Protein Kinases in Testes of Adult and Prepuberal Rats

1974 ◽  
Vol 52 (7) ◽  
pp. 563-569 ◽  
Author(s):  
E. A. Bernard ◽  
G. F. Wassermann
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Enzymatic Activity ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Seminiferous Tubules ◽  
Interstitial Tissue ◽  
Protein Kinase Activity

Cyclic AMP dependence of protein kinase activity (PrK) in testicular homogenates varies according to the substrate used (histone > casein). The enzymatic activity increased during maturation, notably between 35 and 45 days of age. The increment is higher in isolated seminiferous tubules than in interstitial tissue. Upon centrifugation at pH 4.8, two dissimilar PrK preparations can be obtained. The enzymatic activity of both fractions increased with maturation.

scholarly journals Protein kinase activity in rat testis interstitial tissue. Effect of luteinizing hormone and other factors

1976 ◽  
Vol 154 (2) ◽  
pp. 371-378 ◽  
Author(s):  
B A Cooke ◽  
A J. W. C. M van der Kemp
Keyword(s):  
Protein Kinase ◽  
Luteinizing Hormone ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Soluble Fraction ◽  
Subcellular Fractions ◽  
Interstitial Tissue ◽  
Protein Kinase Activity ◽  
Rat Testis ◽  
Triton X

Protein kinase activity was determined in subcellular fractions of rat testis interstitial tissue after incubation of the intact tissue with LH (luteinizing hormone) in vitro. Various factors that might have changed the activity of this enzyme during preparation of the fractions before assay were also investigated. The following results were obtained. 1. LH and 3-isobutyl-1-methylxanthine (a phosphodiesterase inhibitor) added together during incubation of the interstitial tissue caused a twofold increase in the protein kinase activity in the total tissue homogenate and subcellular fractions (12000g X 5 min pellet and 105000g X 60 min supernatant and pellet). 2. A decrease of approx. 40% in the total amount of protein kinase recovered in the soluble fraction (105000g supernatant) occurred in tissue incubated with LH and 3-isobutyl-1-methylxanthine when compared with the controls. No change in total activity was found in the other fractions. 3. LH and 3-isobutyl-1-methylxanthine caused an increase in cyclic AMP concentration in the soluble fraction (from 30 +/- 6 to 450 +/- 40 pmol/mg of protein, means +/- S.E.M., n = 4), but there was little or no increase in the particulate fractions [from 9 +/- 1 to 13 +/- 3 pmol/mg of protein (n = 3) and from 6 +/- 2 to 23 +/- 11 pmol/mg of protein (n = 3) in the 12000g and 105000g pellets respectively]. 4 Addition of 3-isobutyl-1-methylxanthine alone had little effect on protein kinase activity or cyclic AMP concentrations. 5. Little or no protein kinase activity could be demonstrated in subcellular particulate fractions unless Triton X-100 was added; the effect of this detergent was shown to be at least partly due to the inhibition of adenosine triphosphatase activity. 6. In the presence of Triton X-100 approx. 57% of the total protein kinase activity in the homogenate was found in the 105000g supernatant compared with 11% in the 105000g pellet and 32% in the 12000g pellet. 7. In contrast with adipose-tissue protein kinase [Corbin et al. (1973) J. Biol. Chem. 248, 1813-1821] the relative amounts of cyclic AMP-dependent and -dependent enzyme were not affected by dilution of the interstitial-tissue fractions. NaCl (0.5 M) decreased the estimated total amount of protein kinase activity.

scholarly journals Evidence for Ecto-Protein Kinase Activity That Phosphorylates Kemptide in a Cyclic AMP-dependent Mode

1989 ◽  
Vol 264 (24) ◽  
pp. 14549-14555 ◽  
Author(s):  
D Kübler ◽  
W Pyerin ◽  
O Bill ◽  
A Hotz ◽  
J Sonka ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Protein Kinase Activity

Regulation of protein kinase by vasopressin in renal medulla in situ

1977 ◽  
Vol 232 (1) ◽  
pp. F50-F57
Author(s):  
T. P. Dousa ◽  
L. D. Barnes
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Renal Medulla ◽  
Protein Kinase Activity ◽  
Kinase Activation ◽  
Heat Stable ◽  
Protein Kinase Activation ◽  
Heat Stable Protein

Results of this study demonstrate that vasopressin activates protein kinase in intact renal medullary cells as detected by measurement of the (-cyclic AMP/+cyclic AMP) protein kinase activity ratios in freshly prepared tissue extracts (40,000 X g supernates) from bovine renal medullary slices. The activation of protein kinase was specific for vasopressin since parathyroid hormone, histamine, angiotensin II, or the inactive analog of vasopressin did not activate protein kinase. There was a direct correlation between the extent of protein kinase activation and the elevation in tissue levels of cyclic AMP elicited by increasing doses of vasopressin or with an increase in incubation time. The elevation of tissue cyclic AMP level and maximum activation of protein kinase reached maximum level at a vasopressin concentration of about 2 X 10(-9) M. Incubation of slices with vasopressin caused a dose-dependent decrease in the cyclic AMP-dependent protein kinase activity in the 40,000 X g supernate of homogenate from the renal medullary slices. This effect of vasopressin was specific for protein kinase since activity of lactate dehydrogenase or a specific [3H]colchicine-binding activity was not affected, and the decrease in the protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase activity extracted from 40,000 X g pellets of homogenate prepared from slices exposed to vasopressin. Results thus provide evidence that cyclic AMP-mediated protein kinase activation in the intact cells is an integral part of cellular response of the mammalian renal medulla to vasopressin.

scholarly journals The role of protein kinase activation in the control of steroidogenesis by adrenocorticotrophic hormone in the adrenal cortex

1973 ◽  
Vol 136 (4) ◽  
pp. 993-998 ◽  
Author(s):  
Malcolm C. Richardson ◽  
Dennis Schulster
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Adrenal Cortex ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Adrenocorticotrophic Hormone ◽  
Dependent Protein Kinase ◽  
Kinase Activation ◽  
Protein Kinase Activation ◽  
Dependent Protein

A method has been developed for investigation of the effect of adrenocorticotrophic hormone (ACTH) on the state of activation of a cyclic AMP-dependent protein kinase within cells of the adrenal cortex. Enzyme activity was measured in terms of the quantity of32P transferred from [γ-32P]ATP to histone under conditions in which bound cyclic AMP did not dissociate from the regulatory subunit of the protein kinase ACTH (1×10-2i.u./ml) caused a rapid and complete activation of the cyclic AMP-dependent protein kinase activity within 2min of hormone addition to the isolated cells. In response to a range of ACTH concentrations a sigmoid log dose–response curve for protein kinase activation was obtained, with half-maximal stimulation attained at about 1×10-3i.u./ml. However, some low doses of ACTH that elicited a marked (but submaximal) steroidogenic response failed to cause a clear stimulation of protein kinase activity in isolated adrenal cells. Theophylline (2mm) potentiated the effect of ACTH on protein kinase activity. The results implicate an important role for protein kinase in ACTH action on the adrenocortical cell.

scholarly journals S183. ABNORMALITIES IN SERINE/THREONINE SIGNALING NETWORKS IN SEVERE NEUROPSYCHIATRIC ILLNESS: DEVELOPMENT OF A PIPELINE TO EXPLORE ABNORMALITIES OF KINASE ACTIVITY AND IDENTIFY NOVEL TREATMENT STRATEGIES FOR SCHIZOPHRENIA

2020 ◽  
Vol 46 (Supplement_1) ◽  
pp. S107-S108
Author(s):  
Robert McCullumsmith ◽  
Khaled Alganem ◽  
Nicholas Henkel ◽  
Abdul Hammoud ◽  
Rammohan Shukla ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Biological Pathways ◽  
Signaling Networks ◽  
Protein Kinase Activity ◽  
Drug Candidates ◽  
Group 10 ◽  
Disease Signatures ◽  
Neuropsychiatric Illness

Abstract Background Abnormalities of cellular signaling are well characterized in neuropsychiatric illnesses, including schizophrenia. Changes in signaling pathways reflect the underlying genetic, environmental, and epigenetic perturbations driving disease phenotypes. A shortcoming of most signaling studies is a focus on one or a few protein kinases at a time, a limitation since protein kinases work in networks with other kinases, phosphatases, and regulatory molecules to effect signaling events. We addressed this challenge by employing a kinome array platform that simultaneously measures protein kinase activity at hundreds of reporter peptide substrates. We then developed a novel bioinformatics pipeline to identify protein kinase nodes, signaling networks, upstream biological pathways, and drug candidates that “reverse” kinomic disease signatures. Methods Postmortem DLPFC brain samples from subjects with schizophrenia (n = 20 per group, 10 males and 10 females per group), were compared to age, PMI and pH matched control subjects (n = 20 per group, 10 males and 10 females per group) using the Pamgene12 serine/threonine kinome array chip. Samples were pooled by diagnosis and gender, and run in triplicate. The R-shiny app KRSA was created to automate assignment of kinases, perform permutation analyses, identify biological pathways, and connect to iLINCs for identification of drugs that reverse kinomic disease signatures. We also performed targeted confirmation studies using specific kinase activity assays, QPCR, and western blot analyses. Results We identified unique and common kinase nodes for each diagnostic group. Several of the nodes (for example AKT) are well characterized in schizophrenia, while others have not previously been identified (such as AMPK). We used AMPK KD cultures and AMPK KO brain tissues to demonstrate the validity if the kinome array for this protein kinase. We used standard kinase activity assays for AMPK and found decreased activity for AMPK (P < 0.05). We also found decreased expression of transcripts for the regulatory subunits of AMPK (P < 0.05). We identified several unique biological pathways, as well as candidate drugs, associated with the disease signature in schizophrenia. Discussion Our results confirm well characterized signaling defects in severe neuropsychiatric illness, and identify novel signaling nodes for further study. Confirmation studies for AMPK kinase show significant changes in expression and activity of this kinase, suggesting perturbation of energy sensing and production pathways in schizophrenia. Bioenergetic pathways may be targeted by myriad mechanisms, and we identified several drug candidates that might help restore this pathway in afflicted persons. Overall our novel workflow and pipeline provides a promising new avenue for understanding the complex signaling perturbations found in brain diseases and may provide new leads for developing treatments for schizophrenia and other cognitive disorders.

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