Influence of the morphology of poly(EGDMA-co-HEMA) on the chemical modifications and retention of O-phosphothreonine.

The influence of the morphology of ethylene glycol dimethacrylate-hydroxyethyl methacrylate copolymer [poly(EGDMA-co-HEMA)] base support to obtain different Fe(3+)-containing sorbents and their properties in retention of O-phosphothreonine [Thre(P)] is examined in this paper. Three base supports poly(EGDMA-co-HEMA) (I-III) were obtained using different quantities of initiator in suspension polymerization reactions. These products were submitted to chemical modifications using 1,4-butanediol diglycidyl ether (BDGE) in activation reactions and different chelating agents (iminodiacetic acid, IDA; disodium ethylenediamine tetraacetate, EDTA; and hexamethylenediamine tetrapropanoic acid, HMDTP) in coupling reactions to attain Fe(3+)-containing sorbents. Properties such as specific surface area (S(s)), specific pore volume (V(p)), scanning electron microscopy (SEM), IR spectroscopy, quantity of functional groups (oxirane and carboxyl), amount of chelated metal ion, ligand occupation (L), swelling studies as well as quantity of O-phosphoamino acid retained were used as comparative parameters for matrices. In general, the derivatization reactions proved to be more efficient when higher S(s) of macropores (50-400,000 nm) were available in the matrix. In our case, it was observed when highest percentage of initiator was used. On the other hand, the effect of accessibility of surface area on the yield of coupling reactions was noticed when comparing the different chelating agents since the number of carboxyl groups present in products was higher when the molecular size of the chelating agent was lower. Although all Fe(3+)-containing sorbents resulted efficient to retain Thre(P), the values of retention of the amino acid were slightly higher when IDA-containing matrices were used irrespective of the quantity of metal chelated. This could be probably due to the fact that the IDA ligand could be bounded to the matrix in sites that though accessible for the center of adsorption were hard for Thre(P) to access.

Purification and characterization of protein kinase CK2 from Candida albicans: evidence for the presence of two distinct regulatory subunits beta and beta'.

Protein kinase CK2 of Candida albicans has been purified to near homogeneity by a procedure which involves chromatography on DEAE-cellulose, phosphocellulose, Q-Sepharose, and heparin-agarose. The purified enzyme has the characteristic properties of animal and yeast CK2, i.e., it utilizes ATP as well as GTP as phosphate donor, phosphorylates serine and threonine residues on casein, is inhibited by low concentrations of heparin, and is stimulated by NaCl and polycationic compounds such as polylysine, spermine, and spermidine. The native form of the enzyme exhibits a molecular mass of 159 kDa, and SDS-PAGE analysis indicates that it is composed of four polypeptides with relative molecular masses of 44, 39, 37 and 36 kDa. The 39- and 37-kDa polypeptides were identified as distinct catalytic subunits alpha and alpha' on the basis of in situ phosphorylation assays and immunological recognition with heterologous antibodies. The purified kinase undergoes autophosphorylation on the 44- and 36-kDa polypeptides, a characteristic of the beta subunits from other species. Antibodies raised against the beta subunit of Drosophila melanogaster and human CK2 crossreact only with the 36-kDa polypeptide. The 44-kDa polypeptide was identified as an unusually large beta' subunit by Western blotting with an antibody raised against the beta' subunit of Saccharomyces cerevisiae. All these data suggest that C. albicans CK2 has an alpha alpha' beta beta' heterotetrameric composition similar to that found in S. cerevisiae.

An alternative method for a fast separation of phosphotyrosine.

A simple and rapid procedure is described for fully separating phosphotyrosine from phosphoserine and phosphothreonine through one-dimensional thin-layer chromatography. The migration properties of these phosphoamino acids are compared with those of CMP, UMP, ATP, ribose phosphate, and inorganic orthophosphate, considered the most frequent comigrating products derived from 32P-labeling experiments. We demonstrate that Rf values for the three phosphoamino acids differ from those displayed by the mentioned contaminating compounds. One of the most relevant advantages of this procedure is that a complete separation of phosphotyrosine can be achieved in only 90 min.