Properties Of Protein Kinase Research Articles

31] Calcium-activated, phospholipid-dependent protein kinase (protein kinase C) from rat brain

Ca 2+- activated, phospholipid-dependent protein kinase (protein kinase C) is a new species of protein kinase that is activated by diacylglycerol in the presence of both membrane phospholipid and physiological concentrations of Ca 2+ . Protein kinase C was originally found as an inactive enzyme that could be activated by limited proteolysis with Ca 2+ dependent neutral thiol protease. The enzyme was subsequently shown to be alternatively activated in a reversible manner by attachment to membrane phospholipid in the presence of Ca 2+ . Further analysis revealed that a small amount of diacylglycerol dramatically increases the affinity of the enzyme for Ca 2+ and for phospholipid, and thereby causes activation of the enzyme at physiological concentrations of Ca 2+ . This chapter describes the purification and properties of protein kinase C from a rat brain soluble fraction. The enzyme lacks tissue and species specificities, in the physical and kinetic properties that are described in this chapter.

Functional Properties of Protein Kinase(s) and Phosphatase(s) Converting Quinate:NAD<sup>$</sup> Oxidoreductase into Active and Deactivated Forms in Carrot Cell Suspension Cultures

Functional Properties of Protein Kinase(s) and Phosphatase(s) Converting Quinate:NAD<sup>$</sup> Oxidoreductase into Active and Deactivated Forms in Carrot Cell Suspension Cultures

Distribution and properties of protein kinase and protein phosphatase activities in synaptosomal plasma membranes and synaptic junctions

Some characteristics of the protein kinase activity associated with a synaptosomal plasma membrane (synaptic membrane) fraction and a synaptic junction fraction have been compared. Autoradiography of the phosphorylated fractions separated on sodium dodecyl sulfate polyacrylamine gels showed that cyclic AMP stimulates the phosphorylation of five polypeptides in synaptic membranes, whereas no cyclic AMP dependency could be detected in synaptic junctions. Kinetic studies demonstrated that synaptic junctions contain at high K m and a low K m protein kinase activity while only the high K m activity could be detected in synaptic membranes. The intrinsic ATPase activity of synaptic membranes was shown to strongly interfere with measurements of protein kinase activity. Cyclic AMP binding experiments revealed a 2.6-fold enrichment of cyclic AMP binding capacity in synaptic junctions as compared to synaptic membranes. Protein phosphatase activity was not detected in synaptic junctions but was associated with synaptic membranes, where cyclic AMP was shown to either stimulate or inhibit the dephosphorylation of different polypeptides.

Properties of protein kinase and adenylate cyclase‐deficient variants of a macrophage‐like cell line

Stable variants of the macrophage-like cell line J774.2, defective in adenylate cyclase and protein kinase activities, were selected by cloning cells resistant to the growth-inhibitory effect of cholera toxin and 8-bromo-adenosine 3':5' cyclic monophosphoric acid (8 Br-cAMP), respectively. These variants were analyzed for their ability to respond to cyclic AMP-mediated enhancement of phagocytosis and cyclic AMP-mediated inhibition of plasminogen activator secretion and growtn. The adenylate cyclase variants were unaffected by cholera toxin but were sensitive to 8 Br-cAMP-mediated inhibition of plasminogen activator secretion and growth. One of these variants exhibited a defect in phagocytosis that could be corrected by 8 Br-cAMP. The protein kinase variants exhibited normal basal phagocytosis that could not be stimulated by either 8 Br-cAMP or cholera toxin; they were also insensitive to cyclic AMP-mediated inhibition of plasminogen activator secretion and growth. The studies demonstrate that the three effects of cyclic AMP in J774.2--inhibition of growth and plasminogen activator secretion, and enhancement of basal Fc-mediated phagocytosis--are mediated by a cyclic AMP-dependent portein kinase. The results support the usefulness of variants in cyclic nucleotide metabolism in understanding the regulation of differentiated cell function by cyclic AMP.

Gonadotropin Induced Stimulation and Desensitization of Cyclic 3′,5′-Adenosine Monophosphate Dependent Protein Kinase(S) in the Rat Ovary

Incubation of rat ovarian slices with choriogonadotropin resulted in stimulation of cyclic AMP dependent protein kinase activity in a time and dose dependent manner. However, prior exposure of animals to choriogonadotropin in vivo resulted in a decreased sensitivity to subsequent in vitro stimulation by the same hormone. This decreased sensitivity was due to a lesion in the hormone receptor adenylate cyclase system rather than a change in the property of protein kinase with respect to its stimulation by cyclic AMP. Unchanged levels of cyclic AMP formation in these ovaries were evidenced by the lack of inhibition of [3H] cyclic AMP binding. In the untreated controls, however, choriogonadotropin in vitro resulted in inhibition [3H] cyclic AMP binding activity suggesting endogenous occupation parallel to protein kinase activation. These results are indicative of the linear coupling of hormone receptor-adenylate cyclase-protein kinase system and point out the in vivo role of protein kinase activation in choriogonadotropin mediated regulation of ovarian function.

Cyclic nucleotides and nervous system function.

Cyclic nucleotides and nervous system function.

Developmental changes in the subcellular distribution of endogenous protein kinase in the liver of the domestic chick ( Gallus domesticus)

1. 1. The distribution and some properties of protein kinase in nuclear, mitochondrial, microsomal and cytosolic liver fractions were studied in 15-day-old embryo, and in 1- and 7-day-old chicks ( Gallus domesticus). 2. 2. The protein kinase activity in all subfractions studied with endogenous protein substrate as well as with histone or casein as exogenous substrate was not affected by 0.5-5 μM cyclic adenosine 3', 5'-monophosphate. 3. 3. Phosphorylation of endogenous membrane protein of nuclear, mitochondrial and microsomal fractions was highest in 7-day-old embryo and gradually declined during development. 4. 4. Phosphorylation of exogenous substrate, histone and casein, was higher in nuclear and mitochondrial fractions than in microsomal and cytosolic ones and gradually increased during development. It was greater in 7-day-old chick than in 15-day-old embryo by about 3- and 4-fold with casein and histone respectively. 5. 5. In the presence of 1 % Triton X-100, membrane-bound protein kinase activity was stimulated about 4-, 4.5- and 3-fold in nuclear, mitochondrial and microsomal fractions respectively. 6. A decrease of the phosphorylation activity of endogenous protein substrate and a concomitant increase of the phosphorylation of exogenous substrates seems to suggest that differentiation of the protein kinase pool occurs in liver during chick development.