Different Levels Of Phosphorylation Research Articles

Potential Uses of Caseinophosphopeptides

Abstract Caseinophosphopeptides (CPPs) are phosphorylated casein-derived peptides which possess the ability to bind and solubilise minerals, such as Ca2+. Consumption of high concentrations of Ca2+ in early life contributes to the development of maximal bone density, which in turn can prevent osteoporosis in later life. Furthermore, a recent report has shown a positive correlation between Ca2+ intake and the prevention of hypertension. The high bioavailability of Ca2+ from milk and dairy products has, in part, been attributed to the production of CPPs which have different levels of phosphorylation and are produced in vivo following digestion of α s 1 -, α s 2 - and β-casein by the action of gastrointestinal proteinases. CPPs which appear to be resistant to extensive proteolytic degradation, accumulate in the distal small intestine where they are purported to play a role in enhancing the passive absorption of Ca2+ and other trace elements. CPPs have also been produced in vitro using a range of commercially available proteinases of pancreatic origin. Several CPP enrichment procedures from casein hydrolysates have been reported, generally involving Ca2+ induced aggregation followed by ultrafiltration. These CPP enriched preparations have been used to characterise their interaction with Ca2+ and trace elements and to determine their effect on the bioavailability of dietary Ca2+ during animal and human feeding trials. There are conflicting reports, arising from the results of animal feeding studies, on the effectiveness of CPPs in enhancing Ca2+ bioavailability. However, a recent human feeding trial reported improved Ca2+ and Zn2+ absorption following CPP incorporation into a rice-based infant food. In vitro produced CPPs may also find application in the prevention and treatment of dental calculus. This review summarises the production, characterisation and potential applications of CPPs.

Effect of unphosphorylated smooth muscle myosin on caldesmon-mediated regulation of actin filament velocity

The effect of smooth muscle myosin at different levels of light chain phosphorylation on caldesmon-mediated movement of actin filaments was investigated using an in vitro motility assay. Myosin at different levels of phosphorylation was obtained by mixing different proportions of fully phosphorylated and unphosphorylated myosin in monomeric form, while keeping the total myosin concentration constant. The average velocity of actin filaments containing tropomyosin was 1.20 +/- 0.046 microns s-1 at 30 degrees C with fully phosphorylated myosin. This velocity was not altered when the percentage of unphosphorylated myosin coated on the nitrocellulose surface was increased to 80%; further increases lowered the velocity. When the actin filaments with caldesmon bound at stoichiometric levels were used, filament velocity was unaffected until 50% of the myosin was unphosphorylated, but further increases in the percentage of unphosphorylated myosin induced a decrease in the velocity, and at 95% unphosphorylated myosin, filament movement had ceased. The decreased filament velocity in the presence of caldesmon was also observed when phosphorylated myosin was mixed with myosin rod instead of unphosphorylated myosin, but was not observed when the 38 kDa caldesmon C-terminal fragment, which lacks the myosin-binding domain, was used instead of intact caldesmon. These data indicate that the decreased filament velocity in the presence of caldesmon reflects the mechanical load produced by the tethering of actin to myosin through the interaction of the caldesmon N-terminal domain and the myosin S-2 region. The tethering effect mediated by caldesmon may play a role in smooth muscle contraction when a large number of myosin heads are dephosphorylated, as in force maintenance.

Application of PhastSystem® to the Resolution of Bovine Milk Proteins on Urea-Polyacrylamide Gel Electrophoresis

Optimal conditions were established for alkaline urea-PAGE using modified precast, ultrathin gradient gels on the automated PhastSystem®. Profiles of milk proteins showed that the caseins and whey proteins resolved extremely well. Major bands were observed for αs1-casein and β-casein, and αs2-casein appeared as a well-resolved doublet. In contrast, κ-casein separated from other caseins as a faint doublet, and purified κ-casein appeared as one major and one minor band. Whey proteins (serum albumin, α-lactalbumin, β-lactoglobulin) separated into broad bands resolved from each other and from the caseins. Partially (40%) dephosphorylated whole casein showed multiple bands for αs1-casein and β-casein at different levels of phosphorylation. Separation of genetic phenotypes was observed for β-lactoglobulin A and B; αs1-casein A, B, and C; and β-casein A, B, and C. Electrophoretic patterns of milk proteins extracted from cheese samples varied among the different types of cheeses. Our modified procedure provides researchers with a rapid technique to separate both caseins and whey proteins on the same urea gel according to their charge to mass ratios.

Expression and segregation of nucleoplasmin during development in Xenopus.

The spatial segregation of informational molecules in the unfertilized egg and embryo has been hypothesized to be a necessary phenomenon for the normal progression of development leading to the determination of cellular phenotypes. This study describes the selection of a monoclonal antibody (Mab: 2G6) that identifies an antigen (Ag: 2G6) which is localized in the germinal vesicle of oocytes and has a discrete pattern of inheritance during embryogenesis. The antigen displayed biochemical and physical characteristics very similar to nucleoplasmin, which is the histone-binding and nucleosome-assembly protein previously described. Immunoblot analysis with purified oocyte nucleoplasmin confirmed this relationship. Indirect immunofluorescence was used to study the temporal expression and spatial distribution of nucleoplasmin. From early cleavage stages through gastrulation, it is preferentially localized in nuclei of blastomeres at the animal pole. By tadpole stages, it was detected only in nuclei of postmitotic cells of the central nervous system and in nuclei of striated muscle. It was not detected in adult tissues. Western blot analysis during embryogenesis revealed at least five immunologically related polypeptides that displayed distinct patterns of expression during development. The different species observed most likely represent different levels of phosphorylation of nucleoplasmin. The more acidic forms, known to be more active in nucleosome assembly, were present during cleavage stages. Analysis of labelled oocyte proteins by two-dimensional immunoblots and autoradiography revealed that synthesis of nucleoplasmin was first detected in stage-2 oocytes, reached 60% maximum levels at stage 3, peaked at stage 4 and was undetectable in stage-6 oocytes. The amount of nucleoplasmin message present does not follow a similar pattern during oogenesis. These results suggest that the message undergoes pronounced changes in translational efficiency during oogenesis. A comparative immunoblot analysis using proteins from a variety of adult tissues revealed that, whereas the polyclonal antisera against amphibian vitellogenic oocyte nucleoplasmin recognized several different, tissue-specific polypeptides, two different monoclonal antibodies (Mab: b7-1D1, Mab: 2G6) failed to recognize any of the adult tissues tested. We conclude that nucleoplasmin is a family of closely related proteins with distinct embryonic and adult members.

Correlation between phosphorylation and kinase activity of a tyrosine protein kinase: P56 lck

P56lck is the product of a cellular oncogene highly expressed in lymphoid cells. Tyrosine kinase activity was measured by using an exogenous substrate: polyamino acid glutamic acid-tyrosine (4:1) (PGT). Different levels of phosphorylation of p56lck were achieved by the utilisation of SH reagents and different lengths of incubation time The phosphorylation of PGT was proportional to the level of phosphorylation of p56lck. Identical results were obtained with crude membrane preparations and with p56lck partially purified on immunecomplexes.

The nature of the transport ATPase—Digitalis complex: III. Rapid binding studies and effects of ligands on the formation and stability of magnesium plus phosphate-induced glycoside—Enzyme complex

The [ 3H]ouabain-Na +,K +-ATPase complex formed in the presence of magnesium and inorganic phosphate (Complex II) may represent a different conformation than that formed in the presence of ATP, magnesium, and sodium (Complex I). The nomenclature, Complex I and II, are operational in nature. The differences in binding may, e.g., be due to different levels of phosphorylation and/or to different conformations of the enzyme prior to binding. The kinetics of binding under conditions that lead to Complex II formation can be characterized by a hyperbolic curve arbitrarily divided into three phases. Sodium and potassium exert differential effects on these three phases of binding, with sodium having a greater inhibitory effect than potassium. Potassium primarily retards the reaction. When added to the binding medium (Complex II) after maximal binding, sodium and potassium are found not only to dissociate a portion of the glycoside-enzyme complex, but also appear to convert the unbound enzyme into a conformation which may bind only a small amount of glycoside.