Mary Spencer

Purification and characterization of barley-aleurone xylanase

Xylanase (β-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The Km was 0.86 mg xylan·ml-1.

Ethylene Production during Development of Mustard (Brassica juncea) and Canola (Brassica napus) Seed.

An open, continuous flow system was used to investigate ethylene production during degreening of maturing seed of mustard (Brassica juncea cv Cutlass and cv Lethbridge 22A) and canola (Brassica napus cv Westar and cv Alto). Isolated mustard seed evolved higher amounts of ethylene than those of canola, and this was particularly evident both early in embryogeny and later during the desiccation phase of seed maturation. The silique walls produced negligible amounts of ethylene in both species. The concentrations of ethylene surrounding seed as they matured within siliques were significantly higher in mustard than in canola, and this interspecies difference was greatest during the seed desiccation phase. In mustard, a 4-fold increase in silique internal ethylene levels was apparent during desiccation. In comparison, only a moderate increase in silique-derived ethylene occurred in canola.

Conversion of l-leucine to certain keto acids by a tomato enzyme preparation

Abstract An enzyme preparation from fresh tomato fruit catalyzed the degradation of l -leucine. The reaction products were separated as their 2,4-dinitrophenylhydrazones and analyzed by TLC. α-Ketoisocaproic acid, the keto acid corresponding to l -leucine, was identified. The conversion was confirmed by studies with l -leucine-U- 14 C as substrate. Enzyme activity was found to occur predominantly in the supernatant fraction and to decrease with ripening of fruit.

Effects of cycloheximide, d-threo-chloramphenicol, erythromycin and actinomycin D on De-novo synthesis of cytoplasmic and mitochondrial proteins in the cotyledons of germinating pea seeds

SummaryInhibitors of, and radioactive substrates for, protein synthesis were introduced into germinating pea (Pisum sativum L.) seeds, and protein synthesis was allowed to proceed in vivo. Subsequent analyses of subcellular fractions showed the following: Cycloheximide strongly inhibited the incorporation of [14C]leucine into both mitochondrial and cytoplasmic proteins. d-Threo-chloramphenicol and erythromycin did not affect cytoplasmic protein synthesis, but partially inhibited mitochondrial protein synthesis. These results suggest that most of the new mitochondrial proteins were originally synthesized in the cytoplasm. Actinomycin D did not appreciably affect the initial incorporation of [14C]leucine into either mitochondrial or cytoplasmic proteins, suggesting that information (mRNA) concerning the initially synthesized proteins may be present in the quiescent seeds. The lack of appreciable incorporation of [3H]thymidine into mitochondrial DNA supported our previons report that mitochondria may not be synthesized de novo in pea cotyledons.

Energy sources for photosynthetic carbon dioxide fixation.

The high-energy intermediates, or high-energy states, involved in photophosphorylation (PSP), may be used directly to drive CO2 fixation, without the intercession of ATP. This paper reports an investigation on the amounts of CO2 fixed in isolated spinach chloroplasts, when ATP synthesis was inhibited, but when electron flow and high-energy intermediate synthesis was unaffected. As a corollary to this work, the role of pyrophosphate (PPi) as a source of energy was studied. Izawa, Winget and Good (1966) reported that phloridzin inhibited ATP formation in PSP, in a manner similar to oligomycin inhibition of mitochondrial ATP synthesis. Nobel (1967) noted that phloridzin, while strongly inhibiting PSP had only a marginal effect on the uptake of Ca++ by chloroplasts. It was hoped that the use of this inhibitor would point to a role for high energy states other than for ATP formation. The second inhibitor used in this investigation was quinacrine. This compound was shown by Izawa (1965) and Dilley and Vernon (1966) to inhibit the synthesis of ATP, while allowing high energy intermediates to perform mechanical work in the chloroplasts.

α-Alanine aminotransferase from tomato fruit.

Abstract The α-alanine aminotransferase, l -alanine:2-oxoglutarate aminotransferase EC 2.6.1.2, has been demonstrated in tomato fruit. The enzyme was found in both the supernatant and the mitochondrial preparations; under the experimental conditions used, activity in the mitochondria decreased with development of fruit. The α-alanine aminotransferase was more active than leucine aminotransferase from the same source.

Changes in respiration of mitochondria isolated from cotyledons of ethylene-treated pea seedlings.

Treatment of intact, germinating pea (Pisum sativum L. cv. Homesteader) seedlings with ethylene enhanced the cyanide-resistant respiration of mitochondria isolated from the cotyledons. The level of enhancement depended on the concentration of ethylene. Thus, exposure to 0.9 μl·l-1 of ethylene in air for days 4–6 of germination had little effect on cyanide-resistant respiration, while exposure to 130 μl·l-1 increased it from 10 to 50 nmol O2·min-1·(mg protein)-1. The length of exposure to ethylene also affected the degree of enhancement. According to some literature data, lipoxygenase (EC 1.13.11.12) activity can be mistaken for cyanide-resistant respiration, but in our preparations of purified pea mitochondria ethylene had no effect on lipoxygenase activity, nor did the gas disrupt the outer mitochondrial membrane. Bahr and Bonner plots of respiration in the presence of salicylhydroxamic acid (SHAM) indicated that ethylene did not affect respiration proceeding via the cytochrome pathway. Thus, increases in total respiration in mitochondria from cotyledons of ethylene-treated pea seedlings reflect increases in cyanide-resistant respiration.

Studies on ethylene production by a subcellular fraction from ripening tomatoes—II. : Effects of several inhibitors

Abstract A study was conducted on the effects of several inhibitors on ethylene production by a subcellular fraction from tomatoes. Results disclosed that sulfhydryl groups were essential for the synthesis of the volatile, and that a metalloprotein, or a cation, or both were involved. Carbonyl groups associated with an enzyme or substrate were also shown to be necessary for the biogenesis of the olefin. Results indicated that a considerable portion of the Krebs cycle was not directly involved in the biosynthesis of ethylene. The study also suggested that transamination has a role in the production of the volatile.

Studies on ethylene production by a subcellular fraction from ripening tomatoes—I. : Effects of several substrates, cofactors and cations

Abstract Studies were made of the effects of several substrates, cofactors and cations on the production of ethylene by a subcellular fraction from ripening tomatoes. Compounds found to be effective were ethanol, aspartic acid, glutamic acid, serine, γ-aminobutyric acid, propionic acid and galactose. Those found to exert little influence on ethylene evolution were β-alanine, α-ketoglutaric acid and xylose. Inhibitory to ethylene production were glycolaldehyde, pyruvate, acrylic acid, and glyoxylate. None of the added cations stimulated ethylene evolution, and copper, zinc and cobalt reduced it. Cofactors found to be stimulatory were: reduced α-lipoate, nicotinamide adenine dinucleotide phosphate, and thiamine pyrophosphate. Coenzyme A, reduced glutathione, nicotinamide adenine dinucleotide, bovine serum albumin, flavin adenine dinucleotide, flavin mononucleotide and pyridoxal phosphate, were either inhibitory or had little effect on ethylene production.

Ethylene Analysis — Tricks Played by Plants

The lack of appropriate techniques for working with ethylene has frequently frustrated our efforts to understand ethylene metabolism. While gas chromatography enables good separation of ethylene from other gases, and while sensitive detection systems are now available, there are still many pitfalls and needed improvements in our procedures and capabilities. With knowledge gained from even the presently available techniques, some of which will be highlighted, we can see that some of our old conclusions with respect to ethylene production and action may need re-investigation.