Daphne C. Elliott

Nucleotide Biosynthesis from Preformed Purines in Mammalian Cells: Regulatory Mechanisms and Biological Significance

Publisher Summary This chapter discusses that all cells need a balanced supply of nucleotides as precursors of DNA, RNA, and the nucleotide coenzymes involved in group transfer and bioenergetic processes. The study of purine nucleotide biosynthesis does not reveal the ultimate control points of the normal cells economy, but clarifies the picture already emerging of a highly efficient production line using regulation of enzyme activity by metabolites to ensure adequate provision of essential building blocks, with minimum waste of energy or of nutrients. The chapter deals with the catalytic arid regulatory properties of purine-utilization enzymes and includes a comparison of natural and artificial regulators. Except in the special case of nucleotidases, nucleoside phosphorylases and aminopurine hydrolases, the enzymes that make purine fragments available by degradation of DNA, RNA, and nucleotide coenzymes are not discussed here, and the reader is referred to recent, reviews on nucleases. It discusses the availability of purines and purine nucleosides with particular reference to the availability of these compounds in mammalian plasma for utilization by specialized cells. The function of purine-utilization enzymes in the conversion of purine and purine nucleoside antimetabolites into active nucleotide derivatives is discussed, as is the significance of this activation in immunosuppressive and cancer chemotherapy. Recent studies on defects in function or regulation of purine utilization and de novo pathways are also discussed.

Calcium-dependent, phospholipid-activated protein kinase in plants

Abstract A phospholipid-stimulated protein kinase has been demonstrated in plant extracts which is similar to protein kinase C in its behaviour on a DE-52 cellulose column, its substrate specificity and its calcium dependence. Nonspecific enzyme activation has been discounted by the inability of sodium dodecyl sulphate to mimic the effect of phophatidylserine plus diacylglycerol. However the lack of specific phorbol ester binding by the enzyme fractions makes a complete identity with protein kinase C doubtful.

Partial purification and properties of a protein kinase C type enzyme from plants

Partial purification of a protein kinase with a dependence on micromolar concentrations of free calcium has been achieved from seedlings of Amaranthus tricolor. The enzyme has a Mr of 77 600 as determined by gel filtration and 84 500 by SDS-PAGE analysis. Interaction of the enzyme with membranes (inside-out erythrocyte vesicles) is regulated by calcium, a characteristic of animal protein kinase C. Phospholipid and diolein activation of the enzyme is markedly dependent on the phospholipid used and on both calcium and phospholipid concentration. Km values for Ca2+ in the absence of phospholipid was 20–40 μM and in the presence of phosphatidylserine 5–10 μM. Diolein plus phosphatidylserine lowered the Km to < 1.5 μM. The best activation was achieved at 1OOμM calcium with 40μg/ml phosphatidylserine and 8μg/ml diolein. These properties indicate a protein kinase C type enzyme. The plant enzyme reacted with antiserum directed against the regulatory domain of bovine brain protein kinase C in an immunoblot experiment.

Calcium Involvement in Plant Hormone Action

Many hormones and extracellular stimuli in animal systems regulate cell function by inducing an increase in calcium concentration in the cytoplasm. The evidence that plant hormones also work through calcium as a second messenger is reviewed in this paper. Modulation of hormone responses by added calcium, by inhibitors of calcium transport or by calcium ionophores indicates calcium involvement and there are many instances of this phenomenon in plants. Hormone effects on intracellular calcium levels have not been so widely studied, but some effects on calcium transport have been reported. The current model of how the calcium messenger system works is examined with particular reference as to where the gaps are in applying this model to transmembrane signalling in plants.

Phosphatidylserine activation of plant protein kinase C

Abstract The activation of plant protein kinase C by phosphatidylserine is dependent on whether the enzyme has been isolated from the cytosol of plant cells (extracted in a buffer containing EDTA and EGTA), or eluted from a membrane preparation isolated in the presence of calcium. Both preparations of the enzyme react with antiserum directed against animal protein kinase C, although with some differences in the relative amounts of the immunoreactive species.

Measurement of cytoplasmic free calcium in plant protoplasts

Abstract Control and regulation of cytoplasmic free [Ca2+] are prerequisites for Ca2+-mediated modulation of cellular processes. The recent discovery of the series of calcium-sensitive, fluorescent, tetracarboxylate dyes such as quin-2, fura-2, and indo-1 has transformed calcium measurement in animal cells, but so far in plants the use of these dyes has met with limited success. Problems have included difficulties in getting the ester form of the dyes taken up by plant cells, extracellular hydrolysis of the esters, lack of hydrolysis of the ester after loading into cells and compartmentation of the dyes into vacuoles. In this paper various methods of loading indo-I/AM (the acetoxymethyl ester), and free indo-I, into protoplasts have been investigated, together with the effect of these methods on the viability of the protoplasts. Protoplasts can be loaded at pH 5.5 with indo-1/AM but the ester is not converted to a calcium-sensitive form. At pH 4.5 free indo-1 is loaded, but the protoplast membranes become leaky at this pH. At pH 6.8 indo-1/AM is taken up by protoplasts and hydrolysed. Indo-1 formed is located in the cytoplasm and is responsive to changes in cytoplasmic calcium. The free intracellular calcium of unstimulated Amaranthus tricolor protoplasts measured 2–4 h after isolation was determined to be 113 ± 12 nM (n = 4).

Identification of products of protein phosphorylation in T37-transformed cells and comparison with normal cells

Abstract Proteins from crown gall tissue labelled in vivo with [32P]orthophosphate were analysed by SDS-polyacrylamide gel electrophoresis. The major phosphorylated proteins were of 50.6 and 48.3 kDa, with minor bands at 80.1, 73.9, 68, 40.4, 30, 21.5, 20.2 and 15.2 kDa. Partial hydrolysates of total 32P-labelled proteins were analysed in a number of ways. A two-dimensional separation on paper by electrophoresis in pyridine/acetic acid at pH 3.5 followed by chromatography in isobutyric acid/0.5 M ammonia revealed radioactive spots coincident with phosphoserine and phosphothreonine markers and only partially coincident with the phosphotyrosine marker. Two-dimensional electrophoresis at pH 1.9 followed by pH 3.5, however, unequivocally showed the presence of phosphotyrosine after elution of the phosphotyrosine marker. Phosphoserine, phosphothreonine and phosphotyrosine were present in the ratio 89.4:8.5:2.1. This is a much higher level of phosphotyrosine than normally found in animal cells. The three phosphoamino acids were confirmed by chromatography with authentic samples in four solvent systems on cellulose or silica TLC, and by dansylation followed by silica TLC. The radioactive compound running almost coincident with phosphotyrosine on two-way electrophoresis, pH 3.5, followed by chromatography in isobutyric acid/0.5 M ammonia was identified tentatively as uridine 5′-monophosphate on the basis of electrophoretic and chromatographic behaviour. Further experiments to compare normal (growing and non-growing) tobacco callus and T37-transformed cells did not give markedly different ratios of the three phosphoamino acids, although the rapidly-growing normal tobacco (i.e., plus cytokinin) appeared to have a greater abundance of the two minor phosphoamino acids (approx. 2-times). The lack of effect of transformation is in contrast to animal cells where transformation results in a 10-fold increase in the virally affected cells.

Aluminium Inhibits Net 45Ca2+ Uptake by Amaranthus Protoplasts

Summary Calcium ions serve as a second messenger in the transduction of environmental and hormonal signals to the responsive elements of cell metabolism. Effects of Al on the cell Ca2+ homeostasis remain to be elucidated. Short-term net 45Ca2+ uptake by Amaranthus tricolor protoplasts was measured in media prepared to test the influence of various buffers, pH values, Ca2+ and Al concentrations. A modified GEOCHEM program was used to calculate activities of Ca2+ and all monomeric Al species. Net 45Ca2+ uptake was smaller at pH 4.5 compared to uptake at pH 5.5 and increased linearly with an increase in Ca2+ activity in the uptake media from 0.23 to 2.0 mM. Aluminium caused a significant decrease in net 45Ca2+ uptake. This Al-related decrease was less expressed at higher Ca2+ activities. Summed activities of monomeric Al species (Al3+ and hydroxy-Al) were significantly related to the reduction of net 45Ca2+ uptake. The equilibrium constant (Ki) of the reversible association of the Ca2+ membrane-transporter and monomeric Al species was calculated from Dixon plots to be equivalent to 4 and 30 micromolar activity of total monomeric Al for uptake media with pH values of 4.5 and 5.5, respectively. It is suggested that the primary Al effects are generally exerted through reducing inward Ca2+ fluxes across the plasma membrane.

Betacyanin decolourizing enzyme in Amaranthus tricolor seedlings

Abstract The characterization and partial purification of an enzyme from Amaranthus tricolor which decolourizes betacyanin are described. The enzyme occurs in greatest amounts in the roots and in 3.5-4-day-old seedings. Preparation from an acetone powder of roots results in a more active and more stable enzyme than that obtained from crude buffer extraction. The activity is in the 130 000 g supernatant from sucrose-buffer extraction. It has a pH optimum of 3.4 K m towards amaranthin of 3.1 × 10 −6 M and towards betanin of 3.5 × 10 −6 M, and is inhibited by lack of oxygen, and by azide, diethyldithiocarbamate, thiourea, dithiothreitol and cysteine. The product of the reaction has the spectral and electrophoretic properties of betalamic acid. The possibility of enzymic decolourization of betacyanin during acetic acid extraction used for assay of the pigment in the Amaranthus bioassay for cytokinins needs to be recognized.

Differential effects on fusicoccin- and cytokinin- stimulated betacyanin synthesis by ATPase inhibitors and uncouplers

Abstract Data on the concentration/response relationships of fusicoccin (FC) and benzyladenine (BA) in the induction of betacyanin synthesis in Amaranthus seedlings support an additional role for cytokinins quite separate from any effect on potassium transport. This conclusion is reinforced by differential effects of various APTase inhibitors and uncouplers on endogenous, FC-dependent and cytokinin-dependent betacyanin accumulation, and on FC-cytokinin synergism. In particular oligomycin inhibits BA-induced betacyanin synthesis (−34% at 4 μM) but not FC-stimulated synthesis. In the presence of oligomycin, FC exhibits a greater effect on BA-dependent synthesis which is seen as increased BA/FC synergism. A study of the APTases in Amaranthus seedlings shows that this can not be explained in terms of FC reversing the inhibitory effect of oligomycin on mitochondria. Crude plasma membrane APTase is not inhibited by oligomycin. Significant fusicoccin activation of membrane ATPase was not observed. A general conclusion would be that BA-induced betacyanin synthesis is dependent on oligomycin-sensitive energy coupling and FC stimulation is not.