Metal Ion Preference Research Articles

Chimeric reverse transcriptase of Moloney murine leukaemia virus, having the YXDD box of a Drosophila retrotransposon, 17.6

All reverse tanscriptases (RTs) so far examined are known to possess in common a stretch of 7 amino acids called the YXDD box. Various mutations were introduced in vitro into the nucleotide sequence coding for this box of Moloney murine leukaemia virus (Mo-MuLV) RT. The YXDD box of Mo-MuLV could be replaced by the counterpart of a Drosophila retrotransposon, 17. 6, without loss of activity, while virtually complete loss of activity was detected in the case of repalcement of the YXDD box of Rous sarcoma virus RT. Amino-acid substitution at position X resulted in significant change in divalent metal ion preference and enzyme activity, thus indicating that the YXDD box possibly constitutes a part of the active center of RT through metal ion binding.

Purification and biochemical characterization of non-myristoylated recombinant pp60c-src kinase.

To obtain sufficient material for the biochemical and biophysical study of pp60c-src, we have utilized a recombinant pp60c-src baculovirus lacking the myristoylation site at codon 2. On infection of Sf9 cells, this virus produced large amounts of soluble non-myristoylated pp60c-src. The use of non-myristoylated pp60c-src (1) increases production of pp60c-src compared with the wild-type protein, (2) facilitates purification, (3) yields a stable product and (4) allows biochemical studies in the absence of detergents. Up to 20 mg of pp60c-src of greater than 95% purity has been purified from 6 litres of Sf9 cells grown in a bioreactor. One major and multiple minor forms of pp60c-src were separated by Mono Q f.p.l.c. Isoelectric focusing of purified pp60c-src species revealed heterogeneity, some of which could be attributed to differences in the tyrosine phosphorylation state of the enzyme. Kinetic analysis of non-myristoylated pp60c-src kinase in the presence of Mg2+ gave Km values for angiotensin II and ATP of 2 mM and 30 microM respectively and a Vmax. of 620 nmol/min per mg. The kinetic constants and metal ion preferences of a number of copolymers and peptide substrates have been compared. Polylysine and poly(GLAT), which was not phosphorylated by the pp60c-src kinase, dramatically activated autophosphorylation of Tyr-416, suggesting a conformation modulation of pp60c-src by charged polymers. This finding implies that Tyr-527 dephosphorylation is not sufficient for full activation of pp60c-src in vitro.

Large-scale purification and characterisation of a recombinant epidermal growth-factor receptor protein-tyrosine kinase. Modulation of activity by multiple factors.

The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor alpha. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 61 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent Vmax value of 20 nmol min-1 mg-1 and Km values of 4.5 microM for ATP and 1.43 mM for angiotensin II. This corresponds to a turnover number of 1.4 mol min-1 mol-1. Ammonium sulfate (1 M) resulted in an eightfold stimulation of kinase activity when assayed using angiotensin II as substrate. The specific activity of the intracellular domain of the EGF-R, when assayed at 20 degrees C in the presence of 1M ammonium sulfate, was 160 nmol min-1 mg-1. Activation of the EGF-R kinase by ammonium sulfate was found to be substrate-specific. No activation was found when assayed using polymeric substrates. Addition of Me(2+)-ATP to the purified enzyme resulted in autophosphorylation and was accompanied by retardation of SDS/PAGE migration. Kinetic constants and metal ion preferences of a number of co-polymers and peptide substrates have been compared. Dramatic differences in kinetic constants were found which were dependent on both the substrate and metal ion used. Activation of EGF-R autophosphorylation was found to be influenced by the use of charged polymers. The random polymer of Glu, Lys, Ala, Tyr (2:5:6:1), which was not phosphorylated by the EGF-R kinase, dramatically activates autophosphorylation of the EGF-R. Thus the intracellular domain of the EGF-R appears to be in a low-activity conformation which, under appropriate assay conditions, can be activated to a similar specific activity to that reported for the purified EGF-R holoenzyme.

Development of solid-phase enzyme-linked immunosorbent assays for the determination of epidermal growth factor receptor and pp60 c-src tyrosine protein kinase activity

Solid-phase ELISAs for the determination of EGF receptor (EGF-R) and pp60 c-src tyrosine protein kinase activity are described. The methods were developed and optimized using purified recombinant EGF-R intracellular domain (ICD) and pp60 c-src tyrosine protein kinases. A standardized assay that utilizes poly (GluNa-Tyr)4:1 as substrate and a monoclonal antiphosphotyrosine antibody for detection is described. Assay conditions for both enzymes were optimized with respect to substrate and ELISA plate-coating condition, divalent metal ion preferences, enzyme concentration, apparent kinetic constants for ATP, and reaction linearity. Following standardization, a number of reference tyrosine protein kinase inhibitors were tested in the ELISAs and compared to results obtained using solution-phase radioactive tyrosine protein kinase assays, which are based on the transfer of 32P from [γ- 32P]ATP to synthetic substrate. To enable a comprehensive comparison, IC 50 values obtained in the ELISA were compared with values obtained in radioactive assays using both the holo-EGF-R and EGF-R ICD kinases. No substantial qualitative differences between these assays were seen. For many routine tyrosine protein kinase assays, semiquantitative or qualitative measurement of TPK activity is adequate. For such purpose, the ELISAs would be an attractive alternative to radioactive assays.

Characterization of phosphatidylinositol kinase activity associated with the insulin receptor

Various lipids were tested as substrates for the insulin receptor kinase using either receptor partially purified from rat hepatoma cells by wheat-germ-agglutinin-Sepharose chromatography or receptor purified from human placenta by insulin-Sepharose affinity chromatography. Phosphatidylinositol was phosphorylated to phosphatidylinositol 4-phosphate by the partially purified insulin receptor. In contrast, phosphatidylinositol 4-phosphate and diacylglycerol were not phosphorylated. In some, but not all preparations of partially purified insulin receptor, the phosphatidylinositol kinase activity was stimulated by insulin (mean effect 33%). Phosphatidylinositol kinase activity was retained in insulin receptor purified to homogeneity. Insulin regulation of the phosphatidylinositol kinase was lost in the purified receptor; however, dithiothreitol stimulated both autophosphorylation of the purified receptor and phosphatidylinositol kinase activity in parallel about threefold. (Glu80Tyr20)n, a polymeric substrate specific to tyrosine kinases, inhibited the phosphatidylinositol kinase activity of the purified receptor by greater than 90% and inhibited receptor autophosphorylation by 67%. Immunoprecipitation by specific anti-receptor antibodies depleted by greater than 90% the phosphatidylinositol kinase activity in the supernatant of the purified receptor and the phosphatidylinositol kinase activity was recovered in the precipitate in parallel with receptor autophosphorylation activity. These characteristics of the phosphatidylinositol kinase activity of the purified insulin receptor and its metal ion preference paralleled those of the receptor tyrosine kinase activity and differed from bulk phosphatidylinositol kinase activity in cell extracts, which was not significantly inhibited by (Glu80Tyr20)n, stimulated by dithiothreitol or depleted by immunoprecipitation with anti-(insulin receptor) antibody. These results suggest that the insulin receptor is associated with a phosphatidylinositol kinase activity; however, this activity is not well regulated by insulin. This kinase appears to be distinct from the major phosphatidylinositol kinase(s) of cells. Its relationship to insulin action needs further study.

A hypothesis for the selection of chelate antidotes for toxic metals

The problem of finding antidotes for toxic metal species for which antidotes have not been previously reported can be approached via a consideration of the metal-ion preferences of chelating agents already in clinical use. The metal ion preferences of six types of chelating agents together with information on antidotes used for other elements in the same family in the periodic table can be used to obtain a preliminary notion of which of these six structural types might furnish efficacious antidotes. The results of animal tests on a few of these chelate types can then serve as a guide in the selection of further compounds for testing. The data from the literature on antidotes for 20 toxic metal ions is summarized to assist the selection process.

ChemInform Abstract: CRYSTALLINE INSOLUBLE ACID SALTS OF TETRAVALENT METALS, PART 18, ION EXCHANGE PROPERTIES OF CRYSTALLINE ZRNAH(PO4)2.5H2O TOWARDS ALKALI METAL IONS

AbstractEs zeigt sich, daß im pH‐Bereich 3 bis 5 das H ‐ Ion nicht ausgetauscht wird und nur das Na ‐ Ion gegen Alkalimetalle ausgetauscht wird.

Crystalline insoluble acid salts of tetravalent metals—XVIII: Ion exchange properties of crystalline ZrNaH(PO 4) 2. 5H 2O towards alkali metal ions

The ion exchange behaviour of crystalline ZrNaH(PO 4) 2. 5H 2O towards alkali metal ions has been investigated. It was found that, in the pH range 3–5, hydrogen ion is not exchanged and only Na +-alkali metal ion exchanges take place. Forward and reverse Na +-K +, Na +-Li +, K +-Li + and Na +-Cs + ion exchange isotherms have been determined and discussed comparatively with the results obtained previously with fully-substituted alkali metal ionic form of zirconium phosphate. The exchanges occur with phase transitions and small hysteresis loops have been observed between the isotherms of the two opposite processes. The sequence of decreasing alkali metal ion preference is K + > Cs + > Na + > Li + and a very high selectivity towards K + or Cs + ion has been found. Thus, differently from Zr(HPO 4) 2. H 2O, the monosodium form exhibits preference for large cations and this different behaviour may be due to the large difference in the interlayer distances of the two ionic forms (7·56 and 11·8 Å respectively). The uptake of large ions by enlarged crystalline zirconium phosphate discloses new practical applications for such an exchanger.

Stereochemical control in the choice of donor atoms in metal complexes of a ‘bifunctional’ bidentate ligand

Metal complexes of two phosphorus–nitrogen ligands, 2-bis(diphenylphosphino)methyl-6-methylpyridine (ppn) and 2-[2-diphenylphosphino-1-(diphenylphosphinomethyl)ethyl]-6-methylpyridine (ppn′), are described. Although each ligand contains one pyridine and two tertiary phosphine groups, not more than two of the three potential donor atoms co-ordinate to the same metal atom. Both modes of bidentate behaviour (‘PN’ and ‘PP’) are found for complexes of ppn but only ‘PP co-ordination’ for those of ppn′. The choice of donor atoms is interpreted in terms of chelate ring size and the stereochemical preferences of the various metal ions. The latter include FeII, CoII, NiII, PdII, Zn, Cd, and HgII. The structures of the complexes are deduced from their vibrational and electronic spectra and other physical properties.

Site preferences of transition metal ions in silicate crystal structures

The structures of many silicate minerals contain one or more crystallographic positions which may be occupied by transition metal ions. The coordination sites differ in the number, symmetry and type of oxygen atoms, metal-oxygen distances, and extent of distortion from regular octahedral or tetrahedral symmetry. These factors affect the crystal field within each coordination site, and influence the distributions of transition metal ions within a particular structure and between two or more coexisting phases. Site preferences of individual transition metal ions in the olivine, orthopyroxene and clinopyroxene structures have been predicted from the mean metal-oxygen distances, site distortion, cation size and crystal field criteria. The predicted enrichments are in good agreement with available data on cation ordering in silicate structures and ion enrichments in coexisting minerals.

Electrostatic and non-electrostatic contributions to ion association in solution

Abstract