David R. Murray

The seed proteins of chick pea: Comparative studies ofCicer arietinum, C. reticulatum andC. echinospermum (Leguminosae)

Seed proteins fromCicer arietinum L.,C. reticulatumLadiz. andC. echinospermumDavis were extracted and separated into water soluble (albumin) and water insoluble (globulin) fractions. These were analysed using three polyacrylamide gel systems: uniform pore slab gels, gradient gels and SDS disc gels. For all three species, albumins constitute just over one-third of total protein. Minor differences in the composition of this fraction were observed. Within the globulin fraction, seven disulphide-linked polypeptides were found. Four of these resemble the major polypeptide of legumin, consisting of constant small subunit (21,000 daltons) linked to variable large subunit (46,000, 41,000, 39,000 or 36,000 daltons), forming polypeptides of 67,000 (I), 62,000 (II), 60,000 (III) and 57,000 (IV) daltons respectively. Polypeptide I was prominent in both wild species, but absent fromC. arietinum. Polypeptides II and III were equally prominent inC. arietinum andC. reticulatum. Polypeptide IV was more prominent inC. echinospermum, which was deficient in polypeptide III. Polypeptides V (45,000 daltons) and VI (43,000 daltons), apparently composed of two equal subunits, were present in trace amounts in both wild species, but well represented inC. arietinum Polypeptide VII of 45,000 daltons (31,000 + 14,000) was present in all three species.

Changes in Polypeptide Composition During Seed Development in Chickpea, Cicer arietinum L.

Summary Proteins were extracted from seeds of Cicer arietinum L. at different stages of seed development and analysed by pore gradient electrophoresis and by SDS-disc gel electrophoresis. In contrast to developing pea seeds, there were no marked differences in the times of first appearance of the major albumins and globulins. There were differences in the periods of most rapid net synthesis, those for «vicilin» and most albumins preceding those for three forms of legumin. These differences, together with differences in seed anatomy, support the removal of Cicer L. from the Vicieae to Cicereae.

Electrophoretic Studies of the Seed Proteins of Cowpea, Vigna unguiculata (L.) Walp.

Summary The globulin fraction accounted for the major proportion (72%) of the protein extracted from mature cotyledons of cowpea, Vigna unguiculata (L.) WALP. cv. Vita 3. The globulin and albumin fractions were compared by pore-gradient polyacrylamide gel electrophoresis, and component polypeptides were analysed by SDS polyacrylamide disc gel electrophoresis. The major globulin, vignin, consists mainly of non-covalent associations of polypeptides of MW 56,000 and 52,500 daltons. Under reducing conditions, globulin polypeptides of MW 80,000 and 43,000 dissociate and polypeptides of 61,000, 26,500 and 19,000 daltons are formed. Under the same conditions, the major polypeptides of the albumin fraction are distinct from those of the globulin fraction, having sizes estimated to be ca. 100,000, 70,000, 43,000, 35,000, 32,000 and 25,000 daltons. During embryo development there is no clear division into a period of albumin synthesis followed by a period of globulin synthesis; polypeptides representing both fractions were present from the earliest stage analysed, 7 days after anthesis. There are, however, differences in the periods of most rapid net synthesis for individual polypeptides of each fraction.

Albumins from Pea Seeds are Distinct From Vicilin

Summary Of three forms of vicilin known to be present in pea seeds, one form is water soluble and may therefore contribute to the «albumin» (water soluble) fraction. Using gradient polyacrylamide gel electrophoresis, we have shown that water soluble vicilin is a very minor component of the total water soluble fraction of pea seed proteins. The major albumin proteins are confined to the water soluble fraction and range in size from 36,000 to 135,000 daltons. In the variety studied, P. sativum cv. Melbourne Market, the albumin fraction exceeded 50% of total extractable protein and showed enhanced content of polypeptides of 23,000 and 34,000 daltons, but the seed protein content did not exceed 17 % of dry matter.

Acid Glycosidase Isoenzymes and Activities in Developing Hulls and Seeds of Pisum sativum L.

Summary The changes in β-glucosidase (EC 3.2.1.21), β-galactosidase (EC 3.2.1.23), α-mannosidase (EC 3.2.1.24) and α-glucosidase (EC 3.2.1.20) activities that occur during development of the pea seed have been measured. The highest tissue specific activity of β-glucosidase is found in the seedcoats, whereas the highest specific activities of the other enzymes occur in the cotyledons. Glycosidase isoenzymes were separated by ammonium sulphate fractionation followed by anion exchange chromatography and gel filtration. The α-mannosidase and β-galactosidase activities of the cotyledons are attributed mainly to single forms of each specific enzyme of MW 70,000 and 46,000 daltons respectively. By comparison, a wider spectrum of isoenzymes was observed in the hull and seedcoats. For seedcoats, two forms of β-glucosidase, three forms of β-galactosidase and four forms of α-mannosidase were distinguished. The main form of β-glucosidase from the seedcoats, isoenzyme 2, had a MW estimated to be 65,000 daltons.

Variation in the Sizes of Both Large and Small Disulphide-Linked Subunits of Legumin in Representatives of Vicieae and Cicer

Summary The distribution of seed globulins has been studied in several members of Vicieae, namely Vicia faba L., Lens culinaris Medik., Lathyrus odoratus L. and Pisum sativum L., and compared with seed globulins of three species of chickpea: Cicer arietinum L., C. reticulatum Ladiz. and C. echinospermum Davis. Globulin samples were analysed using pore-gradient polyacrylamide gel electrophoresis, and their constituent polypeptides examined by disc gel electrophoresis after dissociation with SDS in either the absence or presence of 2-mercaptoethanol to reduce disulphide bonds. The disulphide-linked polypeptides comprising legumin varied in size from 57,000 (36,000 + 21,000) daltons up to 71,000 (50,000 + 21,000) daltons. Of the nine different pairs of disulphide-linked polypeptides detected belonging to legumin, none was held in common by all the Vicieae, and only one was held in common by all three species of Cicer and any member of Vicieae: that of 62,000 (41,000 + 21,000) daltons was also prominent in Lens culinaris . Differences were observed in the sizes of both large and small subunits in most of the Vicieae, but only in the size of the large subunit in all three species of Cicer . The maximum number of legumin components coexisting in a single genotype was found to be four for both wild chickp as and three for most members of Vicieae and C. arietinum .

Localization of acid glycosidases in protein bodies of pea cotyledons

Summary Protein bodies collected by centrifugation after cold water extraction of imbibed pea cotyledons were obtained in good yield and free of intact mitochondria, but they were practically devoid of α-mannosidase activity (EC 3.2.1.24). If 0.25 M sucrose was provided as an osmotic support, then mitochondria could be sedimented in good yield, accounting for at least 20% of total malate dehydrogenase activity (EC 1.1.1.37). When protein bodies were recovered from sucrose-Percoll gradients, or from seed meal extracted with 25% (w/v) sucrose in Na acetate buffer at pH 5.0, then up to 86% of total α-mannosidase activity was associated with them. By comparison, only about 30% of α-galactosidase (EC 3.2.1.22) and β-galactosidase (EC 3.2.1.23) activities could be recovered associated with protein bodies. The conditions of extraction clearly influence the composition and recovery of protein bodies and procedures for the complete recovery of undamaged protein bodies separate from mitochondria have yet to be developed for seed storage tissues.

Peroxidase Isoenzymes and Leaf Senescence in Sunflower, Helianthus annuus L

Summary Changes in the activities of acid phosphatase (EC 3.1.3.2) and peroxidase (EC 1.11.1.7) have been measured during development of the first leaves of sunflower seedlings. Acid phosphatase activity increased to a maximum early during expansion growth. This maximum activity coincided with the maximal leaf content of free amino acids (23 days after seed imbibition). Acid phosphatase activity declined throughout subsequent leaf development and senescence. Peroxidase activity remained low until after 30 days, then a 10-fold increase in activity occurred between 30 and 35 days, coincident with a fall in leaf chlorophyll content. Seven anionic isoenzymes of peroxidase were resolved when leaf extracts were analysed by pore-gradient polyacrylamide gel electrophoresis. Only two of these were prominent in young leaves: isoenzymes 2 (MW 42,000) and 7 (MW 81,000). The increase in total peroxidase activity in senescing leaves was brought about by increases in the activities of isoenzymes 1, 2, 3 and 6, of MW 40,000, 42,000, 46,000 and 68,000 daltons respectively.

Effects of Cycloheximide and Actinomycin D on Glycosidase Activities in the Cotyledons of Legume Seeds Following Imbibition

Summary The activities of α-galactosidase (EC 3.2.1.22), β-galactosidase (EC 3.2.1.23), α-mannosidase (EC 3.2.1.24) and N-acetyl β-glucosaminidase (EC 3.2.1.30) were assayed in extracts from the cotyledons of pea ( Pisum sativum L.), cowpea ( Vigna unguiculata [L.] Walp.) and mung bean ( Vigna radiata [L.] Wilcz.) following imbibition of seeds for periods up to 3 days at 24 °C. Cycloheximide or actinomycin D provided over the first 24 h did not inhibit the development of any of these enzyme activities, except for α-galactosidase from pea (cv. Telephone). Cycloheximide inhibited the incorporation of label from 14 C-labelled amino acids into protein by 93.4 % (pea) up to 98.0 % (cowpea). We conclude that acid glycosidase activities in imbibing cotyledons of these species generally result entirely from rehydration of enzyme molecules formed during seed development.

The Occurrence of Disulphide-Linked Polypeptides in Helianthin, the Major Reserve Globulin of Sunflower Seed

Summary The salt-extractable proteins from embryos of Helianthus annuus L. were separated into albumins (20%) and globulins (80%). Each fraction was analysed by pore-gradient electrophoresis. The predominant globulin, helianthin, has a molecular weight estimated to be 270,000 daltons. SDS-disc gel electrophoresis revealed two major pairs of disulphide-linked polypeptide subunits: I, 59,000 = 37,000 + 22,000 daltons, and II, 51,000 = 29,000 + 22,000 daltons. The data suggest that these linked polypeptide pairs associate non-covalently in the ratio 2:3 (I:II respectively).