteolysis with trypsin of the novel peptide reveals a striking similarity to the fragments produced by plasmin cleavage of the rabbit plasma protein called histidine-proline-rich glycoprotein (HPRG). However, some divergence is observed between the sequence of one of the fragments liberated from AMP deaminase by a more extensive trypsinization and rabbit plasma HPRG in the region containing residues 472-477. A fragment with a blocked N-terminus, which was found among those liberated by proteolysis with pepsin of either whole AMP deaminase or the novel component of the enzyme, shows an amino acid composition quite different from that of the N-terminus of the known subunit of AMP deaminase. By coupling this observation with the detection in freshly prepared AMP deaminase of a low yield of the sequence (LTPTDX) corresponding to that of HPRG N-terminus, it can be deduced that in comparison with HPRG, the putative HPRG-like component of AMP deaminase contains an additional fragment with a blocked N-terminus, which is liberated by a proteolytic process during purification of the enzyme. The implications of the association to rabbit skeletal-muscle AMP deaminase of a HPRG-like protein species are discussed.
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Project description:AMP deaminase isoforms from human skeletal muscle can be separated chromatographically [Kaletha, Spychała & Nowak (1987) Experientia 43, 440-443]. In adult tissue nearly all the AMP deaminase activity was eluted from phosphocellulose with 0.75 M-KCl ('adult' isoform), and the remaining activity could be eluted with 2.0 M-KCl. Conversely, most of the AMP deaminase activity from 11-week-old fetal tissue was eluted from phosphocellulose with 2.0 M-KCl ('fetal' isoform). In the present paper the kinetic and regulatory properties of AMP deaminase extracted from 11- and 16-week-old fetal skeletal muscle are reported. The two isoforms from 11-week-old human fetus differed distinctly in these properties. The 'fetal' isoform had about 5-fold higher half-saturation constant (S0.5) value than the 'adult' form. It was also more sensitive to the influence of some important regulatory ligands (ADP, ATP and Pi), and exhibited a different pH/activity profile. The 'adult' isoform of AMP deaminase from fetal muscle and the enzyme from mature muscle possessed similar kinetic and regulatory properties. This isoform seems not to be subject to any major modifications during further ontogenesis. This is not true, however, for the 'fetal' isoform. In the muscle of 16-week-old human fetus, the 'fetal' isoform showed a peculiar, biphasic, type of substrate-saturation kinetics. This phenomenon may reflect appearance of the next, developmentally programmed, isoform of human skeletal-muscle AMP deaminase.
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