Phyrochemistry, Vol. 31, No. 10, pp. 3455-3459,1992

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Printed in Great Britain .

0031-9422/92 $5 .00 + 0.00 1992 Pergamon Press Ltd

RICE SEED GLOBULIN : A PROTEIN SIMILAR TO WHEAT SEED GLUTENIN and H .

S . KOMATSU

Department of Molecular Biology,

HIRANO

Institute of Agrobiological Resources, Kannondai 2-1-2, Tsukuba, Ibaraki 305, Japan (Received in revised form I1 March

Key Word Index-Oryza

sativa;

1992)

Gramineae; rice; seed endosperm protein ; globulin ; amino acid sequences .

Abstract-The globulin of the seed endosperms of rice has an isoelectric point of 6 .4 and a M, of 26000. There is one intra-disulphide bond, but no N-linked oligosaccharide chain . The amino acid sequence of the N-terminal and internal regions of the globulin was determined and found to be homologous with that of glutenin, the storage protein in the seed endosperms of wheat . Rice globulin and wheat glutenin were rich in glycine, and glutamic acid or glutamine, and in addition, wheat glutenin cross-reacted with antibody raised against rice globulin, These results suggest that rice seed globulin represents a protein similar to wheat seed glutenin.

INTRODUCTION

The major storage protein in endosperms of rice (Oryza sativa) is an acid and/or alkaline-soluble protein, glutelin, which accounts for 80% of the total protein [1] . Saltsoluble protein, globulins and alcohol-soluble protein, prolamins, are also present in relatively low amounts in rice endosperms and account for 2-8% and 1-5% of the total protein in the endosperms, respectively [2]. Rice storage proteins are accumulated in two different kinds of protein bodies during seed development with both globulins and glutelins being stored in protein body type II and prolamins in protein body type I [3]. The amino acid sequences of the glutelin and prolamin have been determined by protein sequencing and have also been deduced from nucleotide sequence data of the DNA encoding these proteins [4, 5] . Although rice prolamin has been found to be structurally homologous with the prolamin from seed endosperms of both wheat and barley, rice glutelin is highly homologous in amino acid sequence with IIS globulin, named legumin, which is a major seed storage protein in legumes [4, 6] . Thus, a question arises as to which category the rice globulin is structurally classified. In the present study, we have analysed the amino acid sequence, amino acid composition and immunological properties of rice globulin, and found that the rice globulin is structurally similar to the glutenin wheat seed storage protein .

RESULTS AND DISCUSSION

Proteins extracted from the endosperms of mature rice seeds were separated by 2D-PAGE (Fig . 1). The globulin which had a M, of 26000 [7] was found to have an isoelectric point of 6 .4 (Fig. 1, Spot 8) . Based on the intensity of Coomassie blue staining, this protein consisted of 6 .3% of the total seed endosperm protein in var . Norin 29 . The proteins of spots 1-7 shown in Fig. 1 were glutelin a subunits [8] .

After separation by SDS-PAGE, the globulin was electroblotted and directly sequenced by Edman degradation in a gas-phase sequencer, but was found to have a blocking group at the N-terminus . Consequently, the electroblotted globulin was digested on the polyvinylidene difluoride (PVDF) membrane with pyroglutamyl peptidase and subjected to N-terminal amino acid sequence analysis, to enable the sequence determination of the globulin by Edman degradation . The N-terminal pyroglutamic acid of globulin was thought to be formed by cyclization of either glutamine or glutamic acid . The sequences of 10 residues from the N-terminus of globulin were determined (Fig . 2). The endosperm proteins were separated by 2D-PAGE, and the globulin was electroeluted from the gel . After the electroelution, the globulin was digested with Staphylococcus aureus V8 protease on the SDS-PAGE gel and electroblotted from the gel on to the PVDF membrane to determine the internal amino acid sequences (Fig . 2). Four major fragments of globulin were obtained ; one was from the N-terminal region (Fig . 2, No. 1), the others (Fig. 2, Nos 1-3) from the internal regions of the globulin, In total, the sequences of 39 amino acid residues were determined and a structural homology search indicated that the amino acid sequence of globulin was homologous with that of the high-molecular-weight (HMW) subunit of the glutenin storage protein in wheat seeds, which was deduced by the cDNA sequences [9]. The wheat seed glutenin HMW subunit is one of the important determinants of the bread-making quality of wheat flour [11]. The amino acid composition of rice globulin is shown in Table 1 . The globulin was rich in glycine (ca 76 residues), and glutamic acid or glutamine (ca 32 residues), accounting for 20.6 and 1.6 .9%, respectively . The glutenin HMW subunit is also rich in glycine (20%) and glutamine (36%) [9] and both proteins can thus be considered to be glycine-rich, and glutamic acid- or glutamine-rich . In addition, the wheat endosperm proteins were separated by SDS-PAGE and electroblotted on to a PVDF membrane, the wheat glutenin HMW subunit was shown to

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S. KOMATSU

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AGE attern of total proteins of rice (var . Norin 29) seed endosperms . Right to left, electric fo the first dimension; top to bottom, SDS-PAGE for the second dimension . Detected by Coomassie blue staining . (1-7) glutelin a subunits; (8) globulin.

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Rice seed globulin: a protein similar to wheat seed glutenin.

The globulin of the seed endosperms of rice has an isoelectric point of 6.4 and a M(r) of 26,000. There is one intra-disulphide bond, but no N-linked ...
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