Barminomycin functions as a potent pre-activated analogue of Adriamycin.

The anthracycline Adriamycin is known to form adducts with DNA, but requires prior activation by formaldehyde. In contrast, the anthracycline barminomycin is also able to form adducts with DNA, but does not require activation by formaldehyde. Barminomycin, therefore, appears to function as a pre-activated form of Adriamycin. The DNA adducts formed by both anthracyclines are bound covalently to only one strand of DNA, but both also stabilise duplex DNA sufficiently that they can be detected as virtual interstrand crosslinks in heat denaturation electrophoretic crosslinking assays. The barminomycin-DNA adducts form extremely rapidly with DNA, and at exceedingly low concentrations (approximately 50-fold lower than with Adriamycin in the presence of excess formaldehyde), both characteristics consistent with barminomycin being in a pre-activated state, hence, undergoing a bimolecular reaction with DNA compared with the trimolecular reaction (drug, formaldehyde and DNA) required with Adriamycin. Surprisingly, barminomycin-DNA adducts are substantially more stable (essentially irreversible) than Adriamycin-DNA adducts (half life of approximately 25 h at 37 degrees C). Due to this understanding of the reactivity of barminomycin and its exceptional cytotoxicity (1000-fold more cytotoxic than Adriamycin), detailed structural studies of barminomycin-DNA adducts are now warranted, both in vitro and in tumour cells.

O-glycosylation of insulin-like growth factor (IGF) binding protein-6 maintains high IGF-II binding affinity by decreasing binding to glycosaminoglycans and susceptibility to proteolysis.

Insulin-like growth factor binding protein-6 (IGFBP-6) is an O-linked glycoprotein which specifically inhibits insulin-like growth factor (IGF)-II actions. The effects of O-glycosylation of IGFBP-6 on binding to glycosaminoglycans and proteolysis, both of which reduce the IGF binding affinity of other IGFBPs were studied. Binding of recombinant human nonglycosylated (n-g) IGFBP-6 to a range of glycosaminoglycans in vitro was approximately threefold greater than that of glycosylated (g) IGFBP-6. When bound to glycosaminoglycans, IGFBP-6 had approximately 10-fold reduced binding affinity for IGF-II. Exogenously added n-gIGFBP-6 but not gIGFBP-6 also bound to partially purified rat PC12 phaeochromocytoma membranes. Binding of n-gIGFBP-6 was inhibited by increasing salt concentrations, which is typical of glycosaminoglycan interactions. O-glycosylation also protected human IGFBP-6 from proteolysis by chymotrypsin and trypsin. Proteolysis decreased the binding affinity of IGFBP-6 for IGF-II, even with a relatively small reduction in apparent molecular mass as observed with chymotrypsin. Analysis by ESI-MS of IGFBP-6 following limited chymotryptic digestion showed that a 4.5-kDa C-terminal peptide was removed and peptide bonds involved in the putative high affinity IGF binding site were cleaved. The truncated, multiply cleaved IGFBP-6 remained held together by disulphide bonds. In contrast, trypsin cleaved IGFBP-6 in the mid-region of the molecule, resulting in a 16-kDa C-terminal peptide which did not bind IGF-II. These results indicate that O-glycosylation inhibits binding of IGFBP-6 to glycosaminoglycans and cell membranes and inhibits its proteolysis, thereby maintaining IGFBP-6 in a high-affinity, soluble form and so contributing to its inhibition of IGF-II actions.

Biochemical analysis and crystallisation of Fc gamma RIIa, the low affinity receptor for IgG.

Fc gamma RIIa is one of a family of specific cell surface receptors for immunoglobulin. Fc gamma RIIa, which binds immune complexes of certain IgG isotypes, plays important roles in immune homeostasis. However, the precise characteristics of IgG binding and three-dimensional structure of Fc gamma RIIa have not been reported. This study describes the affinity of the Fc gamma RIIa:IgG interaction as well as biochemical characterisation of recombinant Fc gamma RIIa that has been used to generate high quality crystals. Equilibrium binding analysis of the Fc gamma RII:IgG interaction found, IgG3 binds with an affinity of K(D) = 0.6 microM, as expected. Unlike other Fc gamma R, IgG4 also bound to Fc gamma RIIa, K(D) = 3 microM, clearly establishing Fc gamma RIIa as an IgG4 receptor. Biochemical analysis of mammalian and insect cell derived Fc gamma RIIa established the genuine N-terminus with Q being the first amino acid in the sequence Q, A, A, A, P... extending the N-terminus further than previously thought. Furthermore, both potential N-linked glycosylation sites are occupied. Electrospray ionisation mass spectrometry (ESMS) indicate that the N-glycans of baculovirus derived Fc gamma RIIa are core mannose oligosaccharide side chains. Finally, we describe the first crystallisation of diffraction quality crystals of soluble Fc gamma RIIa. Orthorhombic crystals diffract X-rays beyond 2.1 A resolution in the space group P2(1)2(1)2 with cell dimensions a = 78.8 A, b = 100.5 A, c = 27.8 A. This marks a significant advance towards understanding the three-dimensional structure of Fc gamma RIIa and related FcR proteins that share high amino acid identity with Fc gamma RIIa.

Binding of pepsinogen to the 78 kDa gastrin binding protein.

An endogenous ligand of the 78 kDa gastrin-binding protein (GBP) has been purified from detergent extracts of porcine gastric mucosal membranes by ion exchange chromatography and preparative gel electrophoresis. The ligand bound to the GBP with high affinity (mean IC50 value of 0.31+/-0.09 microgram/ml, or 8 nM), as assessed by inhibition of cross-linking of iodinated gastrin2,17 to the GBP. Both the N- and C-terminal halves of the GBP, which had been expressed individually as glutathione-S-transferase fusion proteins in Escherichia coli, and purified on glutathione-agarose beads, bound the ligand. Two peptides derived from the ligand were purified by reversed-phase high-performance liquid chromatography (HPLC), and characterised by mass spectrometry and Edman sequencing. The peptides were 97% and 100% identical, respectively, to amino acids 119-157 and 199-219 of porcine pepsinogen A. Commercial samples of pepsinogen also bound to the GBP, with a mean IC50 value of 3.9+/-1. 2 micrograms/ml (100 nM). We conclude that the ligand is closely related, but not identical, to pepsinogen A.

The N-terminal disulfide linkages of human insulin-like growth factor-binding protein-6 (hIGFBP-6) and hIGFBP-1 are different as determined by mass spectrometry.

The actions of insulin-like growth factors (IGFs) are modulated by a family of six high affinity binding proteins (IGFBPs 1-6). IGFBP-6 differs from other IGFBPs in having the highest affinity for IGF-II and in binding IGF-I with 20-100-fold lower affinity. IGFBPs 1-5 contain 18 conserved cysteines, but human IGFBP-6 lacks 2 of the 12 N-terminal cysteines. The complete disulfide linkages of IGFBP-6 were determined using electrospray ionization mass spectrometry of purified tryptic peptide complexes digested with combinations of chymotrypsin, thermolysin, and endoproteinase Glu-C. Numbering IGFBP-6 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys2, Cys3-Cys4, and Cys5-Cys6. The next two linkages are Cys7-Cys9 and Cys8-Cys10, which are analogous to those previously determined for IGFBP-3 and IGFBP-5. The C-terminal linkages are Cys11-Cys12, Cys13-Cys14, and Cys15-Cys16, analogous to those previously determined for IGFBP-2. Disulfide linkages of IGFBP-1 were partially determined and show that Cys1 is not linked to Cys2 and Cys3 is not linked to Cys4. Analogous with IGFBP-3, IGFBP-5, and IGFBP-6, Cys9-Cys11 and Cys10-Cys12 of IGFBP-1 are also disulfide-linked. The N-terminal linkages of IGFBP-6 differ significantly from those of IGFBP-1 (and, by implication, the other IGFBPs), which could contribute to the distinctive IGF binding properties of IGFBP-6.

Identification of O-glycosylation sites and partial characterization of carbohydrate structure and disulfide linkages of human insulin-like growth factor binding protein 6.

The actions of insulin-like growth factors (IGFs) are modulated by a family of high-affinity binding proteins (IGFBPs), including IGFBP-6, which preferentially binds IGF-II and is O-glycosylated. Glycosylated and nonglycosylated recombinant human IGFBP-6, expressed in Chinese hamster ovary cells and Escherichia coli, respectively, were purified using IGF-II affinity chromatography and reverse-phase medium-pressure chromatography. Electrospray ionization mass spectrometry (ESMS) of glycosylated IGFBP-6 revealed considerable heterogeneity of carbohydrate composition. Major glycoforms contained 8-16 monosaccharides, including N-acetylhexosamine, hexose, and N-acetylneuraminic acid. Glycosylation sites of IGFBP-6 were identified as Thr126, Ser144, Thr145, Thr146, and Ser152 by using a combination of ESMS and Edman sequencing of tryptic fragments separated by reverse-phase high-pressure liquid chromatography. One oligosaccharide chain contained 5-6 monosaccharides, whereas the others contained 2-4 monosaccharides. Glycosylated IGFBP-6 exhibited greater resistance to proteolysis by chymotrypsin and trypsin than nonglycosylated IGFBP-6. Native disulfide bond positions in IGFBP-6 were localized by means of observed disulfide-linked tryptic fragments, revealing that there are two disulfide-linked subdomains within each of the N- and C-terminal regions and confirming a previous suggestion that the latter regions are not interconnected. A model of IGFBP-6 is developed in which these distinct domains are separated by a central region which is O-glycosylated.

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 Ca(2+)-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.

Purification and sequencing of multiple forms of Brassica napus seed napin small chains that are calmodulin antagonists and substrates for plant calcium-dependent protein kinase.

Six napin small (S) subunits and six napin large (L) subunits were resolved from the seeds of kohlrabi (Brassica napus var. rapifera) by a procedure involving extraction, batchwise elution from carboxymethylcellulose (CM52) and reverse-phase HPLC after treatment with guanidine hydrochloride and 2-mercaptoethanol. The precise average molecular masses of the ca. 4.5 kDa small subunits and the ca. 10 kDa large subunits were determined by electrospray ionisation mass spectrometry (ESMS). The amino-acid sequences of six small subunits (S1A, S1B, S2, S3A, S3B and S4) were deduced from the ESMS-based masses of tryptic fragments, Edman sequencing and previously published data. The deduced structures were precisely consistent with this data and with the ESMS-based average molecular masses of these polypeptides. The structures of the small subunits (39-41 residues) are very similar with variations involving single substitutions at or near the N-terminus and 1 to 3 changes within the last 7 amino acids. Particular B. napus small and large chains are phosphorylated by plant Ca2+-dependent protein kinase (CDPK). The best site of phosphorylation on small chains is inferred to be either S34 or S39 of S1B. The napin-containing basic protein fraction from B. napus seeds largely abolishes the Ca2+-dependent fluorescence enhancement of dansyl-calmodulin and also inhibits calmodulin (CaM)-dependent myosin light chain kinase (MLCK). The resolved napin small chains also inhibit MLCK. All of the kohlrabi napin small chains, as well as homologous Brassicaceae small chains, have a central 23 amino-acid sequence that can potentially form an alpha-helix in which all the basic residues are located on one side. This structural element may be involved in the interaction of these proteins with CaM and the biological activity of antifungal proteins of this kind.

Purification and sequencing of multiple forms of Brassica napus seed napin large chains that are calmodulin antagonists and substrates for plant calcium-dependent protein kinase.

Six napin large (L) chains (as well as six napin small chains) were resolved from the seeds of kohlrabi (Brassica napus var. rapifera) by a procedure involving extraction, batchwise elution from carboxymethylcellulose (CM52) and reversed-phase HPLC after treatment with guanidine hydrochloride and 2-mercaptoethanol. The precise average molecular masses of the circa 4.5 kDa small subunits and the circa 10 kDa large subunits were determined by electrospray ionisation mass spectrometry (ESMS). Of six large subunits resolved (L1A), L1B, L1C, L2A, L2B and L2C), the complete amino acid sequences of four (L1A, L2A, L2B and L2C) and the near-complete sequences of two (L1B and L1C) were deduced from the ESMS-based masses of tryptic fragments, Edman sequencing and previously published data. The deduced structures are precisely consistent with this data and with the ESMS-based average molecular masses of these polypeptides. ESMS analysis of unreduced napin extract revealed only seven circa 14.5 kDa complexes, the observed masses being in close agreement with those calculated for 1:1 complexes of particular small and large subunits assuming four disulfides in each napin complex. The structures of the napin large subunits (86-91 residues) are very similar and all amino acid differences observed are confined to only 25 positions. The L2A, L2B AND L2C large chains (but not the L1A, L1B and L1C large chains) are phosphorylated well by plant Ca2+-dependent protein kinase (CDPK). The CDPK-catalyzed phosphorylation site on the large chain L2A is inferred to be S57 within the sequence LQQVIS57RIYQT (the site being S60 within the same sequence in L2B and L2C). The napin-containing basic protein fraction from B. napus seeds largely abolishes the Ca2+-dependent fluorescence enhancement of dansyl-calmodulin and also inhibits calmodulin (CaM)-dependent myosin light chain kinase (MLCK). The resolved napin long chains also inhibit MLCK. Each kohlrabi large chain contains 2 sequences (corresponding to L(10)-Q(20) and Q(51)-L(64) of L1A) which have the potential to form amphipathic alpha-helices. Each large chain also contains a Q-rich 19 amino acid sequence (corresponding to L(30)-Q(48) of L1A) which has the potential to form a '2-sided' alpha-helix with basic residues confined to one side. These structural elements may be involved in the inferred interaction of these proteins with CaM and may be relevant to the biological activity of antifungal proteins of this kind.

Purification and mass spectrometry-based sequencing of yellow mustard (Sinapis alba L.) 6 kDa proteins. Identification as antifungal proteins.

Three basic proteins, M1, M2A and M2B, that are substrates for plant Ca(2+)-dependent protein kinase (CDPK) were purified from seeds of yellow mustard (Sinapis alba L.) by a protocol involving batchwise chromatography on carboxymethylcellulose (CM52), cation-exchange HPLC on an SP5PW column and reversed-phase HPLC on a C18 column. The complete amino-acid sequences of these proteins have been determined employing Edman sequencing and electrospray ionization mass spectrometry (ESMS) applied to the proteins and their tryptic and chymotryptic fragments. M1 (observed mass 5676.8 +/- 1.0 Da; calculated mass 5677.57 Da), M2A (observed mass 5704.8 +/- 0.8 Da; calculated mass 5704.60 Da) and M2B (observed mass 5839.5 +/- 1.2 Da; calculated mass 5838.78 Da) have been identified as gamma-thionins, which are potent antifungal proteins. M1, M2A and M2B are phosphorylated by plant CDPK on Ser residues, the site of phosphorylation on M2A being S8 as directly confirmed by Edman sequencing and mass spectrometry of the chymotryptically generated phosphopeptide CQRPS(HPO3)GTW11. M1 and M2A have apparent calmodulin (CaM) antagonist activity with IC50 values of 4.8 +/- 1.3 microM and 5.5 +/- 1.5 microM, respectively, for inhibition of CaM-dependent myosin light chain kinase (MLCK). M2A and/or M2B interacts with dansyl-CaM in both the presence and absence of calcium.

Purification and characterization of a heat-stable wheat substrate for wheat embryo calcium-dependent protein kinase.

A heat-stable wheat protein (WP) that is a good substrate for wheat embryo Ca(2+)-dependent protein kinase (CDPK) was purified from wheat embryo by a procedure involving batchwise anion exchange chromatography on DEAE-cellulose (DE52), passage through Phenyl-Sepharose CL-4B, heat and acid treatment and anion exchange HPLC on a DEAE-5PW column. WP is phosphorylated by CDPK to a stoichiometry of about 0.8 mol phosphoryl per mol WP. The Km for WP is 3.5 microM. WP is phosphorylated by CDPK on Ser residues. [32P]phosphoWP exactly copurifies on SDS-PAGE with WP (59 kDa). Phosphorylation of WP by CDPK is largely Ca(2+)-dependent. The N-terminal amino acid sequence of WP has homology with bacterial azurins. Evidence for two serine phosphorylation sites was obtained from sequencing of phosphopeptides derived from tryptic and chymotryptic digests of phosphoWP. One major site of phosphorylation is inferred to be on a serine within the sequence KKMASMK. WP is one of the best endogenous protein substrates yet found for wheat embryo CDPK. A 59kDa protein is phosphorylated in vivo in sprouting wheat.

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).

Purification and characterization of wheat and pine small basic protein substrates for plant calcium-dependent protein kinase.

A wheat basic protein (WBP) was purified to homogeneity from wheat germ by a protocol involving extraction, centrifugation, batchwise elution from carboxymethylcellulose (CM-52), acidification with trifluoroacetic acid, neutralization and HPLC on a SP5PW cation exchange column. WBP is a 10 kDa protein and is phosphorylated on serine residues by wheat germ Ca(2+)-dependent protein kinase (CDPK). [32P]phosphoWBP exactly comigrates with WBP on SDS-PAGE. WBP does not inhibit either wheat germ CDPK or calmodulin-dependent myosin light chain kinase. Apart from histone H1, WBP is the best endogenous substrate yet found for wheat embryo CDPK. A 12 kDa pine basic protein (PBP) was purified to homogeneity from seeds of stone pine (Pinus pinea L.) by a simple procedure involving batchwise elution from carboxymethylcellulose and cation exchange HPLC. PBP is also a good substrate for CDPK and is phosphorylated on Ser residues. N-terminal sequencing of WBP and PBP revealed that these proteins are homologous to a family of small basic plant proteins having a phospholipid transfer function.