A. Yarwood

Purification and characterization of the major storage proteins of Phaseolus vulgaris seeds, and their intracellular and cotyledonary distribution

Abstract Several extraction and fractionation procedures have been employed to isolate the major storage proteins of mature seeds of Phaseolus vulgaris cv. “Seafarer”; three proteins which were soluble at pH 4,7, and one that was insoluble at that pH were identified. The characteristic subunits of the three pH 4.7 soluble proteins had MWs 50000 and 47000, 32000, and 23000 respectively; those of the pH 4.7 insoluble fractions had MW 60000 and 20000. Amino acid compositions, N -terminal amino acid residues and the presence of carbohydrate in these proteins have been determined. All these proteins occurred in the protein body fraction and their relative amounts were different in the outer and central parts of the cotyledons.

The amino acid sequence and reactive (inhibitory) site of the major trypsin isoinhibitor (DE5) isolated from seeds of the Brazilian Carolina tree (Adenanthera pavonina L.)

Abstract Eight iso-inhibitors of trypsin were isolated from seeds of the Brazilian Carolina tree ( Adenanthera pavonina L.) by precipitation with ammonium sulphate, gel filtration, affinity chromatography on enzymatically inert anhydrotrypsin Sepharose 4B, and separated by ion-exchange chromatography on DEAE-Sepharose. The p I values of the isoinhibitors (DE1-DE8) ranged from 5.10 to 4.40. Each isoinhibitor had an M r of approx. 21 000 and was composed of a large α chain ( M r 16 000) and a smaller β chain ( M r 5000) linked together by a disulphide bond. The complete amino acid sequence of isoinhibitor DE5 (p I 4.75) was deduced by analysis of peptides and fragments derived from the separated α and β chains by digestion with trypsin, chymotrypsin, pepsin, thermolysin, the Staphylococcus aureus V8 proteinase and iodosobenzoic acid. The sequence of the Carolina DE5 isoinhibitor and the location of its reactive (trypsin-inhibitory) peptide bond showed clear homology with the Kunitz-type proteinase inhibitors from soybean, winged bean and a number of other legume seeds.

The complete amino acid sequences of the β1- and β2-subunits of the isolectins LoL1 and LoL11 from seeds of Lathyrus ochrus (L.) DC

The complete amino acid sequences of the β1‐ and β2‐subunits of the isolectins (LoL1 and LoL11) from seeds of Lathyrus ochrus were determined by analysis of peptides derived from the proteins by digestion with trypsin, chymotrypsin, pepsin and the S. aureus V8 protease, as well as fragments produced by cleavage with iodosobenzoic acid. Both β‐subunits consisted of singlepolypeptide chains of 181 amino acids, which differed from one another in only 3 positions. The homology of the Lathyrus ochrus isolectins with the other two‐chain lectins of the tribe Vicieae, and the single‐chain lectins of other tribes of the Leguminosae is discussed.

The amino acid sequences of the α 1 and α 2 subunits of the isoelectins from seeds of Lathyrus ochrus (L) DC

Lathyrus ochrus isoelectin α Subunit Amino acid sequence Homology Legume lectin

Synthesis of saporin gene probes from partial protein sequence data: Use of inosine-oligonucleotides, genomic DNA and the polymerase chain reaction

SummaryA strategy employing the polymerase chain reaction to synthesize gene-specific probes suitable for genomic Southern analyses and for screening genomic libraries is described. The method utilizes partial amino acid sequence data from the protein of interest, genomic DNA and inosine-containing oligonucleotide primers. An example of its application for the isolation of plant gene sequences encoding saporin, a ribosome inactivating protein, is described.

Changing protein synthetic machinery during development of seeds of vicia faba

Abstract Microsomal preparations isolated from seeds at various stages of development have been assayed for amino acid incorporation in vitro, and have also been fractionated on isokinetic sucrose gradients. The changes in protein synthetic activity in vivo are reflected in the composition and activity of the preparations in vitro.

Methionyl-tRNAs and initiation of protein synthesis in Vicia faba (L.)

Abstract Developing seeds of Vicia faba (L.) contain two major and one minor tRNAMet species. Only one of the major species (tRNA1Met) is charged by E. coli enzyme and neither can be formylated. The minor species (tRNA3Met) is charged and formylated by bean or E. coli enzyme. Results of AUG dependent binding, release of methionyl-puromycin, and N-terminal analysis of the products of endogenous messenger, poly-AUG and poly-UG directed incorporation, all implicate tRNA1Met in protein chain initiation; tRNA3Met is possibly the initiator tRNA of cell organelles

Complete amino acid sequences of two trypsin inhibitors from buckwheat seed

The major trypsin isoinhibitors from seed extracts of buckwheat (Fagopyrum esculentum Mönch) were purified by affinity chromatography, anion exchange chromatography, anion exchange HPLC and reversed-phase HPLC, and the complete amino acid sequences of two isoinhibitors, BTI-1 and BTI-2, were established by automated Edman degradation. Each isoinhibitor consists of a single polypeptide chain of 69 amino acids, including two Cys residues. The N-terminal sequence of a third isoform, BTI-3, was also determined. The buckwheat trypsin isoinhibitors exhibit clear sequence similarities with the potato chymotrypsin inhibitor I family of serine proteinase inhibitors.

The amino acid sequence of an atypical single-chain lectin from seeds of Lathyrus sphaericus (Retz)

The major lectin from seeds of Lathyrus spaericus (Retz) was purified by fractional precipitation with (NH4)2SO4, affinity chromatography on Sephadex G‐100, chromatofocusing on PBE 94 and gel filtration on Biogel P‐100 in the presence of 6 M guanidine HCl. The protein was found to be atypical of the lectins found in the Vicieae in that it was a dimer of two identical single polypeptide chains whose monomer molecular mass was estimated to be 27–28 kDa by SDS‐PAGE. The complete amino acid sequence of the monomer was determined by analysis of peptides derived from the protein by digestion with trypsin, chymotrypsin, pepsin, the S.aureus V8 protease and a lysine‐specific protease from Lysobacter enzymogenes, as well as fragments produced by cleavage with iodosobenzoic acid. The polypeptide chain contained 244 amino acids and exhibited overlapping homology with the β (heavy) and α (light) chains of the two‐chain lectins found in other Lathyrus species and in genera belonging to the tribe Vicieae, but was unusual in containing additional amino acids at the N‐terminus and in two other regions (insertions), some deletions, and alterations in 44 previously conserved amino acid positions.

A polyuridylic acid-directed cell-free system from 60 day-old developing seeds of vicia faba

Abstract The components and conditions of an active poly U-directed cell-free system from developing beans have been described. Isokinetic sucrose gradients have been used to show that, in the absence of poly U the membrane-bound ribosomal fraction was labelled, whereas in the presence of poly U a monosome fraction was the most active, with a small amount of radioactivity associated with the polysome area.