Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Limited proteolysis of rabbit skeletal-muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) with trypsin results in conversion of the enzyme into a species which over the pH range 6.5-7.1 exhibits hyperbolic kinetics at low K+ concentration even in the absence of ADP, but shows a 20% decrease in activity at saturating substrate concentration. Analysis by sedimentation-equilibrium techniques reveals the proteolysed enzyme to be homogeneous and to have a molecular mass of 222,000 Da, indicative of a trimeric structure with a subunit molecular mass of 72,000 Da, in contrast with the tetrameric structure of the native enzyme, composed of four 79,000-Da subunits. These observations suggest a role of the 7,000-Da fragment which is removed by proteolysis in the maintenance of the three-dimensional structure of the subunit that causes the enzyme at low K+ concentration to show homotropic positive co-operativity. Study of the influence of pH, isolated from that of K+, on the kinetics of AMP deaminase reveals a highly pH-dependent inhibitory effect by ATP which is completely absent at acid pH values and abruptly manifests itself just above neutrality. This phenomenon may have significance in the metabolism of exercising muscle, in connection with the pH-dependent interaction of AMP deaminase with the thick filament.
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PMID:Regulation of skeletal-muscle AMP deaminase. Evidence for a highly pH-dependent inhibition by ATP of the homogeneous derivative of the rabbit enzyme yielded by limited proteolysis. 226

Limited proteolysis of rabbit skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) with trypsin results in conversion of the enzyme to a form which is no longer inhibited by ATP and exhibits hyperbolic kinetics even at low K+ concentration and in the absence of ADP. The interaction with troponin T from white skeletal muscle or with the phosphorylated 42-residue N-terminal peptide of troponin T restores in the trypsin-treated AMP deaminase the sensitivity to adenine nucleotides and increases the KA for K+ activation of the enzyme from 1 mM to 12 mM, this effect being diametrically opposite to that exerted by limited proteolysis on the native enzyme. Treatment of the N-terminal peptide of troponin T with alkaline phosphatase abolishes the modulating properties of the peptide, suggesting that phosphorylation-dephosphorylation processes may be involved in the regulation of the enzyme.
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PMID:Interaction with troponin T from white skeletal muscle restores in white skeletal muscle AMP deaminase those allosteric properties removed by limited proteolysis. 396 31

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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PMID:Regulation of skeletal-muscle AMP deaminase: involvement of histidine residues in the pH-dependent inhibition of the rabbit enzyme by ATP. 763 1

Rabbit skeletal muscle AMP deaminase was submitted to limited proteolysis by trypsin that converts the native 80 kDa enzyme subunit to a stable product of approx. 70 kDa, which, in contrast to the native enzyme, is not sensitive to regulation by ATP at pH 6.5. Tryptic peptide mapping indicates that proteolysis is confined to the N-terminal region of the molecule, identifying in this region of AMP deaminase a non-catalytic, 95 residue regulatory domain that stabilises the binding of ATP to a distant site in the molecule. Protein sequence analysis reveals a marked degree of divergence between rat and rabbit skeletal muscle AMP deaminases in the regions containing residues 7-12 and 51-52, giving molecular basis to the hypothesis of the existence of isoenzymes of AMP deaminase in the mature skeletal muscle of the mammals.
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PMID:Evidence of a species-differentiated regulatory domain within the N-terminal region of skeletal muscle AMP deaminase. 794 74

Adenylate deaminase (EC 3.5.4.6) may help to regulate the adenine nucleotide catabolism characteristic of such disease states as myocardial ischaemia. We report analysis of the molecular, kinetic and allosteric properties of rabbit heart adenylate deaminase when extracted and purified under phosphate-free conditions (i.e., with Hepes/KOH). The enzyme's subunit molecular mass (approximately 81 kDa), pI (6.5), substrate specificity for 5'-AMP, and activation by K+ were identical in the absence or presence of phosphate. At each chromatographic step during isolation without phosphate, cardiac adenylate deaminase showed a lower apparent activity as compared with the enzyme prepared with phosphate present. Kinetic constants for the phosphate-free rabbit heart adenylate deaminase preparation (Km 0.54 mM AMP; Vmax. 1.4 mumol/min per mg of protein) were approximately 10-fold lower than those of the enzyme isolated with phosphate. The same irreversible decrease in kinetic constants could be achieved by dialysing phosphate from the phosphate-containing enzyme preparation. The relationship between enzyme activity and substrate concentration was sigmoidal in the presence of phosphate, but hyperbolic in its absence. Cardiac adenylate deaminase under phosphate-free conditions was no longer allosterically activated by ATP and ADP, yet remained inhibitable by GTP. Enzyme inhibition by the transition-state mimic coformycin was not influenced by phosphate status. The phosphate-free preparation of rabbit heart adenylate deaminase was markedly labile and extremely susceptible to proteolysis by trypsin or chymotrypsin. The inactivation kinetics and fragmentation pattern in response to controlled proteolysis depended on whether the enzyme had been isolated with or without phosphate present, suggesting a conformational difference between the two enzyme preparations. These data constitute direct evidence that the absence of phosphate irreversibly converts cardiac adenylate deaminase into a pseudo-isoenzyme with distinct kinetic, regulatory and stability properties.
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PMID:Cardiac adenylate deaminase: molecular, kinetic and regulatory properties under phosphate-free conditions. 800 40

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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PMID:Association of purified skeletal-muscle AMP deaminase with a histidine-proline-rich-glycoprotein-like molecule. 930 11

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.
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PMID: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. 1548 71