Enzymatic binding of aminoacyl-tRNA and peptide chain elongation by ribosomes and ribosome subunits in pea

In vitro aminoacyl transfer from aminoacyl-tRNA to elongating peptide chains and binding of aminoacyl-tRNA to ribosomes were studied with n     

Inhibition of protein synthesis by products of lipid peroxidation

Effects of lipid peroxidation products on in vivo and in vitro protein synthesis have been studied. Malondialdehyde (MDA), a product, and a routinely used index of lipid peroxidation, inhibits in vivo protein synthesis in the two mosses, Tortula ruralis and Cratoneuron filicinum, and in pea (Pisum sativum) leaf discs. When wheat germ supernatant or poly(A)-rich mRNA of T. ruralis was incubated with MDA its subsequent activity in a cell-free protein-synthesizing system was reduced. When MDA was added directly to the in vitro protein-synthesizing mixture containing moss polyribosomes, the inhibition of amino acid incorporation was small. However, when simultaneous lipid peroxidation was allowed to occur along with in vitro protein synthesis there was a strong inhibition of amino acid incorporation and MDA accumulated in the reaction mixture indicating that products of lipid peroxidation other than, and apparently more toxic than, MDA were involved. It was concluded that lipid peroxidation inhibits protein synthesis probably by releasing toxic products which may react with and inactivate some components of the protein-synthesizing complex.     

Inhibition of chloroplast development by tentoxin

Light-dependent chloroplast development in detached pea shoots was measured in terms of chlorophyll synthesis and the synthesis of Fraction 1 protein. Both synthetic processes were inhibited more than 90% by the fungal metabolite, tentoxin (1 or 10 μg/ml). These results place Pisum sativum in the class of tentoxin-sensitive higher plants. Tentoxin, actinomycin D, lincomycin, D-threo-chloramphenicol and carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) were compared in their ability to inhibit RNA and protein synthesis by isolated pea chloroplasts. Energy for the synthetic reactions was supplied either by light or by added ATP. Only CCCP gave the same pattern of inhibition as tentoxin, i.e. inhibition of both RNA and protein synthesis in the light-driven system but no inhibition in the ATP-driven system. It is concluded that chloroplast developmental processes are inhibited by tentoxin through the inhibition of photophosphorylation.     

Magnesium replacement by polyamines in higher plant cell-free polyphenylalanine synthesis

The characteristics and specific requirements for the formation of polyphenylalanine from Phe-[¹⁴C] in a barley ribosome cell-free incorporation system were detailed. The polyamines spermine, spermidine and putrescine, and the inorganic cations Ca²⁺, Ba²⁺ and Mn²⁺ demonstrated different capabilities for replacing the Mg²⁺ requirement in the incorporation system. Spermine was extremely efficient in this respect, followed by spermidine; all of the cations tested showed discrete concentration ranges of effectiveness. The data supported the suggestion that, at least to a certain extent, the cation requirement for protein syntheis may be non-specific.     

Effect of phenolics on plant amine oxidases

Phenolic compounds at low concentrations decrease pea diamine oxidase activity without affecting growth, but they have no effect on barley polyamine oxidase in spite of a decrease in growth.     

De novo synthesis of acetylcholinesterase in roots of Pisum sativum

Pisum sativum seeds contain a conserved acetylcholinesterase (AChE) which is active during the early stages of germination. The enzyme activity soon disappears and reappears after 72 hr of germination. A protein devoid of catalytic ability, but exhibiting similar chromatographic and electrophoretic properties as the active AChE, could be detected after 24 hr of germination. The pattern of incorporation of labelled amino acids into AChE and the influence of cycloheximide revealed that the AChE found in the roots from 72 hr onwards was entirely new. During this period of growth, the AChE protein accounts for 4–10% of the total proteins in the root tissue.     

Protein synthesis in isolated etioplasts after light stimulation

Grains of Zea mayswere germinated in the dark for 5 days. Etioplasts were then isolated in the dark and exposed to light in the presence of labelled amino acids and various inhibitors. After periods of incorporation, either in the dark or light, proteins were isolated and then examined with the aid of polyacrylamide gel electrophoresis. The highest specific activity of incorporation was found in the lamellar protein fraction. The use of inhibitors enabled the specific products of etioplast incorporation to be identified on the gels. Analyses of radioactivity in protein bands indicate that the plastid is capable of responding to light in vitro in at least two ways: (1) by an increase in the rate of protein synthesis; and (2) by a reproducible control of the various proteins synthesized either in the dark or light, which resulted in the ‘turning off’ of some proteins synthesized in the dark, and the subsequent initiation of the synthesis of others, in response to light. The results presented inthis study indicate that the plastid in vitro is capable of a rather complex response mechanism when subjected to environmental change, such as light stimulation. This suggests that the plastid is capable of a great degree of autonomy, at least when necessary, and is possibly more independent of nuclear control than heretofore suggested in the literature.     

The role of Coenzyme A in lipid synthesis by a particulate fraction from germinating peas

The effect of CoA on fatty acid synthesis by the microsomal fraction from germinating pea (Pisum sativum) was examined. Increasing concentrations of CoA progressively decreased total fatty acid synthesis from [¹⁴C]malonyl-CoA. However, the synthesis of very long chain fatty acids was relatively unaffected so that their proportion in the reaction products increased. Other CoA-esters also decreased total fatty acid synthesis while increasing the relative accumulation of radioactivity in very long chain fatty acids. The addition of CoA also altered the distribution of newly synthesized fatty acids in different lipid fractions. Complex lipid labelling was relatively increased while that of acyl-acyl carrier proteins was decreased. Very long chain fatty acids accumulated in lipids rather than thioesters. The role of CoA in controlling fatty acid synthesis in the pea microsomal fraction is discussed.     

Protein synthesis and changes in nucleic acids during grain development of Sorghum

Changes in DNA, RNA, nitrogen, nucleotide composition and in vitro incorporation of leucine/lysine by polysomes have been studied during sorghum grain development. Both DNA, RNA and protein content increased substantially during grain development. Although RNase activity increased, it did not affect RNA accumulation. Minor changes in the nucleotide composition of rRNA and sRNA were observed during grain development. In vitro incorporation of leucine and lysine by polysomes indicate qualitative change in the mRNA during later stages of grain development and the substantial accumulation of proteins during this period ultimately results in accumulation of proteins rich in leucine and poor in lysine.     

L-acetylcarnitine, a substrate for chloroplast fatty acid synthesis

Abstract. H¹⁴CO₃ was not incorporated into fatty acids by isolated pea leaf chloroplasts, which, therefore, do not possess a self-contained pathway for the synthesis of fatty acids from early intermediates of the Calvin cycle. Citrate, pyruvate, acetate and L-acetylcarnitine were all shown to act as sources of acetyl groups for fatty acid synthesis by pea leaf chloroplasts. L-acetylcarnitine was the best substrate, being incorporated into fatty acids at rates that were at least five-fold higher than those achieved with the other substrates. Citrate was incorporated into fatty acids at the lowest rate, followed by pyruvate, with acetate being incorporated at the second highest rate of all. When the isolated chloroplasts were ruptured, an inhibition of L-acetylcarnitine incorporation into fatty acids was noted, whilst acetate incorporation remained unaffected. L-acetylcarnitine also increased the ratio of monoenoic: saturated fatty acids synthesized, compared with a 1:1 ratio observed when citrate, pyruvate and acetate were supplied as substrates. It is suggested that L-carnitine and carnitine acyltransferases play a central role in plant acyl CoA metabolism by facilitating the transfer of activated acyl groups across membranes (acyl CoA barriers).     

Polyamine oxidation by enzymes from Hordeum vulgare and Pisum sativum seedlings

The properties of the amine oxidases of barley leaves and pea seedling cotyledons have been compared using a colorimetric assay in which the hydrogen p     

Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6- dinitroanilino)-N-methylpropionamide

Selective effects of lincomysin and cycloheximide in detached shoots of Pisum sativum on the synthesis of photosystem I and II proteins, and a chloroplast membrane protein of molecular weight 32000, confirm results obtained from studies of protein synthesis by isolated chloroplasts. A model is proposed in which one role of chloroplast ribosomes is to synthesize membrane proteins required for the immobilization of chloroplast components, such as photosystem I protein, which are synthesized by cytoplasmic ribosomes. 2-(4-Methyl-2,6-dinitroanilino)-N-methylpropionamide rapidly inhibits the synthesis of both the large and small subunits of Fraction I protein in greening detached pea shoots. This observation can be reconciled with the site of synthesis of the large subunit being in the chloroplast by a model which proposes that the small subunit is a positive initiation factor for the synthesis or translation of the messenger RNA for the large subunit.     

Confirmation of the structures of the phytoalexins lathodoratin and methyl-lathodoratin by synthesis

The two chromone phytoalexins were synthesized by condensing 2,4,6-trihydroxybutyrophenone with dimethylformamide.     

Isoxazolin-5-one derivatives in Lathyrus odoratus during development and growth

Variations in the concentrations of isoxazolin-5-ones and of some non-protein amino acids in the dry seeds, seedlings and various parts of mature Lathyrus odoratus plants were examined. The lathyrogenic compounds α-amino-γ-(isoxazolin-5-on-2-yl)-butyric acid, 2-cyanoethyl-isoxazolin -5-one and γ-glutamyl-β-aminopropionitrile were major products during development and growth.     

The synthesis of glutamate and the control of glutamate dehydrogenase in pea mitochondria

The synthesis of glutamate from α-oxoglutarate and NH₄⁺ by pea seedling mitochondria has been demonstrated under certain defined but non-physiological conditions. Malate acts as a hydrogen donor for the synthesis of glutamate but isocitrate is more effective, whilst succinate, in the presence or absence of ATP, is a poor donor of hydrogen. Glutamate dehydrogenase has been purified from pea mitochondria and from the cytosol. The similarities between the two preparations are interpreted to mean that the soluble glutamate dehydrogenase is released from the mitochondria during isolation. The kinetics of the mitochondrial enzyme and the effect of various metabolites on its activity have been examined. The results are discussed in relation to the proposed role of this enzyme and it is suggested that the ratio NADH-NAD⁺ may play a role in the control of glutamate metabolism.     

Amine levels in Lathyrus sativus seedlings during development

In growing Lathyrus sativus seedlings, the levels of DNA, RNA and protein markedly decreased in the cotyledons and progressively increased in the embryo-axis. In cotyledons, spermidine and spermine contents were substantially reduced while those of agmatine and putrescine were sharply increased. By contrast the embryo-axis progressively accumulated relatively larger amounts of agmatine, homoagmatine. putrescine, cadaverine, spermidine and spermine in parallel with similar changes in its DNA, RNA and protein content. While the cotyledons contained ca 50% of the total agmatine and putrescine present in the plant embryo by day 10, the embryo-axis, though representing less than 20% of the dry wt, contained 90 and 75% of total cadaverine and homoagmatine respectively of the seedlings. Spermidine and spermine levels of this tissue were also comparatively higher, being of the order of 80 and 50% respectively of the total. The root and shoot portions of the embryo-axis also exhibited a similar relationship between changes in DNA, RNA and protein and all the above amines during development. However, the polyamine content of the shoots was relatively higher than those of the roots during the growth period.     

Changes in protein fractions and leucine-[14C] incorporation during Sorghum grain development

Incorporation of leucine and changes in different protein fractions have been studied during Sorghum grain development. Most of the label from the injected leucine-[¹⁴C] was found in glutelin and residue fraction towards later stages of maturity. The label in albumin, globulin and prolamin decreased with a concomitant increase in label in glutelin and residue proteins. The concentration of lysine, aspartic acid and glycine decreased while that of leucine, proline, alanine, tyrosine, phenylalanine, and cystine increased during grain development. Increase in serine, methionine, valine and isoleucine was only marginal. The proportion of glutamic acid was high at all stages of grain development. Glutelin fraction resolved into two peaks on gel chromatography, only one of which with higher MW was labelled, while in albumin both the peaks were found to be labelled. Tannin content also increased during grain development.     

Biochemical changes during seed development in sorghum (Sorghum bicolor)

Grain dry weight accumulation, soluble sugars, starch, protein, fat and ash contents were studied in developing grains of eight sorghum cultivars. The     

Protein and free amino acids in a high lysine maize double mutant

A comparative study of free amino acids and protein fractions of normal with a double mutant (su₁ o₂) was made, during endosperm development in segregating ears of a maize synthetic. Zein content showed striking differences in the two genotypes, being 7.7 and 6 times greater in the normal endosperm at 24 and 47 days after pollination respectively. This observed decrease in zein synthesis, coded by sugary-1/opaque-2 genes, causes an accumulation of alanine, glutamic and aspartic acids, glutamine and asparagine in the high lysine endosperm mutant.