Rosemary Condron

Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase

Disulphide bonds in secreted proteins are considered to be inert because of the oxidizing nature of the extracellular milieu. An exception to this rule is a reductase secreted by tumour cells that reduces disulphide bonds in the serine proteinase plasmin. Reduction of plasmin initiates proteolytic cleavage in the kringle 5 domain and release of the tumour blood vessel inhibitor angiostatin. New blood vessel formation or angiogenesis is critical for tumour expansion and metastasis. Here we show that the plasmin reductase isolated from conditioned medium of fibrosarcoma cells is the glycolytic enzyme phosphoglycerate kinase. Recombinant phosphoglycerate kinase had the same specific activity as the fibrosarcoma-derived protein. Plasma of mice bearing fibrosarcoma tumours contained several-fold more phosphoglycerate kinase, as compared with mice without tumours. Administration of phosphoglycerate kinase to tumour-bearing mice caused an increase in plasma levels of angiostatin, and a decrease in tumour vascularity and rate of tumour growth. Our findings indicate that phosphoglycerate kinase not only functions in glycolysis but is secreted by tumour cells and participates in the angiogenic process as a disulphide reductase.

Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin.

A novel trypsin inhibitor was extracted from the seeds of Cassia fistula by a process successively involving soaking seeds in water, extraction of the seeds in methanol, and extraction of the cell wall material at high ionic strength. The protease inhibitor (PI) was subsequently purified by chromatography on carboxymethylcellulose, gel filtration and reversed phase HPLC (RP-HPLC). Electrospray ionization mass spectrometry (ESMS) of the oxidized from of the PI yielded an average molecular mass of 5458.6+/-0.8 Da. Edman sequencing of the PI yielded a full-length 50 amino acid sequence inferred to contain eight cysteines and with a calculated average molecular mass (fully oxidized form) of 5459.3 Da, in agreement with the observed mass. The C. fistula seed PI is homologous to the family of plant defensins (gamma-thionins), which have four disulfide linkages at highly conserved locations. The C. fistula PI inhibits trypsin (IC(50) 2 µM), and is the first known example of a plant defensin with protease inhibitory activity, suggesting a possible additional function for some members of this class of plant defensive proteins. C. fistula seeds also contain a 9378 Da lipid transfer protein (LTP) homologue, other LTPs, a 7117 Da protein copurifying with PI activity and a 5144 Da defensin which does not inhibit trypsin. The complete sequence of the 5144 Da defensin was determined by Edman sequencing, yielding a calculated average molecular mass (oxidized form) of 5144.1 Da, in agreement with the mass observed by ESMS. The likely trypsin inhibitory residue on the 5459 Da defensin is Lysine-25, the corresponding amino acid being Tyrosine-25 in the homologous 5144 Da defensin that is not a trypsin inhibitor.

Purification and sequencing of a family of wheat lipid transfer protein homologues phosphorylated by plant calcium-dependent protein kinase

Four low molecular weight, basic proteins (WBP1A, WBP1B, WBP2 and WBP3) that are substrates for wheat germ Ca(2+)-dependent protein kinase (CDPK) were purified from wheat germ by a procedure involving batchwise cation exchange on carboxymethylcellulose (CM52), acid precipitation, cation exchange HPLC on an SP5PW column and reverse-phase HPLC on a C18 column. While WBP1A, WBP1B and WBP3 are phosphorylated by wheat germ CDPK exclusively on Ser residues, WBP2 is phosphorylated on both Ser and Thr residues. CDPK-catalysed phosphorylation sites on WBP1A and WBP1B were determined. With all four proteins the phosphorylated form comigrates with non-phosphorylated protein (Mr about 9 kDa) on SDS-PAGE. Average molecular masses of reduced WBP1A, WBP1B, WBP2 and WBP3 measured using electrospray ionisation mass spectrometry (ESMS) are 9389 Da, 9274 Da, 9479 Da and 9467 Da, respectively. The complete amino-acid sequences of WBP1A and WBP1B (determined by Edman sequencing and ESMS of proteolytically derived fragments) and N-terminal sequences of WBP2 and WBP3 are highly homologous to each other and to sequences of low molecular weight, basic plant lipid transfer proteins (LTPs).

The complete primary structure of rat chaperonin 10 reveals a putative βαβ nucleotide-binding domain with homology to p21ras

The first complete amino-acid sequence of a mitochondrial chaperonin 10 is reported. The amino-terminal alanine residue is acetylated, a modification that may be required for the interaction with heptameric chaperonin 60. Part of the sequence constitutes a potential dinucleotide binding motif and is identical with 7 out of 10 residues in the GTP-binding site of p21ras. This similarity may be the structural basis for the recently discovered complex between p21ras and chaperonin 60 in intact cells

Purification, characterization and sequencing of a family of Petunia petal lipid transfer proteins phosphorylated by plant calcium-dependent protein kinase

Abstract A number of small basic proteins (Pet1, Pet2, Pet3, Pet4 and Pet5) were purified to homogeneity from petals of petunia (Petunia hybrida var. Old Glory Blue) by a procedure involving batchwise cation exchange chromatography on carboxymethyl-cellulose (CM52) and cation exchange HPLC on a sulphopropyl-based SP5PW column. Pet1 to Pet5 were identified from extensive N-terminal sequencing as having a high degree of sequence homology to each other and to other plant phospholipid transfer proteins. Pet1, Pet2, Pet4 and Pet5 are phosphorylated by wheat embryo Ca2+-dependent protein kinase (CDPK) whereas Pet3 is a very poor substrate for this enzyme. After tryptic digestion of [32P]phosphoPet1 and [32P]phosphoPet2 phosphorylated by CDPK and reversed phase HPLC-based purification of [32P]phosphopeptides, Edman sequencing of the purified [32P]phosphopeptides revealed major Ser phosphorylation sites of S40 and S70 in the sequences SQAS40TTP and GLPS70TCG in Pet1 and Pet2, respectively. These phosphorylation site sequences differ from the Basic-X-X-Ser(Thr) motif found with many synthetic peptide substrates of plant CDPK but are similar to each other and to phosphorylation sites on some other CDPK substrates involving A G , S T and P residues. The complete primary structure of Pet2 (90 residues) has been determined by Edman sequencing and electrospray ionization mass spectrometry of the native Pet2 and of proteolytically-generated fragments. While Pet2(19–90) and Pet2(32–90) retain activity as CDPK substrates, all smaller fragments tested were inactive. A CDPK is present in Petunia petals. After in vivo labelling of Petunia petals with [32P]phosphate the major labelled protein resolved by SDS-PAGE has a mass of 9.4 ± 0.6 kDa, similar to the Mr of Pet1, Pet2 and Pet3.

Phosphorylation of barley and wheat phospholipid transfer proteins by wheat calcium-dependent protein kinase

Abstract Wheat (Triticum aestivum L.) germ Ca2+-dependent protein kinase (CDPK) phosphorylates a wheat basic protein (WBP) which is a putative phospholipid transfer protein and one of the best protein substrates yet found for wheat CDPK. Reversed phase high performance liquid chromatography (HPLC) of the tryptic digest of [32P]phospho WBP resolved two [32P]phosphopeptide peaks. Edman sequencing of one these established that WBP is phosphorylated by CDPK on S17 within the sequence A13PCISYA. WBP may also be phosphorylated at an additional site. Wheat CDPK also phosphorylates barley (Hordeum vulgare) lipid transfer protein (LTP1) and [32P]phosphoLTP1 exactly comigrates with LTP1 on SDS-PAGE. Unlike wheat WBP, LTP1 is a relatively poor substrate for wheat CDPK and is phosphorylated largely on threonine. Reversed phase high performance liquid chromatography (HPLC) of the tryptic digest of [32P]phosphopeptide resolved one [32P]phosphopeptide and subsequent Edman sequencing of this phosphopeptide showed that LTP1 is phosphorylated on T15 within the sequence K11PCLTYVQ. This phosphorylation site amino acid sequence motif differs from the Basic-X-X-Ser(Thr) motif found for a variety of synthetic peptide substrates of wheat germ CDPK but corresponds to the Pro-Cys-X-Ser/Thr phosphorylation site motif found for the phosphorylation by wheat germ CDPK of WBP, which is a homologue of barley LTP1. The phosphorylation sites determined for WBP and LTP1 are similar structurally and are within precisely aligned homologous sequences.

Phosphorylation of a plant protease inhibitor protein by wheat calcium-dependent protein kinase

Abstract Wheat germ ( Triticum aestivum ) Ca 2+ -dependent protein kinase (CDPK) phosphorylates soybean ( Glycine max ) Bowman-Birk trysin/chymotrypsin inhibitor protein (BBI) and [ 32 P]phospho BBI comigrates with BBI on SDS-PAGE. The phosphorylation of S 34 within the sequence RLNSCHS 34 ACK was determined from isolation of [ 32 P]-phospho-LNSCHSACK from tryptic digests of [ 32 P]-phosphoBBI and from subsequent analysis of the Edman degradation products. Confirmation of S 34 phosphorylation was obtained from similar analysis of phosphopeptides purified from the tryptic digest of 2-bromoethylamine-derivatised [ 32 P]phosphoBBI. This phosphorylation site differs from the Basic-X-X-Ser(Thr) phosphorylation site specificity previously found for synthetic peptide substrates of wheat CDPK. The adjacent S 31 of BBI, which does lie within a Basic-X-X-Ser motif, is not phosphorylated. Bovine serum albumin (BSA) is also phosphorylated by wheat germ CDPK on a site not involving a Basic-X-X-Ser motif, namely on S 468 within the sequence K 464 TPVSEK 470 . The site of phosphorylation of BBI by CDPK is located in an exposed central loop lying between the two anti-parallel β-sheet protease inhibitory domains. Two other CDPK protein substrates have phosphorylation site sequences of either Ser-X-X-Basic or Ser-X-Basic as found for BBI and BSA, respectively, suggesting that a C-terminal side basic residue could be a substrate recognition determinant for plant CDPK on some protein substrates.

Purification and sequencing of napin-like protein small and large chains from Momordica charantia and Ricinus communis seeds and determination of sites phosphorylated by plant Ca2+-dependent protein kinase

The basic protein fraction from seeds of castor bean (Ricinus communis L.) contains 4732 Da and 4603 Da proteins phosphorylated in vitro by plant Ca(2+)-dependent protein kinase (CDPK). These proteins, RS1A and RS1B respectively, were purified by cation-exchange HPLC (SP5PW column) and reverse-phase HPLC (C18 column) and identified as napin-like protein small chains by Edman sequencing and electrospray ionization mass spectrometry (ESMS). The other R. communis 4 kDa small chains (RS2A, RS2B, RS2C and RS2D) are not phosphorylated by CDPK and neither is the corresponding 7332 Da large chain (RL) that forms 1:1 disulfide-linked complexes with RS2(A-D). RS1A/B is one of the best substrates found for plant CDPK (K(m) = 1.8 +/- 0.8 microM). RS2(A-D) (but not RL or RS1A/B) strongly inhibit calmodulin (CaM)-dependent myosin light chain protein kinase (MLCK) (IC50 = 0.25 microM) and inhibit the Ca(2+)-dependent enhancement of dansyl-CaM fluorescence. The basic protein fraction from seeds of bitter melon (Momordica charantia) also contains napin-like proteins that are 1:1 disulfide-linked complexes of a small chain (MS1, MS2, MS3 or MS4) and a large chain (ML). The M. charantia small chains were purified and completely sequenced by Edman degradation and ESMS. M. charantia small chains MS1, MS2, and MS4 (but not MS3) are phosphorylated by CDPK to unit stoichiometry on S21 within the sequence R17SCES21FLR. The R. communis small chain RS1A is phosphorylated on S34 within the sequence R31QSS34SRR. Both of these phosphorylation site motifs are consistent with those found for other plant CDPK substrates.

Affinity-purification and identification of GrpE homologues from mammalian mitochondria

We used affinity chromatography on DnaK columns to identify a mitochondrial GrpE homologue from bovine, porcine and rat liver mitochondria. The 24 kDa GrpE homologue bound specifically to the DnaK column and was not eluted with 1 M KCl but readily with 5 mM ATP. Sequence analysis of the bovine homologue (85 residues) revealed 42% positional identity to mitochondrial GrpEp from S. cerevisiae and about 30% identity to the bacterial counterparts. Thus, GrpE homologues from higher and lower eukaryotes are highly conserved.

Purification and sequencing of yellow mustard seed napin small and large chains that are phosphorylated by plant calcium-dependent protein kinase and are calmodulin antagonists

Abstract A multiplicity of small (S) and large (L) napin subunits were purified from yellow mustard (Sinapis alba L.) seeds by a protocol involving extraction, successive batch-wise cation exchange chromatography on carboxymethylcellulose (CM52), cation exchange HPLC on an SP5PW column and reversed phase HPLC on a C18 column. Initial cation exchange HPLC resolved 4 major zones of proteins (M1, M2, N1 and N2) that can be phosphorylated by plant Ca2+-dependent protein kinase (CDPK). Electrospray ionization mass spectrometry (ESMS) revealed that M1 and M2 are 6 kDa proteins, later identified as γ-thionin-related proteins. ESMS of fractions N1 and N2 revealed the presence of 14.5 kDa proteins identified as napin complexes, each composed of a single small subunit linked to a single large subunit and involving 4 disulphide linkages. The napin complexes (N1A, N1B, N1C, N1D, N2A, N2B and N2C) were disrupted and the constituent small subunits (S1, S2 and S3) and large subunits (L1A, L1B, L1C, L2A, L2B and L2C) were resolved by reversed phase HPLC and precise average molecular masses determined by ESMS. The small and large subunits have average molecular masses of about 4.4 kDa and 10.1 kDa, respectively. The masses of each napin complex can be precisely accounted for from the masses of the constituent subunits. Thus the major complex N2A (14 569 ± 3 Da) is evidently composed of S3 (4434.0 ± 1.5 Da) and L2A (10 142.5 ± 1.5 Da) and involves 4 disulphides (loss of 8.0 Da), the expected mass of S3 + L2A-8H being 14 569 ± 2 Da. The yellow mustard napin large chain L2A is phosphorylated by wheat CDPK on Ser60 within the sequence LQHVIS60RIY. The complete sequence of this and other large (and small) napin subunits were determined from Edman sequencing and/or ESMS data by comparison with published napin sequences. Yellow mustard seed CM52-binding fractions decrease the Ca2+-dependent fluorescence emission of dansyl-CaM and yellow mustard small and large chains inhibit CaM-dependent myosin light chain kinase (MLCK).