Lipase-induced alterations of fatty acid synthesis by subcellular fractions from germinating pea (Pisum sativum L.).

1. The effect of exogenous lipases on fatty acid synthesis from [14C]malonyl-CoA by the microsomal and soluble fractions from germinating peas was studied. 2. Addition of phospholipase A2 or the lipase from Rhizopus arrhizus had no effect on total fatty acid synthesis by the soluble fraction but caused severe inhibition of that by the microsomal fraction. 3. The addition of enzymes with phospholipase activity particularly inhibited the microsomal stearate elongase. 4. Control studies indicated that the phospholipase-induced inhibition of fatty acid synthesis was due to the location of fatty acid synthetase, palmitate elongase and stearate elongase on the outside of the microsomal vesicles. 5. Experiments with a trypsin-like proteinase showed that approximately half the microsomal fatty acid synthesis was resistant to proteolysis. 6. Although addition of exogenous phospholipases had no effect on total fatty acid synthesis by the soluble fraction, it did increase alpha-hydroxylation of newly-formed palmitate and stearate. 7. The results provide further evidence for differences between the soluble and particulate fatty acid synthetase and palmitate elongase activities of germinating pea.     

Fatty acid elongation by a particulate fraction from germinating pea.

Rats received two injections (each 2.6 mg/kg body wt.) of CdCl2, and the kidneys were removed 24 h later. Postmicrosomal supernatant fractions of the homogenized kidneys were used as a source of elongation factors 1 and 2 in assays for [14C]phenylalanyl-tRNA binding to ribosomes and for peptide-bond synthesis. After purification of these preparations by precipitation with (NH4)2SO4 and gel filtration on Sephadex G-200 and G-100, elongation factor 1 activity was significantly increased. A significant increase in the activity of purified elongation factor 2 was also found. The results are discussed in relation to the reported effects of CdCl2 and of HgCl2 on renal tissue.     

Age-dependent variation in membrane lipid synthesis in leaves of garden pea (Pisum sativum L.).

To study membrane lipid synthesis during the life-span of a dicotyledon leaf, the second oldest leaf of 10-40-d-old plants of garden pea (Pisum sativum L.) was labelled with [1-(14)C]acetate and the distribution of radioactivity between the major membrane lipids was followed for 3 d. In the expanding second oldest leaf of 10-d-old plants, acetate was primarily allocated into phosphatidylcholine (PC) during the first 4 h of labelling. During the following 3 d, labelling of PC decreased and monogalactosyldiacylglycerol (MGDG) became the most radioactive lipid. In the fully expanded second oldest leaf of older plants, acetate was predominantly allocated into phosphatidylglycerol (PG), which remained the major radiolabelled lipid during the 3 d studied. The proportion of radioactivity recovered in MGDG decreased with increasing plant age up to 20 d, suggesting that, in expanded leaves, MGDG is more stable and requires renewal to a lower extent than PG. When the second oldest leaf approached senescence, labelling of MGDG again increased, indicating an increased need for thylakoid repair. The proportion of acetate allocated into phosphatidylethanolamine and free sterols was largest in leaves of 18-26-d-old plants and in the youngest leaves, respectively. Thus, these results demonstrate that the distribution of newly synthesized fatty acids between acyl lipid synthesis in the chloroplast and extraplastidial membranes strongly varies with leaf age, as do the proportion utilized for sterol synthesis. The findings emphasize the importance of defining the developmental stage of the leaf material used when performing studies on leaf lipid metabolism.     

The post-translational proteolysis of the subunits of vicilin from pea (Pisum sativum L.).

Tryptic-peptide profiles and amino acid sequencing of purified pea (Pisum sativum L.) vicilin subunits were used to show that their sequences were interrelated. Comparison with the nucleotide sequence of a cloned vicilin complementary DNA (mRNA) showed that all vicilin subunits could be derived from 50 000-Mr precursors containing up to two sites for post-translational proteolytic cleavage, and allowed these subunits to be located relative to the precursor.     

Purification, properties and amino acid sequence of a low-Mr abundant seed protein from pea (Pisum sativum L.).

The seeds of pea (Pisum sativum L.) contain several proteins in the albumin solubility fraction that are significant components of total cotyledonary protein (5-10%) and are accumulated in developing seeds concurrently with storage-protein synthesis. One of these proteins, of low Mr and designated 'Psa LA', has been purified, characterized and sequenced. Psa LA has an Mr of 11000 and contains polypeptides of Mr 6000, suggesting that the protein molecules are dimeric. The amino acid sequence contains 54 residues, with a high content (10/54) of asparagine/aspartate. It has no inhibitory action towards trypsin or chymotrypsin, and is distinct from the inhibitors of those enzymes found in pea seeds, nor does it inhibit hog pancreatic alpha-amylase. The protein contains no methionine, but significant amounts of cysteine (four residues per polypeptide), suggesting a possible role as a sulphur storage protein. However, its sequence is not homologous with low-Mr (2S) storage proteins from castor bean (Ricinus communis) or rape (Brassica napus). Psa LA therefore represents a new type of low-Mr seed protein.     

Fatty acid biosynthesis by a particulate preparation from germinating pea

1. Fatty acid synthesis was studied in microsomal preparations from germinating pea (Pisum sativum). 2. The preparations synthesized a mixture of saturated fatty acids up to a chain length of C(24) from [(14)C]malonyl-CoA. 3. Whereas hexadecanoic acid was made de novo, octadecanoic acid and icosanoic acid were synthesized by elongation. 4. The products formed during [(14)C]malonyl-CoA incubation were analysed, and unesterified fatty acids and polar lipids were found to be major products. [(14)C]Palmitic acid represented a high percentage of the acyl-carrier protein esters, whereas (14)C-labelled very-long-chain fatty acids were mainly present as unesterified fatty acids. CoA esters were minor products. 5. The addition of exogenous lipids to the incubation system usually resulted in stimulation of [(14)C]malonyl-CoA incorporation into fatty acids. The greatest stimulation was obtained with dipalmitoyl phosphatidylcholine. Both exogenous palmitic acid and dipalmitoyl phosphatidylcholine increased the amount of [(14)C]-stearic acid synthesized, relative to [(14)C]palmitic acid. Addition of stearic acid increased the amount of [(14)C]icosanoic acid formed. 6. [(14)C]Stearic acid was elongated more effectively to icosanoic acid than [(14)C]stearoyl-CoA, and its conversion was not decreased by addition of unlabelled stearoyl-CoA. 7. Incorporation of [(14)C]malonyl-CoA into fatty acids was markedly decreased by iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid). Palmitate elongation was sensitive to arsenite addition, and stearate elongation to the presence of Triton X-100 or fluoride. The action of fluoride was not, apparently, due to chelation. 8. The microsomal preparations differed from soluble fractions from germinating pea in (a) synthesizing very-long-chain fatty acids, (b) not utilizing exogenous palmitate-acyl-carrier protein as a substrate for palmitate elongation and (c) having fatty acid synthesis stimulated by the addition of certain complex lipids.     

Fatty acid metabolism by a particulate fraction from germinating peas

The incorporation of fatty acids into lipid fractions was studied using a high-speed particulate fraction from germinating peas (Pisum sativum cv Feltham First). The acids were incorporated principally into the acyl-CoA and polar lipid fractions, with unsaturated fatty acids labelling the latter fraction at higher rates than saturated substrates. α-Hydroxylation also occurred. Oleate from oleoyl-CoA or oleoyl-acyl carrier protein was incorporated into polar lipids faster than from ammonium oleate. The incorporation of fatty acids into polar lipids was dependent on the presence of CoA and ATP in the medium and on the consequent generation of acyl-CoA's. Time-course studies and experiments when two fatty acid substrates were added consecutively confirmed the role of acyl-CoA's in the transfer of acyl groups to phospholipids. Although CoA was necessary when acyl-CoA's had to be generated, high concentrations were found to inhibit the rate ofacyl transfer. The results are discussed in terms ofthe ‘witching mechanism’ for controlling the fate of fatty acids in the plant cell.     

The control of CTP:choline-phosphate cytidylyltransferase activity in pea (Pisum sativum L.).

Several possible control mechanisms for CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) activity in pea (Pisum sativum L.) stems were investigated. Indol-3-ylacetic acid (IAA) treatment of the pea stems decreased total cytidylyltransferase activity but did not affect its subcellular distribution. Oleate (2 mM) caused some stimulation of enzyme activity by release of activity from the microsomal fraction into the cytosol, but neither phosphatidylglycerol nor monoacyl phosphatidylethanolamine had an effect on activity or subcellular distribution. A decrease in soluble cytidylyltransferase protein concentrations was found in IAA-treated pea stems, but this was not sufficient to account for all of the decrease in cytidylyltransferase activity. A 50% inhibition of enzyme activity could be obtained with 0.2 mM-CMP, which indicated possible allosteric regulation. Similar inhibition was obtained with 1.5 mM-ATP, but other nucleotides had no effect. The cytidylyltransferase enzyme protein was not directly phosphorylated, and the inhibition with 1.5 mM-ATP occurred with the purified enzyme, thus excluding an obligatory mediation via a modulator protein. The results indicate that the cytosolic form of cytidylyltransferase is the most important in pea stem tissue and that the decrease in cytidylyltransferase activity in IAA-treated material appears to be brought about by several methods.     

The major albumin protein from pea (Pisum sativum L.)

The major albumin protein in storage parenchyma tissue of developing peas has been localised at an ultrastructural level by immunocytochemistry. Tissue was fixed in buffered aldehyde and embedded in LR White resin which was polymerised by addition of catalyst. Sections were labelled by the indirect method of absorption of Protein A-gold to specifically bound antibodies. This method gives high levels of specific labelling on sections which retain good ultrastructural preservation and have high contrast after conventional staining. The albumin is located throughout the cytoplasm although no labelling was found associated with the endoplasmic reticulum, Golgi apparatus, vacuoles-protein bodies or other organelles.Abbreviation PMA pea major albumin protein     

Biosynthesis of alkanes by particulate and solubilized enzyme preparations from pea leaves (Pisum sativum)

Enzymatic activity responsible for the conversion of fatty acids to alkanes catalyzed by pea leaf homogenate was found to be mainly in the microsomal fraction. This particulate preparation catalyzed alkane formation from n-C18, n-C22, and n-C24 acids at rates comparable to that observed with n-C32 acid with O2 and ascorbate as required cofactors. In each case the major alkane contained two carbon atoms less than the precursor acid. Since the preparation also catalyzed alpha-oxidation, it was suspected that some alpha-oxidation intermediate, with one less carbon atom than the substrate acid, might lose another carbon to generate the alkane. Thin-layer and radio-gas-liquid chromatographic analysis of the products generated from [U-14C]stearic acid by the particulate preparation after different periods of incubation showed that, at all time periods, alpha-hydroxy C18 acid, C17 aldehyde, and C17 acid were the major products. Since C16 alkane was the major product even after short periods of reaction, the C17 aldehyde might have been the immediate precursor of the alkane. Exogenous labeled C18 and C24 aldehyde were converted to alkanes. The alkane-synthesizing activity was solubilized from the microsomal preparation using Triton X-100. The solubilized preparation was retarded in a Sepharose 6-B column, but the hydrocarbon-forming activity was not resolved from alpha-oxidation. The solubilized preparation produced alkane with two carbon atoms less than the parent acid in a time- and protein-dependent manner. The soluble preparation also required O2 and ascorbate and, like the microsomal preparation, was inhibited by dithioerythritol and metal ion chelating agents.     

Aminolevulinic acid dehydratase in pea (Pisum sativum L.). Identification of an unusual metal-binding domain in the plant enzyme

Aminolevulinic acid dehydratase (ALA dehydratase) catalyzes the second step of tetrapyrrole synthesis leading to the formation of heme and chlorophyll in higher plant cells. Antibodies elicited against spinach leaf ALA dehydratase were used to immunoscreen lambda gt11 cDNA libraries constructed from etiolated pea (Pisum sativum L.) leaf poly(A)+ RNAs. A set of overlapping cDNAs was characterized that encode the pea enzyme. The predicted amino acid sequence of the pea ALA dehydratase is similar to those reported for other eukaryotic and prokaryotic ALA dehydratases. The pea enzyme has an active site domain centered on lysine that is highly conserved in comparison to other known ALA dehydratases. Consistent with the previously reported requirement of Mg2+ for catalytic activity by plant ALA dehydratases, the pea enzyme lacks the characteristic Zn(2+)-binding domain present in other eukaryotic ALA dehydratases, but contains a distinctive metal ligand-binding domain based upon aspartate. Northern blot analyses demonstrated that ALA dehydratase mRNA is present in leaves, stems, and to a lesser extent in roots. Steady state levels of mRNA encoding ALA dehydratase exhibit little or no change during light-induced greening.     

The purification and characterization of a third storage protein (convicilin) from the seeds of pea (Pisum sativum L.).

A third storage protein, distinct from legumin and vicilin, has been purified from the seeds of pea (Pisum sativum L.). This protein has been named 'convicilin' and is present in protein bodies isolated from pea seeds. Convicilin has a subunit mol.wt. of 71 000 and a mol.wt. in its native form of 290 000. Convicilin is antigenically dissimilar to legumin, but gives a reaction of identity with vicilin when tested against antibodies raised against both proteins. However, convicilin contains no vicilin subunits and may be clearly separated from vicilin by non-dissociating techniques. Unlike vicilin, convicilin does not interact with concanavalin A, and contains insignificant amounts of carbohydrates. Limited heterogeneity, as shown by isoelectric focusing, N-terminal analysis, and CNBr cleavage, is present in convicilin isolated from a single pea variety; genetic variation of the protein between pea lines has also been observed.     

Biochemical changes associated with cadmium and copper stress in germinating pea seeds (Pisum sativum L.)

Seeds of pea (Pisum sativum L.) were germinated for four days over two sheets of filter paper moistened with H2O (control) and 5 mM Cd(NO3)2 or CuSO4 (treated). The relationship between heavy-metal stress and breakdown of storage compounds was studied. Germination rate and growth of radicle decreased, while the water content in stressed seeds remained around the control values. Cotyledons changed their biochemical constituents: disorders in the contents of micronutrients (Fe, Mn, Zn), free amino acids and soluble sugars were found. Decline of alpha-amylase activity as well as acid phosphatase were also observed, whereas beta-amylase and alkaline phosphatase ones were not modified by heavy-metal treatments. These results suggest that the inhibition of seed germinations after exposure to cadmium or copper is not the consequence of starvation in water uptake by seed tissues, but may be due to a failure in the reserve mobilization process from cotyledons.