Gary M. Hathaway

Casein kinases--multipotential protein kinases.

Publisher Summary Casein kinase I and casein kinase II are unique protein kinases that have been described in a number of mammalian and avian cells; an enzyme with properties similar to those of casein kinase I has been described in yeast and plants. The casein kinases prefer acidic substrates and appear to differ from the enzyme endogenous to the mammary gland. Casein kinases I and II are multipotential in the sense that a number of endogenous substrates have been identified for them. Other multipotential protein kinases include the cAMP-dependent and cGMP-dependent protein kinases and phosphorylase kinase. No physiological regulator for casein kinase I has been identified, but the enzyme requires Mg2+ for activity and is stimulated by monovalent cations; the cation requirement is similar for casein kinase II.

[33] Casein kinase I

Publisher Summary Casein kinase I is a multipotential, cyclic nucleotide-independent, Ca 2+ independent protein kinase, which phosphorylates acidic substrates including casein. The type I casein kinases is identified and described in a variety of eukaryotes including yeast, plants, fowl, and mammals. The enzyme is present in nuclei and cytoplasm and found associated with membranes, ribosomes, and mitochondria. The type I casein kinases are termed “multipotential enzymes” because they phosphorylate a number of different substrates. The type I casein kinase is distinct from both the type II casein kinases, which are also present in many eukaryotic tissues, and the mammary gland enzymes, which function in the physiological phosphorylation of casein. Although casein kinase I has been shown to modify a number of different substrates, the functional role for the enzyme in metabolism remains to be elucidated. It is postulated that the casein kinases function to integrate metabolism because of the large number and variety of substrates identified for the enzymes.

[34] Casein kinase II

Publisher Summary This chapter describes casein kinase II. A multipotential casein kinase, casein kinase II, has been identified in both the nucleus and cytoplasm of a wide variety of eukaryotes. Casein kinase II is structurally and chromatographically distinct from casein kinase I, but like the latter enzyme, phosphorylates acidic substrates. Casein kinase II phosphorylates both seryl and threonyl residues; the recognition sequence for the enzyme two acidic residues following the phosphorylatable residue and is identified as Ser(Thr)- Glu(Ser-P)-Asp(Glu). Casein kinase II utilizes both adenosine triphosphate (ATP) and guanosine triphosphate (GTP) as phosphoryl donors in the phosphotransferase reaction. Regulation of the type II casein kinase appears to be quite complex. Monovalent cations stimulate casein kinase activity with most of the substrates and polyamines are shown to increase activity. Casein kinase II is shown to phosphorylate translational initiation factors, mRNP particles, spectrin, glycophorin, RNA polymerase, high-mobility-group protein 17, glycogen synthase, troponin T, and the regulatory subunit of the type II, cAMP-dependent protein kinase. In addition, it is postulated that casein kinase II may function to integrate total cell metabolism by regulating phosphorylation of a number of proteins in different metabolic pathways.

Interaction of polyamines and magnesium with casein kinase II

In reticulocytes, polyamines appear to be physiologically relevant activators of casein kinase II [Hathaway, G. M. and Traugh, J. A. (1984). J. Biol. Chem. 259, 7011-7015]. The mechanism by which polyamines and Mg2+ interact to activate casein kinase II has been investigated. These studies were conducted by holding ionic strength constant at 0.10 M. At low Mg2+ (2.5 mM), activation by spermine resulted in a 33% decrease in the apparent Km for casein. Under these conditions, a 2.3-fold increase in the maximum velocity of the reaction was observed, and half-maximal stimulation was obtained with 275 microM spermine. At a kinetically optimal Mg2+ concentration of 12.5 mM, the effects of spermine on Km and Vmax were reduced, and the concentration of spermine required to give 50% of maximal stimulation was increased to 750 microM. Kinetic data obtained at the two Mg2+ concentrations indicated that Mg2+ and spermine competed for the same form of the enzyme. Double-reciprocal plots of velocity versus Mg2+ concentration showed downward curvature at Mg2+ concentrations higher than 1 mM, and these results were interpreted as evidence for two binding sites on the enzyme with an apparent Km of 0.5 and 2.5 mM. Experiments carried out with ATP-Mg2+ in the absence of excess MgCl2 gave results consistent with an absolute requirement of the enzyme for the metal ion which could not be replaced by spermine. These results are consistent with the formation of an enzyme-activator complex. A model is proposed where spermine activates casein kinase II at one site on the enzyme at which MgCl2 can also bind, while a second, high-affinity site exists exclusively for the metal ion.

Preparative capillary electrophoresis for off-line sequence, composition, and mass analysis of peptides

Abstract Capillary electrophoresis was used to separate and collect peptides for multiple, off-line analytical techniques employed in many modern core-resource facilities. Peptides separated peak to peak by less than 30 s were successfully collected. Starting with a mixture of peptides at 0.1 to 0.3 mg/ml, enough material was obtained for off-line mass determination by laser desorption, time-of-flight analysis, peptide sequencing, and even the relatively sample-demanding analysis of amino acid composition. Preparations were readily reanalyzed by capillary electrophoresis in the analytical mode without the need for further sample handling. Details of the methodology and some of its limitations are examined and discussed.

Molecular characterization of pig muscle phosphoglucose isomerase

Abstract As a corollary to X-ray crystallographic work performed by H. Muirhead, detailed studies on crystalline pig muscle phosphoglucose isomerase have been conducted to establish its basic physical and chemical properties. The enzyme species being investigated by X-ray diffraction has been determined to be isoenzyme III. Its molecular weight in the native state was found to be 132,000, its s020,w value to be 7·25 S. The enzyme is composed of two subunits of equal molecular weight (66,000). Its amino acid composition is largely similar to that of rabbit muscle phosphoglucose isomerase, with the significant exception that the pig muscle isomerase contains only three sulfhydryl groups per polypeptide chain (two of them accessible to titration with p-mercuribenzoate) as compared with twice that number for the rabbit muscle enzyme. This low number of sulfhydryl groups is interpreted as being responsible for the ease with which heavy-atom, isomorphous derivatives could be prepared for the pig muscle enzyme by Shaw & Muirhead (1977).

Evidence for two independent mechanisms in the pyridoxal 5′-phosphate-mediated photoinactivation of phosphoglucose isomerase

Abstract Pyridoxal 5′-phosphate, which is believed to bind to the active site of phosphoglucose isomerase, has been shown to sensitize the enzyme toward irradiation by white light. During the process of photoinactivation, the substituted pyridine moiety and radioactivity from the tritium label at C4′ were incorporated into the protein. The presence of oxygen was not essential for either the inactivation or the incorporation processes. Although inactivation occurred at a faster rate when molecular oxygen was present, incorporation of label did not increase proportionately. A substrate analog (5-phosphoarabinonate), added to the enzyme pyridoxal 5′-phosphate reaction mixture, afforded complete protection against photoinactivation in the absence of oxygen. However, only partial protection was obtained when oxygen was present. Photoinactivation experiments performed in solutions enriched with D2O or containing sodium azide suggest that under aerobic conditions, singlet oxygen was inactivating the enzyme. Inactivation which occurred in the absence of oxygen proceeded with the incorporation of one pyridoxal 5′-phosphate molecule per inactivated subunit. The process of photoinactivation is believed, therefore, to involve two mechanisms which are capable of proceeding simultaneously and independently.

CYCLIC NUCLEOTIDE-INDEPENDENT PROTEIN KINASES FROM RABBIT RETICULOCYTES AND PHOSPHORYLATION OF TRANSLATIONAL COMPONENTS

Five different cyclic nucleotide-independent protein kinases have been isolated from rabbit reticulocytes. The enzymes are distinct from the type I and type II cAMP-dependent protein kinases and the free catalytic subunit of these enzymes. Three of the cyclic nucleotide-independent protein kinases, casein kinase I, casein kinase II and the hemin controlled repressor, have been obtained in highly purified form. The physical and chemical properties of these enzymes have been studied and the possible modes of regulation examined. In addition, two protease-activated kinases have also been observed in reticulocytes. These five cyclic nucleotide-independent protein kinases have different substrate specificities with respect to histone and casein. All of the protein kinases, cyclic nucleotide-independent and cAMP-dependent, differentially phosphorylate components of the protein synthesizing system including 40S ribosomal subunits and initiation factors 2, 3, 4B and 5. Each of the protein kinases phosphorylates two or more of these components and the initiation factors and ribosomal subunits are in turn modified by at least two different protein kinases. The result is a multiply phosphorylated system. Alterations in the phosphorylation state of these components can be observed under conditions which activate or inhibit specific protein kinase activities.