Purification and properties of iminopeptidase from peanut seeds

Ovando, Mariana Eugenia Oviedo; Isola, María Clara; Maldonado, Antonio; Franzoni, Luis

doi:10.1016/j.plantsci.2003.12.009

Cited by 11 publications

(7 citation statements)

References 18 publications

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“…This data ensure, that stud ied en zyme is a proline imino peptidases show ing high pref er ences to wards sub strates with proline at N-ter mi nus. It is known that iminopeptidases pre fer sub strates with proline at the N-ter mi nus, but in the case of en zyme from rye shoots the re place ment of Pro-β-NA with Leu-β-NA or Ala-β-NA al lows for achiev ing only 5-6 % ac tiv ity, whereas from pea nut seeds -12 and 37 %, re spec tively (Oviedo Ovando et al 2004). The above ob ser va tions may in di cate the im por tant met a bolic func tions of this en zyme, as spe cific release of proline serves the plant growth and de velop ment pro cesses.…”

Section: Re Agent Con Cen Tra Tion (Mm)mentioning

confidence: 99%

“…In ad di tion, chro matog ra phy on Sephadex G-200 was a nec es sary step, as it was the only way to sep a rate var i ous aminopep ti das es with broad sub strate spec i fic ity from proline iminopeptidases. As a re sult of us ing partially orig i nal pu ri fi ca tion pro ce dure one of the high est de scribed fac tor of pu ri fi ca tion of proline iminopeptidase in higher plants was ob tained (Ninomiya et al 1982, Wa ters and Dalling 1983, Oviedo Ovando et al 2004. In fact, the real pu ri fica tion of the en zyme seems to be higher than 405-fold be cause of pres ence of other amino pep tidas es in cru de ex tract which use Pro-βNA as a substrate.…”

Section: Pu Ri Fi Ca Tionmentioning

confidence: 99%

“…For the phosphate buffer op ti mum pH was lower (7.5) and the level of en zyme ac tiv ity within tested pH range slightly dif fer en ti ated. Sim i lar re sults were ob tained for apri cot (Ninomiya et al 1982) and pea nut seeds iminopeptidase (Oviedo Ovando et al 2004), whereas op ti mum pH both for the crude and partially pu ri fied en zyme prep a ra tion from wheat leaves was 7.4 (Wa ters and Dalling 1983). Op timum tem per a ture of stud ied en zyme was 37 °C, and its in crease by 5 °C over the op ti mum caused al most 30 % drop of ac tiv ity (Fig.…”

Section: Char Ac Ter Is Tics Of the En Zymementioning

confidence: 99%

“…1996). How ever, up to the pres ent only three iminopeptidases from higher plants have been pu rified: from wheat 127-fold (Wa ters and Dalling 1983), apri cot 1 700-fold (Ninomiya et al 1982) and pea nut 300-fold (Oviedo Ovando et al 2004). Much more ef fort was made to pu rify iminopeptidases in procaryota (Atlan et al 1994, Kitazono et al 1996, Medrano et al1998, Inoue et al 2003, but the ob tained re sults show only sen si ble dif ferences be tween plant and bac te ria en zy mes.…”

Section: Introductionmentioning

confidence: 99%

“…Plant iminopeptidases have cysteine -SH groups in the ac tive site and are char ac ter ized by high sub strate spec i fic ity to wards proline at N-ter mi nus. An im por tant role in en zyme ac tiv ity is also played by group -OH of serine pres ent in enzy matic pro tein (Oviedo Ovando et al 2004). The met a bolic func tions of proline amino pep ti das es have not been in ves ti gated yet.…”

Section: Introductionmentioning

confidence: 99%

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The molecular and biochemical characteristics of proline iminopeptidase from rye seedlings (Secale cereale L.)

Szawłowska¹,

Prus²,

Bielawski³

2006

Acta Physiol Plant

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De part ment of Bio chem is try, War saw Ag ri cul tural Uni ver sity, A proline iminopeptidase (EC. 3.4.11.5) was iso lated from shoots of 3 day old seed lings. The pu ri fi ca tion pro ce dure consisted of 5 steps: acid pre cip i ta tion, gel fil tra tion on Sephadex G-200, ion-ex change chro ma tog ra phy on Sepharose CL 6B, twice re peated hy dro pho bic chro ma tog ra phy on PhenylSepharose HP. The en zyme was pu ri fied 404.8-fold, with the spe cific ac tiv ity of 8.5 units·mg -1 of pro tein with re cov ery yield of 3 %. The pu ri fied en zyme had a mo lec u lar mass of 225 kDa es ti mated by gel fil tra tion and 55.4 kDa by SDS PAGE. This in di cates that na tive en zyme is com posed of four subunits. The en zyme was spe cific for proline β-naphtylamide among var i ous amino acid β-naphtylamides.An op ti mal ac tiv ity was ob served at 37 °C at pH 7.75. The enzyme was thermostable up to 37 °C for 30 min. The en zyme was strongly in hib ited by pHMB, E-64, heavy metal ions and par tially by PMSF, DFP. The re sults sug gest that cysteine and serine res i dues may par tic i pate in the en zyme activity.List of ab bre vi a tions: 2-ME, 2-mercaptoethanol PAGE, polyacrylamide gel elec tro pho re sis; SDS-PAGE, so dium dodecyl sul fate-polyacrylamide gel elec tro pho re sis; EDTA, ethylenediaminetetraacetic acid; E-64, transepoxysuccinyl-L-leucyloamido-(4-gua nidino)bu tane; pHMB, p-hydroxymercuribenzoic acid; βNA, β-naphthylamide; PMSF, phenyl methylsulfonyl flu o ride; DFP, diisopropyl fluorophosphate In tro duc tionPlant amino pep ti das es cat a lyze the hy dro ly sis of pep tide bonds on N-ter mi nus of pep tide sub strates (Varshavsky and Byrd 1997, Schaller 2004). Great di ver sity of these en zymes in terms of ki netic and mo lec u lar prop er ties, sub-cel lu lar lo cal iza tion and met a bolic func tions was a base for in tro duc ing vari ous clas si fi ca tion sys tems. One of them dis tinguishes neu tral, acidic and ba sic amino pep ti das es (Tay lor 1993). Neu tral amino pep ti das es is a common, abun dant en zyme group in all liv ing or ganisms with op ti mum pH 6.5 -7.5, In terms of substrate pref er ences neu tral amino pep ti das es have been di vided into three sub groups. The first consists of en zymes show ing the high est af fin ity to sub strates with leucine, ty ro sine, tryptophane and phenylalanine on N-ter mi nus. They are mono mers with mo lec u lar mass in range of 56 -76 kDa and belong to cysteine en zymes (Couton et al. 1991, Yama oka et al. 1994. The sec ond sub group consists of amino pep ti das es pre fer ring sub strates with alanine or leucine on N-ter mi nus. They are metalloenzymes with mo lec u lar mass vary ing from 517

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Section: Re Agent Con Cen Tra Tion (Mm)mentioning

confidence: 99%

Section: Pu Ri Fi Ca Tionmentioning

confidence: 99%