EFFECTS OF N-TERMINAL TRUNCATIONS UPON CHLOROPLAST NADP-MALATE DEHYDROGENASES FROM PEA AND SPINACH

被引:24
作者
OCHERETINA, O
HARNECKER, J
ROTHER, T
SCHMID, R
SCHEIBE, R
机构
[1] UNIV OSNABRUCK, DEPT PLANT PHYSIOL, BARBARASTR 11, W-4500 OSNABRUCK, GERMANY
[2] UNIV OSNABRUCK, DEPT MICROBIOL, W-4500 OSNABRUCK, GERMANY
[3] UNIV BAYREUTH, DEPT PLANT PHYSIOL, W-8580 BAYREUTH, GERMANY
关键词
CHLOROPLAST NADP-MALATE DEHYDROGENASE; REDOX MODIFICATION; REGULATORY SEQUENCE; LIMITED PROTEOLYSIS; AMINO-TERMINAL TRUNCATION;
D O I
10.1016/0167-4838(93)90272-S
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Using the purification procedure of Fickenscher and Scheibe (Biochim. Biophys. Acta 749 (1983), 249-254) and a modification of the method, we produced a series of NADP-MDH forms from spinach and pea-leaf extracts that were characterized by a stepwise shortening of the N-terminal sequences. Limited proteolysis of the enzymes resulted in the generation of even shorter forms. Immunoprecipitation of the NADP-MDH from crude extracts revealed that the sequences of the intact enzymes from pea, spinach and maize started at a position (Ser) identical with that established for the Sorghum enzyme (Cretin, C., et al. (1990) Eur. J. Biochem. 192, 299-303). Spinach NADP-MDH isolated by conventional methods was shown to represent the intact form. Thus, the kinetic, regulatory and structural properties of the various truncated forms could be compared with those of an intact form. Removal of 5 or 11 amino acids, as occurred during isolation of the pea NADP-MDH, was without any significant effect. The enzymes were all dimeric and still exhibited the characteristic redox-regulatory properties. However, removal of 31 and 37 amino acids using aminopeptidase K resulted in the formation of active monomers characterized by only slightly lowered affinities towards the substrates, a shift of their pH optimum from 8 to 7, the loss of oxaloacetate inhibition and an increased maximal velocity. Although these forms lacked most or all of the N-terminal extra-peptide, including the 2 cysteines involved in redox-modification, they were still sensitive to the redox-potential. However, the low concentration of thiol required for immediate and complete restauration of any lost activity (40 mM beta-mercaptoethanol) suggested that this reaction might not be relevant for redox-regulation in vivo.
引用
收藏
页码:10 / 16
页数:7
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