Characterization of methanol-soluble and methanol-insoluble proteins from developing peanut seed.

The 85% methanol-soluble proteins are known to specifically contribute to the production of flavor of roasted peanut. To determine the nature of the 85% methanol-soluble proteins, they were isolated from the peanut seed, and the 85% methanol-soluble (MS) and 85% methanol-insoluble (MIS) fractions were characterized using polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The results showed that the 85% MS fraction contained lower amounts (9-10%) of protein than the MIS fraction (15-33%). Protein content of the MIS fraction increased more significantly during seed maturation than it did in the MS fraction. Unlike the protein, free amino acids and soluble sugars levels of the MS fraction decreased significantly during seed maturation. The 85% MS fraction contained predominantly low molecular weight (<20 kDa) proteins/polypeptides, whereas the MIS fraction contained a mixture of polypeptides with molecular weight between 14 kDa and 90 kDa. SDS-PAGE showed no major changes in the polypeptide composition of the MS fraction during seed maturation. Capillary electrophoretic analysis revealed major qualitative and quantitative changes in the protein and polypeptide composition of the MS and MIS fractions during seed maturation. Fatty acid analysis of these fractions indicated that the MS fraction is lipoprotein in nature and rich in oleic and linoleic acids.

The Glycoproteins of Plant Seeds : ANALYSIS BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS AND BY THEIR LECTIN-BINDING PROPERTIES.

Protein from the jack bean, peanut, soybean and kidney bean seeds were extracted with a solution containing 9.3 molar urea, 5 millimolar K(2)CO(3), 0.5% dithiothreitol and 2% Nonidet P-40 and then subjected to two-dimensional gel electrophoresis. After electrophoresis, the slab gels were stained with a variety of (125)I-labeled lectins and the lectin-binding proteins were identified after autoradiography. Incubation of slab gels of jack bean with concanavalin A, peanut with peanut agglutinin, soybean with soybean agglutinin, and kidney bean with phytohemagglutinin showed that the majority of the polypeptides in each seed type were able to bind to their homologous lectins. Control slab gels in which incubations were carried out with identical amounts of proteins, (125)I-lectin and an appropriate sugar inhibitor showed little or no lectin binding to the polypeptides. Additionally, incubation of slab gels of peanut proteins with (125)I-ricin, (125)I-wheat germ agglutinin, (125)I-concanavalin A, and (125)I-soybean agglutinin each revealed a clearly distinct binding pattern compared to the one observed with the peanut agglutinin. The results demonstrate that a large number of legume seed polypeptides are glycoproteins and that the carbohydrate groups within a seed species are heterogeneous in structure, thus indicating the existence of complex glycosylating enzyme systems in legume seeds. It is suggested that the high degree of binding between seed proteins and their homologous lectins might have some functional significance in maintaining large aggregates of protein in compact, insoluble form.

Effect of the conceptus on quantitative and qualitative aspects of uterine secretion in pigs.

Endometrial explants were removed from uteri of animals pregnant and pseudopregnant (5 mg oestradiol valerate on Days 11--15) for 60 days, from the gravid and non-gravid horns of unilaterally pregnant pigs at Day 60 of pregnancy and fron non-pregnant animals at Day 12 of the oestrous cycle. The tissues were cultured in the presence of L-[34S]methionine for 24 h, and the tissues and medium were then analysed separately by two-dimensional polyacrylamide gel electrophoresis. Radioactive polypeptides were identified by autoradiography of dried gels. Tissues from all all except the cyclic animals released an identical group of polypeptides into the culture medium: the major radioactive products were 4 acidic polypeptides of low molecular weight and several basic proteins, which included the purple phosphatase uteroferrin, and lysozyme. In separate experiments explants from 3 unilaterally pregnant pigs were cultured with L-[3H]leucine, and, on a fresh weight basis, the tissue from the non-gravid horn released significantly less radioactive macromolecular material into the medium in 24 h than did tissue from gravid horns. It therefore appears that although the nature of the secretion produced by the pregnant uterus is a consequence of maternal hormonal regulation alone, the tissue underlying a conceptus is quantitatively more active than that from unoccupied regions of a uterus.

Method for obtaining ovine uterine secretions from unilaterally pregnant ewes.

Development of the ovine conceptus was confined to the uterine horn ipsilateral to the corpus luteum (CL) by placing a ligature around that uterine horn at a point near the uterine body on day 5 of pregnancy. On day 140 of gestation, seven of 10 ewes were still pregnant and from 21 to 815 ml of uterine fluid (488 +/- 94 ml, X +/- SEM) were collected from the nongravid uterine horn. Total recoverable protein (X +/- SEM) was 13.4 +/- 3.4 grams. Polyacrylamide gel electrophoresis of the reduced proteins in presence of sodium dodecyl sulfate indicated that protein composition of uterine fluid was distinct from that of colostrum, serum, amniotic fluid, and allantoic fluid, and revealed the presence of two major polypeptides with molecular weights of about 57,000 and 58,500, respectively, plus numerous other minor components. Gel filtration on columns of Sephadex G-200 and Sepharose CL-6B suggested that these polypeptides formed a series of aggregates of high molecular weight when kept under nonreducing conditions. Glucose (.18 +/- .03 mg/ml), but not fructose, was present in uterine fluid. In addition, high levels of prostaglandin F (451.4 +/- 83.3 ng/ml) were present.

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

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 K(2)CO(3)-9.5 molar urea. After addition of Nonidet P-40 (2%, v/v) and dithiothreitol (0.5%, w/v) the solution was centrifuged at 25,000 g. This procedure led to solubilization of more than 95% of the total protein. The clear supernatant fraction was then subjected to two-dimensional polyacrylamide gel electrophoresis, employing isoelectric focusing in the first dimension and electrophoresis in presence of sodium dodecyl sulfate in the second. After examining several cultivars, it was possible to construct a composite map to include all of the polypeptide species found among all of the cultivars examined. At least 74 major and between 100 and 125 minor components were detectable by Coomassie blue staining. The majority of these had isoelectric points between pH 4.4 and 8.0, and molecular weights between 16,000 and 75,000. Several different cultivars have been compared using this method and it has been shown that considerable variation exists among the major polypeptides present. The method should prove valuable for analyzing different genotypes and selecting varieties with a particular storage protein make-up, as well as for following compositional changes that occur during seed development and germination.

Glycoprotein Metabolism in the Cotyledons of Pisum sativum during Development and Germination.

The glycoprotein nature of legumin and vicilin, the reserve globulins in the cotyledons of Pisum sativum was studied. Legumin from mature seed was found to contain 1% neutral sugars (mannose and glucose) and 0.1% amino sugar (glucosamine), whereas vicilin contained 0.3% neutral sugar (mannose) and 0.2% amino sugar (glucosamine). On the basis of the incorporation of (14)C-labeled glucosamine, it appeared that not all of the component subunits of the reserve proteins are glycosylated to the same extent. In addition, it has been established that glycosylation occurs after peptide synthesis. During seed development there was a change in neutral sugars and amino sugar ratio in vicilin. During germination, the neutral sugars and the amino sugar content of the glycoproteins declined. These findings are discussed in relation to the synthesis and degradation of the glycosyl component of the glycoproteins.

The development of proteolytic activity and protein degradation during the germination of Pisum sativum L.

The change in protein content and composition of the cotyledons of Pisum sativum L. cv. Burpeeana during germination was studied. Protein depletion from the cotyledons was slow during the first 4 days of germination but became rapid on the 5th day and by the 16th day the majority of the protein had disappeared. During the first 4 days the depletion of the globulins exceeded that of the albumins; legumin appeared to be degraded slightly more rapidly than vicilin during the early phase of germination. Sodium-dodecylsulfate (SDS) electrophoresis of SDS- and dithiothreitol-dissociated globulins indicated that before rapid protein depletion there were marked changes in the component composition of the major globulins legumin and vicilin. The onset of rapid protein depletion was associated with an increase in the level of an acid-sulfhydryl protease in the cotyledons. These findings indicate that the reserve globulins undergo modifications prior to their eventual hydrolysis.