Identification of Cultivar Differences in Seed Polypeptide Composition of Peanuts (Arachis hypogaea L.) by Two-Dimensional Polyacrylamide Gel Electrophoresis

Abstract

Seed polypeptides from several cultivars of peanut (Arachis hypogaea L.) have been compared by means of a two-dimensional polyacrylamide gel electrophoresis. Protein was extracted from the defatted peanut meal by homogenizing in 5 millimolar K2CO3-9.5 molar urea. After addition of Nonidet P40 (2%, v/v) The peanut (Arachis hypogaea L.) is an economically important source of vegetable protein, but is relatively low in its content of methionine, lysine and tryptophan (1, 11). There are a few reports suggesting that certain genotypes exist within the species that contain a better balance of nutritionally important amino acids than found in commercial cultivars (1,3,11).Current methods for screening variants for differences in protein composition are generally tedious and often inaccurate. For example, one-dimensional polyacrylamide gel electrophoresis, although widely employed, resolves only a limited number of polypeptides (4,8,10,11,19). In the peanut, the proteins are very poorly defmed and are contained mainly in two major globulin fractions, arachin and conarachin, which together constitute 97% of the protein of the meal. Each of these globulins consists of several polypeptide species, which are thought to exist together as subunits of large multimeric complexes. In addition, many of the polypeptides appear to have similar molecular weights (2, 20). In this study I have characterized the proteins of peanut meal using a two-dimensional electrophoretic technique which separates the 'This research was supported in part by National Science Foundation Grant PCM 76-81071 to R. M. Roberts. polypeptides according to their isoelectric point in the first dimension and by subunit size in the second. The method has been employed to distinguish different cultivars according to their polypeptide composition. It also allows application of up to 500 ,ug of protein on a single slab gel, thus providing the potential for purifying significant quantities of individual polypeptide components. MATERIALS AND METHODS SEED MATERIALSeeds from the following peanut (A. hypogaea L.) cultivars were provided by A. J. Norden of the University of Florida from their breeding lines: Florunner, Florigiant, Early Bunch, Jenkins Jumbo Runner, Altika, UF 75102, UF 77318, 439-16-10-3, 439-16-10-3-1, 439-16-10-3-2, and 439-16-10-1-1. After removal of skins and embryonic axes, the cotyledons were ground in a mortar with a pestle. The ground material was then defatted with cold ethyl ether by repeated extractions until the peanut meals were essentially fat-free. The defatted meals were stored at -20 C. CHEMICALS SDS (specially pure) was from British Drug House, Poole, UK.; ampholines were purchased from LKB, Uppsala, Sweden; urea was bought from Pierce Chemical Co., Rockford, Ill., Nonidet P-40 was obtained from Particle Data Laboratories, Ehmhurst, Ill. Coomassie brilliant blue R-250 and N,N'diallyltartardiamide (DATD) were products of Bio-Rad Laboratories, Richmond, Calif. Protein standards and agarose were from Sigma Chemical Co. Reagents f...

The Glycoproteins of Plant Seeds

“…As pointed out earlier, it is likely that the horizontal rows of proteins noted for each seed type on the two dimensional gels are not artifacts resulting from charge modifications during and after extraction because: (a) they were consistent from experiment to experiment; (b) they were not progressive upon storage of the sample; and (c) different cultivars of peanut (1) and soybean (unpublished results) showed distinct and consistent differences in many ofthese protein spots. The inclusion of the protease inhibitor phenylmethanesulfonyl fluoride in the extraction medium did not influence the polypeptide patterns (unpublished results).…”

“…Comparable results were obtained with the other legumes. The advantages of the method for extraction and maintaining solubility of proteins has been discussed elsewhere (1,7).…”

“…Seed proteins were solubilized by homogenizing the defatted meals in a 9.3 M urea, 5 mm K2CO3 (pH 10.2) solution, followed by the addition of Nonidet P-40 (to 2% v/v) and DTT (to 0.5% w/v) (7). This procedure solubilized approximately 95% of the total protein, most of which is considered to be storage material (1).…”

“…The protein extracts were subjected to two-dimensional polyacrylamide gel electrophoresis by a modification (1) tion for 15 h, a corner of the bag was cut, overlay solution drained off and replaced with 20 ml TBS-azide. The gel was rinsed, drained, removed from the bag, and soaked in about six to eight changes of TBS-azide (250 ml) until radioactivity in the buffer reached background.…”

Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis

“…In 1975, O'Farrell (24) introduced a highly sensitive 2D-technique which separates proteins by pI on an IEF gel in the first dimension and then by molecular weight on an SDS-polyacrylamide slab gel in the second dimension. Recently, 2D-techniques, including that of O'Farrell (24), have been used with plants to compare the composition of seed polypeptides in different peanut cultivars (4) and to analyze the protein composition of various chloroplast subfractions from spinach (9,10,23,26,28), Chlamydomonas (9,10), pea (16) and bean (2).…”