Affinity chromatographic purification of horse muscle acylphosphatase: Evidence of the existence of multiple molecular forms

Manao, Giampaolo; Camici, Guido; Stefani, Massimo; Berti, Andrea; Cappugi, Gianni; Liguri, Gianfranco; Nassi, Paolo; Ramponi, Giampietro

doi:10.1016/0003-9861(83)90310-7

Cited by 26 publications

(10 citation statements)

References 17 publications

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“…MT-ACP, purified by immunoaffinity chromatography, contained three ACP molecular forms that were separated by cation exchange chromatography (28): the first (68%) was the mixed disulfide with glutathione, the second (3%) remained uncharacterized, and the third (29% of total enzymatic protein) was an S-S dimeric form of the enzyme. On the other hand, we demonstrated that the main natural form of the enzyme contained in the freshly excised muscle is the reduced one (29).…”

Section: Discussionmentioning

confidence: 99%

Hydrogen Peroxide Triggers the Formation of a Disulfide Dimer of Muscle Acylphosphatase and Modifies Some Functional Properties of the Enzyme

Paoli

Giannoni

Pescitelli

et al. 2001

Journal of Biological Chemistry

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Acylphosphatase is expressed in vertebrates as two molecular forms, the organ common and the muscle types. The former does not contain cysteine residues, whereas the latter contains a single conserved cysteine (Cys-21). We demonstrated that H 2 O 2 at micromolar levels induces, in vitro, the formation of a disulfide dimer of muscle acylphosphatase, which displays properties differing from those of the reduced enzyme. In particular, we observed changes in the kinetic behavior of its intrinsic ATPase activity, whereas the kinetic behavior of its benzoyl phosphatase activity does not change. Moreover, the disulfide dimer is capable of interacting with some polynucleotides such as poly(G), poly(C), and poly(T) but not with poly(A), whereas the reduced enzyme does not bind polynucleotides. Experiments performed with H 2 O 2 in the presence of increasing SDS concentrations demonstrated that disulfide dimer formation is prevented by SDS concentrations higher than 300 M, suggesting that a non-covalently-linked dimer is present in non-denaturing solvents. Light-induced cross-linking experiments performed on the Cys-21 3 Ser mutant in the pH range 3.8 -9.0 have demonstrated that a non-covalently-linked dimer is in fact present in non-denaturing solutions and that an enzyme group with a pK a of 6.4 influences the monomer-dimer equilibrium. Acylphosphatase (ACP)1 is an enzyme widespread in all organisms; two isoenzymes, the muscle type (MT) and the organcommon type (CT), codified by two distinct genes, are expressed in vertebrates. The MT isoform is prevalently expressed in skeletal muscle and heart, whereas the CT isoform is prevalently expressed in erythrocytes, in brain, and in testis (1, 2). Several reports on the possible physiological roles of the enzyme have been produced: Some indicate ACP as an enzyme involved in the control of membrane ion pumps (Refs. 3, 4, and citations therein), because it displays hydrolytic activity against the aspartyl-phosphate intermediates formed during the action of Na ϩ ,K ϩ -and Ca 2ϩ -ATPases. Other reports involve ACP in cell differentiation and apoptosis (5-7); this is because the enzyme is overexpressed when cells are induced to differentiate by certain agents, and it is able to migrate into the nucleus during both differentiation and apoptosis (8, 9). The primary structure of the two isoenzymes differs in about 50% of amino acid positions, but they display very similar folds: Both consist of a five-stranded antiparallel twisted ␤-sheet flanked on one side by two antiparallel ␣-helices running parallel to the ␤-sheet (10). This peculiar ACP fold is very similar to that found in small RNA-binding domains of several RNA-binding proteins (11, 12); in fact, many RNA-binding proteins have modular structures consisting of RNA-binding modules and auxiliary domains that perform additional functions (13). This prompted us to investigate the involvement of ACP in polynucleotide processing. Chiarugi et al. (14) found that, in vitro, ACP catalyzes the hydrolysis of both DNA and RNA. This pape...

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Section: Discussionmentioning

confidence: 99%

Hydrogen Peroxide Triggers the Formation of a Disulfide Dimer of Muscle Acylphosphatase and Modifies Some Functional Properties of the Enzyme

Paoli

Giannoni

Pescitelli

et al. 2001

Journal of Biological Chemistry

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“…Recombinant wild-type and mutated acylphosphatases were purified by immunoaffinity chromatography using a column filled with an immunoadsorbent prepared by linking purified specific anti-horse muscle acylphosphatase antibodies, raised in rabbits, to a Sepharose 4B matrix, as previously reported [27]. The enzyme purity was assessed by amino acid analysis, reverse-phase chromatography, and SDS-PAGE.…”

Section: Enzyme Purification and Protein Determinationmentioning

confidence: 99%

Properties of N‐terminus truncated and C‐terminus mutated muscle acylphosphatases

et al. 1995

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Enzymatic activity and structure of N-terminus truncated and C-terminus substituted muscle acylphosphatase mutants were investigated by kinetic studies under different conditions and tH NMR spectroscopy, respectively. The N-terminus truncated mutant lacked the first six residues (A6), whereas arginine 97 and tyrosine 98 were replaced by glutamine giving two C-terminus substituted mutants (R97Q and Y98Q, respectively). All acylphosphatase forms were obtained by modifications of a synthetic gene coding for the human muscle enzyme which was expressed in E. coli. The A6 deletion mutant elicited a reduced specific activity and a native-like structure. The kinetic and structural properties of R97Q and Y98Q mutants indicate a possible role of Arg-97 in the stabilisation of the active site correct conformation, most likely via back-bone and side chain interactions with Arg-23, the residue involved in phosphate binding by the enzyme. This study also suggests a possible involvement of Tyr-98 in the stabilisation of the acylphosphatase overall structure.

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“…Amino acid analyses were performed using a Carlo Erba mod. 3A29 apparatus equipped with a computing integrator according to Manao et al [26]. Values for serine and threonine were corrected for degradation during sample hydrolysis.…”

Section: Methodsmentioning

confidence: 99%

Expression, purification and preliminary crystal analysis of the human low M_r phosphotyrosine protein phosphatase isoform 1

et al. 1998

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The genes of the human low M r phosphotyrosine protein phosphatase (PTPase) isoforms 1 (IF1) and 2 (IF2) were isolated by screening a human placenta cDNA library, cloned in pGEX and expressed in E. coli as fusion proteins with glutathione S-transferase. The recombinant proteins were purified by a rapid one-step procedure allowing each enzyme to purify with high final yield and specific activity. This result is important for IF1, whose purification from natural sources is difficult, due to precipitation propensity, thus hindering structural studies. The enzymes obtained showed kinetic parameters very similar to those previously determined for the enzymes purified by classical procedures from both human erythrocytes and rat liver. These recombinant enzymes can therefore be used in place of those purified from natural sources for every purpose. IF1 and IF2 crystals were also grown. IF1 crystals were X-ray-grade, diffracted to better than 2.4 A î and were suitable for high resolution X-ray structure determination.z 1998 Federation of European Biochemical Societies.

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Affinity chromatographic purification of horse muscle acylphosphatase: Evidence of the existence of multiple molecular forms

Cited by 26 publications

References 17 publications

Hydrogen Peroxide Triggers the Formation of a Disulfide Dimer of Muscle Acylphosphatase and Modifies Some Functional Properties of the Enzyme

Hydrogen Peroxide Triggers the Formation of a Disulfide Dimer of Muscle Acylphosphatase and Modifies Some Functional Properties of the Enzyme

Properties of N‐terminus truncated and C‐terminus mutated muscle acylphosphatases

Expression, purification and preliminary crystal analysis of the human low M_r phosphotyrosine protein phosphatase isoform 1

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Affinity chromatographic purification of horse muscle acylphosphatase: Evidence of the existence of multiple molecular forms

Cited by 26 publications

References 17 publications

Hydrogen Peroxide Triggers the Formation of a Disulfide Dimer of Muscle Acylphosphatase and Modifies Some Functional Properties of the Enzyme

Hydrogen Peroxide Triggers the Formation of a Disulfide Dimer of Muscle Acylphosphatase and Modifies Some Functional Properties of the Enzyme

Properties of N‐terminus truncated and C‐terminus mutated muscle acylphosphatases

Expression, purification and preliminary crystal analysis of the human low Mr phosphotyrosine protein phosphatase isoform 1

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Expression, purification and preliminary crystal analysis of the human low M_r phosphotyrosine protein phosphatase isoform 1