Peter D. Shargool

The use of a sulfide ion selective electrode to study O-acetylserine sulfhydrylase from germinating rapeseed

Abstract O-Acetylserine sulfhydrylase was partially purified from germinating seeds of Rape (Brassica napus L. var Target), and a sulfide ion specific electrode used to study the properties of this enzyme. It was demonstrated that the sensitivity of the electrode to sulfide ion concentration was not affected by pH, or by various concentrations of cysteine, sulfate, sulfite, and acetate. Kinetic studies showed that the Km for O-acetyl- l -serine was 1.74 × 10−6 m , while the Km for sulfide was 4.3 × 10−4 m . The implications of the data, together with the methods used to obtain them, are discussed.

The biosynthesis of ornithine from glutamate in higher plant tissues

Abstract The biosynthesis of N-acetyl glutamate and N-acetyl-γ-glutamyl phosphate was investigated using crude enzyme preparations made from tissues of a number of higher plants. The results obtained led to the conclusion that the pathway of ornithine biosynthesis in plants utilizes a pathway which is similar to that found in the non-enteric bacteria, fungi and green algae, but different from that found in Escherichia coli.

Quantitation of the delivery of liposome contents into plant protoplasts

A procedure for the quantitation of the delivery of liposome contents into Catharanthus roseus protoplasts has been developed. The method is based on the uptake of liposome encapsulated methylumbelliferyl beta-D-glucoside and its enzymatic hydrolysis to yield fluorescent methylumbelliferone. Since the free glucoside is not taken up by the protoplasts to a significant extent, the delivery of material in the nanomole range can be measured with ease.

Biotechnological applications of plant cells in culture.

For many workers, the most exciting recent advances in the realm of plant cell biotechnology, center on results obtained from experiments concerned with the genetic engineering of plant cells. Various groups of workers have managed to introduce new genetic material into plant cells, using Ti-plasmids (or modified Ti-plasmids) from Agrobacterium tumefaciens. This genetic material has been expressed (with varying degrees of efficiency), in each case. Thus the way may possibly be coming clear to produce plant cell cultures, or whole plants with entirely new or novel properties. Other areas in which progress has been made, are in the design of media conditions to promote secondary product formation, and in ways of immobilizing plant cells and enzymes, to achieve efficient secondary product formation.

A modified assay system for enzymes involved in N-acetyl group transfer reactions: Its use to study enzymes involved in ornithine biosynthesis in plants

A new method for assaying the enzymes N2-acetyl-L-ornithine:L-glutamate N-acetyltransferase (EC 2.3.1.35) and acetyl-coenzyme A:L-glutamate N-acetyltransferase (EC 2.3.1.1) has been designed. This assay system is based on the separation of N-[14C]acetylglutamate from N-[14C]acetylornithine or [14C]acetyl-coenzyme A by differential absorption of these compounds to DEAE anion-exchange disks. The procedure is simple and sensitive and eliminates the use of ion-exchange resin columns. Using the present method, the inhibition of the enzyme N2-acetyl-L-ornithine:L-glutamate N-acetyltransferase by alpha-methylornithine and N-bromoacetylornithine was studied.

Evidence for the importance of histidine at the active site of argininosuccinate synthetase from soybean.

Studies to determine the role of histidine in catalysis by L-argininosuccinate synthetase (EC 6. 3. 4. 5) were carried out with the enzyme isolated from soybean cell suspension cultures. These experiments utilized analogues of the substrates citrulline and aspartate to investigate substrate binding, and to determine which portion of the molecule were required for binding at the active site of the enzyme. Photooxidation studies using rose bengal were carried out to define the importance of histidine residues for catalysis. These studies suggest that an active site histidine residue has an important role to play in the formation of argininosuccinate by this enzyme.

Effect of the sulfur mutation (Su) on levels of ribulose bisphosphate carboxylase/oxygenase and glutamate synthase activities in tobacco plants

Abstract The heterozygous (Su/su) aurea mutant, the wild-type (su/su) and albino (Su/su) varieties of John Williams Broadleaf tobacco (Nicotiana tabacum L.) were grown as shoot cultures on artificial medium. Leaf extracts of the plants were utilized in determining the specific activities of ribulose 1,5-biphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39) and glutamate synthase (EC 1.4.7.1). The specific activity level of RuBP carboxylase/oxygenase was found to be about 75% lower in the albino variety than in heterozygote or wild type plants, which had approximately similar activities. Levels of glutamate synthase activity were similar in all three types. Glutamate was synthesized at similar rates by chloroplasts isolated from all three varieties. It was found that mitochondria present in crude chloroplast preparations could compensate for the absence of α-ketoglutarate (α-KG) omitted from assay mixtures for the estimation of glutamate production by the chloroplastic glutamate synthase system.