Hexokinase II of Pea Seeds

A second hexokinase (EC 2.7.1.1) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed hexokinase II, had a high affinity (K_m, 48 micromolar) for glucose and a relatively low affinity (K_m, 10 millimolar) for fructose. The K_m for MgATP was 86 micromolar. Mg²⁺ was required for activity, but excess Mg²⁺ was inhibitory. MgADP inhibited hexokinase II. The addition of salts of monovalent cations increased hexokinase II activity. Al³⁺ was a strong inhibitor of the enzyme at pH 6.6 but not at the optimum pH (8.2). Citrate and 3-phosphoglycerate activated pea seed hexokinase II at pH 6.6, probably by coordinating with aluminum present as a contaminant in commercial ATP. The properties of hexokinase II are compared with those of the other three hexose kinases obtained from pea seed extracts. The possible role of these enzymes in plant carbohydrate metabolism is discussed.     

Isolation of Casparian Strips from Pea Roots

Casparian strips were isolated from roots of pea seedlings.The plasma membrane was firmly attached to the wall of the individualcells in the isolated Casparian strip. The outer electrondenselamella of the plasma membrane, which is characteristic of themembrane of the Casparian strip, was not removed from the isolatedCasparian strip by treatment with either 1 M NaCl or 2 M sodiumthiocyanate. Although treatment with 1% Triton X-100 disruptedthe lamellar structure of the membrane and removed most of theelectron-dense material of the outer lamella, no detectablepolypeptides were extracted by treatment of isolated Casparianstrips with 1% Triton X-100. Small amounts of electron-densematerial remained after treatment with Triton X-100 and thismaterial seemed to be removed almost entirely by subsequenttreatment of the same sample with 2% SDS. Since polypeptidesof 46, 30 and 20 kDa, which were detected in the extract obtainedby treatment with SDS, were not detected in an extract of totalcell walls of pea roots, it seems possible that these polypeptidesare specific to the Casparian strip and, therefore, that theymay be responsible singly or together for the adhesion of theplasma membrane to the cell wall at the Casparian strip. (Received January 30, 1992; Accepted April 23, 1992)     

Chemical Composition of Ceramide and Cerebroside in Azuki Bean Seeds

The enzymic properties of three electrophoretically distinct β-glucosidases, β-glucosidase-1, -2 and -3, from Aspergillus aculeatus No. F-50 were investigated. β-Glucosidase-3 had a low optimum pH of 3.0, but the other two enzymes had optimum pHs of 4.0~4.5. Both β-glucosidase-1 and -2 were potently active not only on soluble cellooligosaccharides, such as cellotriose to cellohexaose, but also on insoluble cellooligosaccharide, of which the average degree of polymerization was 20. On the contrary, β-glucosidase-3 was only slightly active on the insoluble substrate. The combined use of either β-glucosidase-1 or -2 and endo-glucanase remarkably stimulated the hydrolysis of amorphous cellulose, yielding glucose only. But β-glucosidase-3 did not show such a synergistic effect, and the glucose content of the hydrolyzate was only about 60%.     

The Effect of Temperature Change on Leakage from Pea Seeds

Intact seeds of P. sativum and P. elatius leaked electrolytes,sugars and proteins for as long as 48 h after the beginningof imbibition. Initial leakage was higher at 25 °C thanat 5 °C, but its extent was much greater at the lower temperature.Transfer of seed from 5 °C to 25 °C after 5 h imbibitionresulted in leakage for 48 h at the initial rate at 5 °C.The transfer from 25 °C to 5 °C gave leakage at a rateequivalent to the initial rate at 25 °C. The results arediscussed in relation to behaviour of membranes and to the sensingof the initial temperature. Key words: Pisum sativum, P. elatius, Leakage, Electrolytes, Sugars, Proteins, Temperature     

The Isolation of Nucleoli from Ungerminated Pea Embryos

A method is described for the isolation of nuclei and nucleoli from ungerminated pea embryos. Electron micrographs of the nucleolar preparation indicate a high degree of purity but only a partial preservation of composition, because of a loss of material from the nucleoli which occurs during the isolation procedure.     

Isozymes of beta-N-Acetylhexosaminidase from Pea Seeds (Pisum sativum L.)

Four isozymes of β-N-acetylhexosaminidase (β-NAHA) from pea seeds (Pisum sativum L.) have been separated, with one, designated β-NAHA-II, purified to apparent homogeneity by means of an affinity column constructed by ligating p-aminophenyl-N-acetyl-β-d-thioglucosaminide to Affi-Gel 202. The other three isozymes have been separated and purified 500- to 1750-fold by chromatography on Concanavalin A-Sepharose, Zn²⁺ charged immobilized metal affinity chromatography, hydrophobic chromatography, and ion exchange chromatography on CM-Sephadex. All four isozymes are located in the protein bodies of the cotyledons. The molecular weight of each isozyme is 210,000. β-NAHA-II is composed of two heterogenous subunits. The subunits are not held together by disulfide bonds, but sulfhydryl groups are important for catalysis. All four isozymes release p-nitrophenol from both p-nitrophenyl-N-acetyl-β-d-glucosaminide and p-nitrophenyl-N-acetyl-β-d-galactosaminide. The ratio of activity for hydrolysis of the two substrates is pH dependent. The K_m value for the two substrates and pH optima of the isozymes are comparable to β-NAHAs from other plant sources.     

Apparent Free Space of Germinating Pea Seeds

The apparent free space (AFS) of pea seeds was determined by measurement of the exodiffusion of solutes from the seeds previously equilibrated with radioactive solutions. AFS (per cent of volume of water taken up by dry seeds) varied with the kinds of solutes used, that is, values of 27–30% were obtained for mannitol or leucine and 12% for malonate, at pH 6. When the seeds were incubated in a radioactive solution at lower pH (3.5), higher AFS values were obtained, especially for malonate. The AFS of imbibing seeds showed a tendency to increase during the course of imbibition.     

Appearance of Three Chloroplast Isoenzymes in Dark-grown Pea Plants and Pea Seeds

Activity peaks characteristic of the chloroplastic Calvin cycle enzymes triose-phosphate isomerase, ribose 5-phosphate isomerase, and fructose 1,6-diphosphate aldolase are found in isoelectric focusing patterns of dark-grown pea (Pisum sativum) seedlings and seeds. Apparently, in this higher plant these three chloroplastic isoenzymes can be formed in the absence of light and of chloroplast formation.     

Isolation and Characterization of Biotin Carboxylase from Pea Chloroplasts

Pea (Pisum sativum L.) leaf acetyl-coenzyme A carboxylase (ACCase) exists as two structurally different forms: a major, chloroplastic, dissociable form and a minor, multifunctional enzyme form located in the leaf epidermis. The dissociable form is able to carboxylate free D-biotin as an alternate substrate in place of the natural substrate, biotin carboxyl carrier protein. Here we report the purification of the biotin carboxylase component of the chloroplastic pea leaf ACCase. The purified enzyme, free from carboxyltransferase activity, is composed of two firmly bound polypeptides, one of which (38 kD) is biotinylated. In contrast to bacterial biotin carboxylase, which retains full activity upon removal of the biotin carboxyl carrier component, attempts to dissociate the two subunits of the plant complex led to a complete loss of biotin carboxylase activity. Steady-state kinetic studies of the biotin carboxylase reaction reveal that addition of all substrates on the enzyme is sequential and that no product release is possible until all three substrates (MgATP, D-biotin, bicarbonate) are bound to the enzyme and all chemical processes at the active site are completed. In agreement with this mechanism, bicarbonate-dependent ATP hydrolysis by the enzyme is found to be strictly dependent on the presence of exogenous D-biotin in the reaction medium.     

Fructokinase (Fraction III) of Pea Seeds

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K_m values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K_m for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg²⁺ was essential for activity but the enzyme was inhibited by excess Mg²⁺ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K⁺, Rb⁺, and NH₄⁺ were the most effective. The possible role of fructokinase (fraction III) is discussed.     

Fructokinase (Fraction IV) of Pea Seeds

A fructokinase (EC 2.7.1.4) was obtained from pea (Pisum sativum L.) seeds. This enzyme, termed fructokinase (fraction IV), was specific for fructose as substrate and had little activity with glucose or mannose. Excess fructose inhibited the enzyme at the optimum pH (8.2) but not at pH 6.6. MgATP was inhibitory at pH 6.6. The apparent Michaelis-Menten constants at pH 8.2 were 0.057 mm for fructose and 0.10 mm for MgATP. Mg(2+) ions were essential for activity; Mn(2+) could partially replace Mg(2+). Fructokinase (fraction IV) had a requirement for K(+) ions which could be substantially replaced by Rb(+) or NH(4) (+) but not by Na(+). The enzyme was inhibited by MgADP. The possible significance of fructokinase (fraction IV) in plant carbohydrate metabolism is discussed.     

In situ Imaging of Pea Starch in Seeds

A method has been developed for the in situ imaging of starch in dry seeds by exploiting the tight packing of the starch and protein storage reserves within the cells of the embryo. The method can be adapted to prepare seed samples which are suitable for light microscopy (birefringence and iodine staining), scanning electron microscopy and atomic force microscopy. Its potential for imaging the internal structure of starch granules without any prior isolation process is demonstrated for round smooth peas. Using a standard ultramicrotome, thin sections were cut directly from selected regions of dry pea seeds and examined by light microscopy before and after hydration. The sectioning procedure left a planed surface with the internal structure of the starch granules exposed. This material was examined by scanning electron microscopy and atomic force microscopy directly or after controlled hydration. In the hydrated pea samples, the growth ring structure and blocklet sub-structure of individual starch granules within the seed were visualised directly by atomic force microscopy. Furthermore, the effects of hydration and staining were monitored and have been used to introduce contrast into the images. The observations have revealed new information on the blocklet distribution within pea starch granules and the physical origins of the growth ring structure of the granules: the blocklet distribution suggests that the granules contain alternating bands with different levels of crystallinity, rather than alternating amorphous and crystalline growth rings.     

Cerebroside analogues from 3-phenylserines

Cerebroside analogues were synthesized from DL-threo-and DL-erythro-3-phenylserines by the following sequence of reactions: estirification, N-acylation, reduction with sodium bis (2-methoxyethoxy)-aluminum hydride (SMEAH), and condensation with acetobromoglucose followed by deacetylation. Mass spectrometry disclosed that the glycosidic bond was formed at the primary hydroxyl group.     

The Influence of Embryo Condition on the Leaching of Solutes from Pea Seeds

Differences in the leaching of solutes from different seed lotsof peas were found to be associated with the condition of theembryos and not the testae. Seeds with areas of dead tissueon the abaxial surfaces of their cotyledons exhibited the highestlevels of leaching but the large range of levels found amongseed lots resulted from differences in leaching from livingtissue. When dry embryos were placed in water a rapid declinein the leaching of solutes was seen over the first 30 min forall lots tested but differences between lots were seen fromthe first minute in water and were related to the ability ofthe embryos to retain solutes rather than their solute content.Differential retention of solutes by the cytoplasm and sensitivityto the damaging effects of rapid imbibition are both discussedas possible explanations for differences in leaching.     

Isolation and Anti-Fatty Liver Activity of a Novel Cerebroside from the Sea Cucumber Acaudina molpadioides

Cerebrosides are a kind of important bioactive substance in sea cucumber. A novel cerebroside, AMC-2, was purified from the less-polar lipid fraction of the sea cucumber Acaudina molpadioides by repeated column chromatography. The major structure of AMC-2 was analyzed by gas chromatography-mass spectra. The amide-linked fatty acid unit was confirmed to be four saturated and monounsaturated α-hydroxy fatty acids, the long-chain base was dihydroxy sphingoid base with one double bond, and the glycosyl group was glucose. We also investigated the anti-fatty liver activity of AMC-2 in rats with fatty liver induced by orotic acid. AMC-2 significantly reduced hepatic triglyceride (TG) and total cholesterol (TC) levels at a diet supplement of 0.03% and 0.006%. The indexes of stearoyl-CoA desaturase (SCD) activity and mRNA expression were significantly decreased by AMC-2. This indicates that AMC-2 ameliorated nonalcoholic fatty liver disease (NAFLD) through suppression of SCD activity and impaired the biosynthesis of monounsaturated fatty acids in the livers of the rats.     

The Movement of Calcium in Germinating Pea Seeds

Pea seeds contain less calcium than phosphorus, potassium ormagnesium; more than half of this calcium is located in thetesta. Peas at either end of a pod have more calcium than thosein the middle. When pea seeds are allowed to germinate in water,less than 30 per cent of the cotyledonary calcium moved to thegrowing axis during the first 15 days of germination, whereas70–90 per cent of magnesium, potassium and phosphate wasexported. Various attempts to increase the amount of calciumexported were not successful. When radioactive calcium was appliedto the cotyledons, essentially no movement to the axis was observedunder conditions where extensive movement of radioactive phosphateoccurred.