Phosphorylase activity in relation to starch synthesis in developing Hordeum distichum grain

Activity, control and primer requirements of starch phosphorylase in developing barley endosperm were investigated. Phosphorylase was detected in endosperm extracts from 3 days after anthesis. Unprimed activity was predominant between 2 and 10 days after anthesis, when it constituted 70–80% of total activity, but this proportion declined rapidly as the grain developed. The existence of at least 2 isoenzymes was indicated by studies of pH dependence and phosphate inhibition, and was further supported by acrylamide gel electrophoresis and column chromatography using DEAE-cellulose. The two isoenzymes which ere possibly both glyco proteins, appear in barley endosperm soon after anthesis. One appears capable of unprimed activity, and may be associated with the initiation of a-1,2 glucans, which then serve as primers for starch synthetase. This disappears by 13–15 days after anthesis. The other isoenzyme is capable of some unprimed activity but undergoes modification between 15 and 20 days after anthesis, resulting in the loss of unprimed activity. The relevance of the results to initiation of starch synthesis and to starch synthetase in amyloplasts is discussed.     

Two types of heat tolerance in FHM-cells. Induction by heat-shock versus elevated culturing temperature

1. 1.Increased heat tolerance in FHM-cells from Pimephales promelas (Pisces) can be induced by culturing the cells at elevated temperatures (heat resistant acclimation) as well as by heat shock (heat hardening).

2. 2.After shift of culturing temperature (CT) from 16 to 32°C both effects are detectable with different temporal patterns.

3. 3.Cellular concentrations of heat-shock proteins correlate with the hardening effect but not with heat resistance acclimation.

4. 4.Several culturing temperature specific proteins were detected. The patterns of some enzymes are also altered by culturing temperature.

5. 5.Heat resistance acclimation is not caused by selection of a thermoresistant subpopulation of cells.

6. 6.Heat hardening and heat resistance acclimation must be distinguished as different phenomena in FHM-cells.

Platelet glycohydrolase activities: Characterization and release

Granules containing acid hydrolases have been detected in human platelets but have not been thoroughly characterized. We have studied the activity and characteristics of glycohydrolases present in normal human platelets, evaluated their release upon stimulation with thrombin, and assessed the contribution of platelet - released lysosomal contents to the glycohydrolase activity present in normal serum. Platelets contained a remarkable glycohydrolase activity with a prevalence of β - N-acetylhexosaminidase. All glycohydrolases were released to some extent upon stimulation with thrombin and contributed to the glycohydrolase activity found in human serum. α-Mannosidase and α-galactosidase were partially inactivated after release by a mechanism as yet undefined. In addition, thrombin stimulation affects the intraplatelet isoenzyme pattern of β-N-acetylhexosaminidase by producing the appearance of a new form.     

Ammonium assimilation in bryophytes. l-glutamine synthetase from Sphagnum fallax

Cytosolic and plastidic l -glutamine synthetase (EC 6.3.1.2) isoenzymes from Sphagnum fallax Klinggr. (Klinggr. clone 1) were separated by size-exclusion and ion exchange chromatography. The cytosolic enzyme (GS₁) was purified to apparent electrophoretic homogeneity. The native enzyme had a molecular mass of 390 ± 20 kDa as estimated by gel filtration and was apparently composed of 8 subunits with molecular masses of 48 kDa. GS₁ activity could be measured from pH 6.8 to 8.6 in 50 m M imidazole buffer, with a broad optimum between pH 7.2 and 8.0. The K_m values were 2.5 m M , 0.5 m M and 0.5 m M for l -glutamate, ammonium and ATP, respectively. The enzyme was inhibited by more than 10 m M ammonium or glutamate. The incorporation of ¹⁵NH₄⁺ into amino acids was observed in vivo using ¹⁵ NMR. Label from ammonium was first detected in the amide N of glutamine, and only subsequently in the amino N of glutamate. Moreover, no assimilation was detected in the presence of the specific GS inhibitor methionine sulfoximine. These observations are consistent with a dominant role for GS in the assimilation of ammonium in Sphagnum .     

Production of peroxidases by hairy roots ofBrassica napus

Hairy roots cultures derived from leaf explants ofBrassica napus L. produced and secreted peroxidases. The enzyme activity in the medium increased with growth but it remained nearly constant in the tissue. The changes in extracellular peroxidase activity seemed to be correlated with the increase in a basic peroxidase of pI: 9.6. Four isoenzymes with pI in the range 8.5–9.6 and a neutral peroxidase of pI 6.3 were the most important peroxidases detected in cell extracts. Ca²⁺ addition at the beginning of the culture stimulated both the excretion of peroxidase to the medium and the enzyme activity in hairy roots but the isoenzyme profiles did not show qualitative changes during the growth cycle for both culture conditions.     

Three differentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization

We describe the molecular and functional characterization of three closely related S-adenosyl-L-methionine synthetase (SAMS) isoenzymes from Catharanthus roseus (Madagascar periwinkle). The genes are differentially expressed in cell cultures during growth of the culture and after application of various stresses (elicitor, nutritional down-shift, increased NaCl). Seedlings revealed organ-specific expression and differential gene regulation after salt stress. A relationship analysis indicated that plant SAMS group in two main clusters distinguished by characteristic amino acid exchanges at specific positions, and this suggested differences in the enzyme properties or the regulation. SAMS1 and SAMS2 are of type I and SAMS3 is of type II. The properties of the isoenzymes were compared after heterologous expression of the individual enzymes, but no significant differences were detected in a) optima for temperature (37 to 45 °C) or pH (7 to 8.3); b) dependence on cations (divalent: Mg2+, Mn2+, Co2+; monovalent: K+, , Na+); c) Kms for ATP and L-methionine; d) inhibition by reaction products (S-adenosyl-L-methionine, PPi, Pi), by the reaction intermediate tripolyphosphate, and by the substrate analogues ethionine and cycloleucine; e) response to metabolites from the methyl cycle (L-homocysteine) or from related pathways (L-ornithine, putrescine, spermidine, spermine); f) native protein size (gel permeation chromatography). The results represent the first characterization of plant SAMS isoenzyme properties with individually expressed proteins. The possibility is discussed that the isoenzyme differences reflect specificities in the association with enzymes that use S-adenosyl-L-methionine.     

β-Galactosidases of Lilium pollen

Lily (Lilium auratum) pollen contains very high levels of β-galactosidase. There are three forms: β-galactosidase I and II differ in M_r, while β-galactosidase III is firmly bound in the pollen wall. The two cytoplasmic forms were separated and partially purified using a combination of chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose 6B. Forms I and II appear to be glycoprotein in nature as shown by binding to Con A-Sepharose. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. The wall-bound enzyme, β-galactosidase III effectively hydrolysed nitrophenyl β-galactosidase but not lactose, and could not be released from the wall polysaccharide matrix by high salt concentrations or detergents. The total β-galactosidase activity of lily pollen remained constant during in vitro germination. A possible role for this enzyme may be in degradation of stylar arabinogalactans providing a carbon source for pollen tube nutrition.     

The involvement of aspartate aminotransferases in ammonium assimilation in lupin nodules

Aspartate aminotransferase (AAT) activity has been detected in the plant and bacteroid fractions of lupin nodules, and in free-living Rhizobium lupini. Two electrophoretically distinct forms of AAT were detected in the plant fraction of the nodule and a third form in the bacteroid fraction. AAT activity increased in the plant fraction during nodule development and this increase may be due to an increase in the activity of one of the AAT forms in this fraction. The single form of AAT detected in the bacteroid fraction had the same electrophoretic mobility as that detected in free-living R. lupini. The nodulated roots of lupins, grown in a media supplemented with nitrate and ammonium, had a 3- and 4-fold lower activity of AAT and nitrogenase activity respectively, compared to the nodulated roots of plants grown in the absence of added nitrogen. A role for the plant AAT in ammonium assimilation in lupin nodules is proposed.     

La phosphoglycolate phosphatase des feuilles de haricot: Deux formes isofonctionnelles

2-Phosphoglycollate phosphohydrolase (EC 3.1.3.18) was isolated and partially purified from leaves of Phaseolus vulgaris L. by acetone fractionation followed by chromatography on DEAE-cellulose. Two chromatographically stable forms could be separated by stepwise elution from DEAE-cellulose; they have been resolved by electrofocusing (pI 4.2 and 5.5) and exhibited two protein bands (R_itfs 0.68 and 0.82) on polyacrylamide gel electrophoresis. These data suggest the isoenzymatic nature of the two forms which are specific for phosphoglycollate.