scholarly journals Isolation of two myosin light-chain kinases from bovine carotid artery and their regulation by phosphorylation mediated by cyclic AMP-dependent protein kinase

1982 ◽  
Vol 203 (3) ◽  
pp. 583-592 ◽  
Author(s):  
Ramesh C. Bhalla ◽  
Ram V. Sharma ◽  
Ramesh C. Gupta
Keyword(s):  
Protein Kinase ◽  
Carotid Artery ◽  
Cyclic Amp ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Type I ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Dependent Protein

Myosin light-chain kinase was purifed from bovine carotid artery. Approx. 90% of myosin kinase was extracted in the supernatant fraction with buffer containing EDTA during myofibril preparation. The soluble fraction yielded two distinct peaks on DEAE-Sephacel chromatography. Peak I was eluted at a conductance of 11–12mmho and was completely dependent on Ca2+–calmodulin for its activity. Peak II was eluted at a conductance of 13–14mmho and showed approx. 15% Ca2+-independent activity. The myosin kinases I and II were further purified by affinity chromatography by using calmodulin coupled to Sepharose 4B, which resulted in 960-and 650-fold purification of type I and type II kinases respectively. Myosin kinase II activity was completely Ca2+-dependent after affinity chromatography on the calmodulin–Sepharose column. Myosin kinases I and II were phosphorylated by cyclic AMP-dependent protein kinase. In the presence of bound calmodulin 0.5–0.7mol of phosphate was incorporated/mol of myosin kinases I and II. On the other hand, in the absence of bound calmodulin 1–1.4mol of phosphate was incorporated/mol of kinases I and II. Phosphorylation in the absence of calmodulin significantly decreased the myosin kinase activity of both enzymes, and the decrease in myosin kinase activity was due to a 3–5-fold increase in the amount of calmodulin required for half-maximal stimulation of both type I and type II kinases. The regulation of myosin kinase activity by cyclic AMP-dependent phosphorylation would suggest that β-adrenergic-mediated relaxation of vascular smooth muscle may be partly due to the direct interaction of cyclic AMP at the site of contractile proteins.

Myosin light chain kinase and cyclic AMP-dependent protein kinase activities and calmodulin levels in placental and non-placental regions of the rabbit myometrium

1986 ◽  
Vol 109 (1) ◽  
pp. 97-100 ◽  
Author(s):  
K. Matsui ◽  
K. Higashi ◽  
T. Yoshimura ◽  
M. Ito ◽  
E. Miyamoto
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Dependent Protein Kinase ◽  
Uterine Contractions ◽  
Dependent Protein

ABSTRACT Myosin light chain kinase activity in the placental region of the rabbit myometrium on day 28 of gestation was 4·7±0·1 (mean ± s.e.m.) nmol/min per mg protein, which was significantly higher than that (3·6 ± 0·1 nmol/min per mg protein) in the non-placental region. The amount of calmodulin in the placental region was 4·2 ± 0·1 μg/mg protein, which was significantly higher than that (3·2 ± 0·1 μg/mg protein) in the non-placental region. In contrast, cyclic AMP-dependent protein kinase activities showed no difference between the two regions. These findings suggest that calcium- and calmodulin-dependent protein phosphorylation is activated mainly in the placental region, and uterine contractions can occur more strongly in this part than in the non-placental region. Such enzymatic phenomena may be related to the mechanism whereby the placenta separates from the myometrium after delivery of the fetus. J. Endocr. (1986) 109, 97–100

scholarly journals Characterization of the protein kinase activities of human platelet supernatant and particulate fractions

1984 ◽  
Vol 218 (2) ◽  
pp. 285-294 ◽  
Author(s):  
S E Salama ◽  
R J Haslam
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Soluble Fraction ◽  
Deae Cellulose ◽  
Type I ◽  
Type Ii ◽  
Dependent Protein Kinase ◽  
Regulatory Subunits ◽  
Dependent Protein ◽  
Triton X

After human platelets were lysed by freezing and thawing in the presence of EDTA, about 35% of the total cyclic AMP-dependent protein kinase activity was specifically associated with the particulate fraction. In contrast, Ca2+-activated phospholipid-dependent protein kinase was found exclusively in the soluble fraction. Photoaffinity labelling of the regulatory subunits of cyclic AMP-dependent protein kinase with 8-azido-cyclic [32P]AMP indicated that platelet lysate contained a 4-fold excess of 49 000-Da RI subunits over 55 000-Da RII subunits. The RI and RII subunits were found almost entirely in the particulate and soluble fractions respectively. Chromatography of the soluble fraction on DEAE-cellulose demonstrated a single peak of cyclic AMP-dependent activity with the elution characteristics and regulatory subunits characteristic of the type-II enzyme. A major enzyme peak containing Ca2+-activated phospholipid-dependent protein kinase was eluted before the type-II enzyme, but no type-I cyclic AMP-dependent activity was normally observed in the soluble fraction. The particulate cyclic AMP-dependent protein kinase and associated RI subunits were solubilized by buffers containing 0.1 or 0.5% (w/v) Triton X-100, but not by extraction with 0.5 M-NaCl, indicating that this enzyme is firmly membrane-bound, either as an integral membrane protein or via an anchor protein. DEAE-cellulose chromatography of the Triton X-100 extracts demonstrated the presence of both type-I cyclic AMP-dependent holoenzyme and free RI subunits. These results show that platelets contain three main protein kinase activities detectable with histone substrates, namely a membrane-bound type-I cyclic AMP-dependent enzyme, a soluble type-II cyclic AMP-dependent enzyme and Ca2+-activated phospholipid-dependent protein kinase, which was soluble in lysates containing EDTA.

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