Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.)

Porphobilinogen deaminase catalyzes the condensation of four porphobilinogen monopyrrole units into hydroxymethylbilane, a linear tetrapyrrole necessary for the formation of chlorophyll and heme in higher plant cells. We report the purification to homogeneity of a chloroplast-localized form of the enzyme from pea (Pisum sativum L.) by a novel purification scheme involving dye-ligand affinity chromatography. The purified chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by size-exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of the protein is acidic. The activity of the enzyme shows different levels of sensitivity to divalent cations and is most sensitive to FE2+. The amino terminus of pea enzyme has been obtained by microsequencing and determined to bear little similarity to the amino acid sequences of porphobilinogen deaminases purified from other organisms. Polyclonal antisera elicited against the purified protein has been used to examine the abundance and cellular distribution of the enzyme.     

Sequence specificity of the post-translational proteolytic cleavage of vicilin, a seed storage protein of pea (Pisum sativum L.).

Amino acid sequence data from vicilin of pea (Pisum sativum L.) were compared with predicted sequences from complementary DNA species. The sites of potential post-translational proteolytic cleavage of vicilin precursor polypeptides were located in polar regions of the polypeptide, at acidic or amide residues. Proteolysis did not take place in precursors containing a functionally distinct sequence: neutral residue-hydrophobic residue-basic residue at the cleavage site. Differences between the genomic sequences encoding vicilin thus specify proteolytic cleavage of vicilin precursor polypeptides.     

Products of fatty acid synthesis by a particulate fraction from germinating pea (Pisum sativum L.).

The synthesis of lipids and acyl thioesters was studied in microsomal preparations from germinating pea (Pisum sativum cv. Feltham First) seeds. Under conditions of maximal synthesis (in the presence of exogenous acyl-carrier protein) acyl-acyl-carrier proteins accounted for about half the total incorporation from [14C]malonyl-CoA. Decreasing the concentrations of exogenous acyl-carrier protein lowered the overall synthesis of fatty acids by decreasing, almost exclusively, the radioactivity associated with acyl-acyl-carrier proteins. A time-course experiment showed that acyl-acyl-carrier proteins accumulated most of the radioactive label at the beginning of the incubation but, eventually, the amount of radioactivity in that fraction decreased, while a simultaneous increase in the acyl-CoA and lipid fractions was noticed. Addition of exogenous CoA (1 mM) produced a decrease of total incorporation, but an increase in the radioactivity incorporated into acyl-CoA. The microsomal preparations synthesized saturated fatty acids up to C20, including significant proportions of pentadecanoic acid and heptadecanoic acid. Synthesis of these 'odd-chain' fatty acids only took place in the microsomal fraction. In contrast, when the 18,000g supernatant (containing the microsomal and soluble fractions) was incubated with [14C]malonyl-CoA, the radioactive fatty acid and acyl classes closely resembled the patterns produced by germinating in the presence of [14C]acetate in vivo. The results are discussed in relation to the role of acyl thioesters in the biosynthesis of plant lipids.     

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.     

The major albumin proteins from pea (Pisum sativum L). Purification and some properties.

A scheme is described for the fractionation of pea (Pisum sativum) albumin proteins. By using this scheme, two closely related major albumin proteins have been isolated and purified to homogeneity. The larger protein, designated PMA-L, has Mr approximately 53 000 and consists of two 25 000-Mr subunits, whereas the smaller, PMA-S, has Mr approximately 48 000 and contains two 24 000-Mr subunits. There was no evidence of mixed dimers of the two subunit sizes, despite their close homology as judged by immunological crossreaction, amino acid composition, N-terminal amino acids, tryptic-peptide mapping and CNBr-cleavage products. Both proteins contained significant amounts of sulphur amino acids. The proteins were shown to be located in the soluble cytosol fraction of cotyledon cells and are not significantly degraded on seed germination. Preliminary screening indicates the presence of homologous major albumin proteins in at least three different, though closely related, legume species.     

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     

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.     

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.     

Nucleotide sequence of phenylalanine transfer ribonucleic acid from pea (Pisum sativum, Alaska).

Phenylalanine transfer ribonucleic acid from peas (Pisum sativum, Alaska) was completely digested with beef pancreatic ribonuclease (RNase I) and with ribonuclease T1. The resulting oligonucleotides were compared with those from the corresponding hydrolyses of phenylalanine transfer ribonucleic acid from wheat germ. The structures of both ribonucleic acids appeared to be identical. This report is the first to show that identical structures for the same specific acceptor transfer ribonucleic acid are present in two different plant species.     

The lectin-binding protein from the pea (Pisum sativum); properties and interactions

The lectin-binding protein (lectin binder) from the garden pea (Pisum sativum) was studied. It is a glycoprotein composed of four subunits of about 50 000 Da. Its amino-acid composition and molecular mass differ from those of lectin and of storage proteins. The interaction between lectin and lectin binder is demonstrated and quantified by several different methods and is shown to be specifically sugar-dependent. A biological function of lectin binders and lectins is discussed.