Affine Case Research Articles

Aflatoxin M1 binding to bovine α- and κ-caseins demonstrated by surface plasmon resonance

A preliminary investigation of the potential binding interaction of isolated whey proteins and casein proteins with aflatoxin M1 (AFM1) using a surface plasmon resonance optical biosensor is described. The experimental conditions were restricted to facilitate a qualitative analysis that, for the first time, has demonstrated that AFM1 differentially binds to α-casein and κ-casein; it has negligible interaction with β-casein and the individual whey proteins. These observations with individual casein proteins can be extrapolated to infer that such differential binding occurs in intact milk, and explains the many previous reports of the heterogeneous distribution of AFM1 in milk and milk products that have only indirectly proposed its affinity for casein.

Phosphorylation of serine 833 in cytoplasmic domain of low density lipoprotein receptor by a high molecular weight enzyme resembling casein kinase II.

A soluble protein kinase that phosphorylates the last serine residue (Ser-833) in the cytoplasmic domain of the low density lipoprotein (LDL) receptor was purified about 1300-fold from the cytosol of bovine adrenal cortex. The LDL receptor kinase shared several properties with casein kinase II: use of either GTP or ATP; phosphorylation of a typical casein kinase II recognition sequence in the LDL receptor (a serine followed by a cluster of three negatively charged amino acids); and inhibition by heparin. The LDL receptor kinase differed from classic casein kinase II in the following respects: its apparent molecular weight on gel filtration was approximately 500,000 as opposed to the usual molecular weight of 130,000 for casein kinase II; its affinity for the LDL receptor (apparent Km approximately 5 nM) was much greater than its affinity for casein (approximately 10 microM); and its activity was inhibited by polylysine, an agent that stimulates casein kinase II. The physiologic role of this unusual kinase, if any, is unknown.

Compartimentalization of spectrin-phosphorylating enzyme in human erythrocytes

Functional characteristics of purified casein kinases from cytosol and membrane of normal human red cell were determined in vitro using pure spectrin as substrate. Both enzyme which differed from each other by their salt solubility, molecular weight and affinity for casein had similar affinity for spectrin and same optimum pH ; they used ATP as phosphoryl donor, were similarly stimulated by monovalent cations and Mg 2+ and inhibited by Ca 2+ and were insensitive to cyclic nucleotides. Cytosol kinase had a poor substrate and phosphoryl donor specificity ; membrane kinase had a much greater affinity for spectrin than for casein and poorly used GTP as phosphoryl donor : it seems to be the physiologically active spectrin kinase of the red cell.

Casein kinase from human erythrocyte membrane, purification characterization and comarison with the cytosolic enzyme

1. 1. A casein kinase was extracted from human erythrocyte membrane in high ionic strength salt solution and purified by ammonium sulfate precipitation and two successive phosphocellulose chromatographies. 2. 2. This enzyme shared some properties with the cytosol casein kinase but clearly differed from this latter by its affinity for casein, GTP utilization, salt solubility and molecular weight.

Protein kinases of Dictyostelium discoideum, strain AX-2

In cell homogenates of Dictyostelium discoideum, strain AX-2, four major soluble protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) and one membrane-associated protein kinase activity were identified. The enzymes showed high affinity for casein. One of the enzymes was purified by affinity chromatography on casein-coated Sepharose. The soluble high molecular weight enzymes phosphorylated histones, whereas the low molecular weight enzymes did not. The same protein kinase species were present in vegetative and aggregation-competent cells. Their specific activity, however, changed during the development to aggregation competence. None of the enzymes was stimulated by cyclic AMP or cyclic GMP, regardless of their origin from vegetative or aggregation-competent cells.

Phosphylation and protein kinase activity of platelet tubulin.

Platelet tubulin isolated by two successive cycles of polymerization-depolymerization was shown to contain protein kinase activity. The phosphorylating activity measured by incorporation of [32P]phosphate from [gamma-32P]ATP was cAMP-independent and behaved with respect to substrate specificity, cation requirement, and maximum incorporation of phosphate similarly to tubulin of brain. Contrary to tubulin from that source, however, platelet tubulin itself, not one of its co-purifying proteins appeared to be the source of the protein kinase activity. This was suggested by assays of tubulin freed from its associated proteins by chromatography on DEAE-Sephadex and on immunosorbent columns containing monospecific antibody to human platelet tubulin. Further corroboration was obtained from experiments in which tubulin was applied to casein affinity columns. No separation of protein kinase from colchicine binding activity could be obtained. Gel filtration showed that all of the in vitro phosphorylated tubulin was aggregated. Tryptic peptide patterns of 32P-labeled alpha- and beta-tubulin subunits were analyzed by ion exchange chromatography. Multiple peptides in both tubulin subunits were identified as acceptors of [32P]phosphate. In vivo phosphorylated tubulin was demonstrated to contain an average of 5 phosphoserine residues/monomer.