scholarly journals Purification, characterization and substrate specificity of calmodulin-dependent myosin light-chain kinase from bovine brain

1987 ◽  
Vol 247 (3) ◽  
pp. 747-756 ◽  
Author(s):  
D C Bartelt ◽  
S Moroney ◽  
D J Wolff
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Bovine Brain ◽  
Major Protein ◽  
Dependent Manner ◽  
Major Protein Band

A substrate-specific calmodulin-dependent myosin light-chain kinase (MLCK) was purified 45,000-fold to near homogeneity from bovine brain in 12% yield. Bovine brain MLCK phosphorylates a serine residue in the isolated turkey gizzard myosin light chain (MLC), with a specific activity of 1.8 mumol/min per mg of enzyme. The regulatory MLC present in intact gizzard myosin is also phosphorylated by the enzyme. The Mr-19,000 rabbit skeletal-muscle MLC is a substrate; however, the rate of its phosphorylation is at best 30% of that obtained with turkey gizzard MLC. Phosphorylation of all other protein substrates tested is less than 1% of that observed with gizzard MLC as substrate. SDS/polyacrylamide-gel electrophoresis of purified MLCK reveals the presence of a major protein band with an apparent Mr of 152000, which is capable of binding 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of MLCK by the catalytic subunit of cyclic-AMP-dependent protein kinase results in the incorporation of phosphate into the Mr-152,000 protein band and a marked decrease in the affinity of MLCK for calmodulin. The presence of Ca2+ and calmodulin inhibits the phosphorylation of the enzyme. Bovine brain MLCK appears similar to MLCKs isolated from platelets and various forms of muscle.

Expression of the catalytic domain of myosin light chain kinase increases paracellular permeability

1996 ◽  
Vol 271 (5) ◽  
pp. C1678-C1684 ◽  
Author(s):  
G. Hecht ◽  
L. Pestic ◽  
G. Nikcevic ◽  
A. Koutsouris ◽  
J. Tripuraneni ◽  
...  
Keyword(s):  
Tight Junction ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Catalytic Domain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Epithelial Tight Junction ◽  
Tight Junction Permeability ◽  
Mlc20 Phosphorylation

Contractile events resulting from phosphorylation of the 20-kDa myosin light chain (MLC20) have been implicated in the regulation of epithelial tight junction permeability. To address this question, Madin-Darby canine kidney cells were transfected with a murine leukemia retroviral vector containing DNA encoding either the catalytic domain of myosin light chain kinase (tMK) or the beta-galactosidase gene (beta-gal). Autoradiograms of sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of myosin immunoprecipitated from 32Pi-labeled transfected cells demonstrated that MLC20 phosphorylation was increased 3.1 +/- 0.9-fold in cells expressing tMK compared with cells expressing beta-gal. Phosphopeptide mapping confirmed that myosin light chain kinase was responsible for the increased MLC20 phosphorylation. Transepithelial electrical resistance, a measurement of barrier function, of tMK cell monolayers was consistently < 10% (123 +/- 20 omega.cm2) of that of monolayers comprised of wild-type cells (1,456 +/- 178 omega.cm2) or cells expressing beta-gal (1,452 +/- 174 omega.cm2). Dual 22Na+ and [3H]mannitol flux studies indicated that the decrease in resistance in tMK cells was attributable to increased paracellular flow. These data support the idea that MLC20 phosphorylation by myosin light chain kinase is involved in regulating epithelial tight junction permeability.

scholarly journals Studies on rat parotid-cell actomyosin

1984 ◽  
Vol 220 (1) ◽  
pp. 291-299 ◽  
Author(s):  
T Kealey ◽  
P J Randle
Keyword(s):  
Affinity Chromatography ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Bovine Brain ◽  
Cell Protein ◽  
Collagenase Digestion ◽  
Heat Treated ◽  
Parotid Cells ◽  
Rat Parotid

Actomyosin was partially purified from rat parotid cells dispersed by collagenase digestion and found to possess different solubility characteristics from that from (undispersed) rat parotid tissue. This is attributed to the decrease in vascular contamination effected by the isolation of parotid cells, yielding a non-muscle actomyosin [Adelstein, Conti, Johnson, Pastan & Pollard (1972) Proc. Natl. Acad. Sci. U.S.A. 69, 3693-3697]. Myosin light-chain kinase was partially purified from dispersed rat parotid cells by calmodulin affinity chromatography and shown to be activated by Ca2+-calmodulin. The calmodulin content of dispersed rat parotid cells was shown to be 6.50 +/- 0.59 ng of calmodulin/micrograms of rat parotid-cell protein (mean +/- S.E.M.), as determined by the activation of purified bovine brain phosphodiesterase by heat-treated extracts of dispersed rat parotid cells.

T1808 Helicobacter pylori Alters Gastric Epithelial Tight Junctions in a Myosin Light Chain Kinase-Dependent Manner

2008 ◽  
Vol 134 (4) ◽  
pp. A-567-A-568
Author(s):  
Lydia E. Wroblewski ◽  
Le Shen ◽  
Toni Nagy ◽  
Seth R. Ogden ◽  
Shannon S. Allen ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Tight Junctions ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Dependent Manner ◽  
Epithelial Tight Junctions

Extracellular calmodulin and its association with epidermal growth factor in normal human body fluids

1988 ◽  
Vol 118 (3) ◽  
pp. 501-NP ◽  
Author(s):  
S. Mac Neil ◽  
R. A. Dawson ◽  
G. Crocker ◽  
C. H. Barton ◽  
L. Hanford ◽  
...  
Keyword(s):  
Growth Factor ◽  
Breast Milk ◽  
Epidermal Growth Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Biologically Active ◽  
Major Protein ◽  
Major Protein Band ◽  
Normal Human ◽  
Epidermal Growth

ABSTRACT In this study we describe the occurrence of a calmodulin-like protein in normal human biological fluids. Extraction of the calmodulin-like protein from breast milk, saliva, serum and urine provided an extract with enhanced calmodulin immunoreactivity which, in the case of milk and saliva, showed a protein band co-migrating with authentic calmodulin (Mr 17 000) on sodium dodecylsulphate-polyacrylamide gel electrophoresis. However, in milk, saliva and serum a major protein band of Mr 14 000–15 000 was always observed, which we speculate may be related to calmodulin, possibly as a partially degraded form. Estimates of biologically active calmodulin in most normal extracellular fluids were of the order which we have found will stimulate cell division when added to the extracellular medium of cells in culture. Levels ranged from 0·03 nmol/l in urine to 18·6 nmol/l in breast milk, and exhibited a quantitative relationship (r = 0·79, P < 0·01) to epidermal growth factor (EGF) levels in fluids. Where EGF concentrations varied from normal (increased in saliva 24 h after oral surgery and reduced in the urine of patients with renal failure) calmodulin concentrations were similarly affected. The presence of calmodulin in serum may in part be attributable to its release from platelets which are particularly rich in calmodulin. Release of calmodulin from the platelet was associated with that of EGF and other platelet products. J. Endocr. (1988) 118, 501–509

Isolation and Characterization of Sepiapterin Reductase from Drosophila melanogaster

Pteridines ◽  
1993 ◽  
Vol 4 (1) ◽  
pp. 43-50 ◽  
Author(s):  
K.H. Yoon ◽  
K.W. Cha ◽  
S.I. Park ◽  
J.J. Yim
Keyword(s):  
Drosophila Melanogaster ◽  
Carbonyl Compounds ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Major Protein ◽  
Sepiapterin Reductase ◽  
Isolation And Characterization ◽  
Major Protein Band

Summary Sepiapterin reductase, an enzyme that catalyses the synthesis of tetrahydrobiopterin (BH4). was partially purified from Drosophila melanogaster using ammonium sulfate fractionation. Affi-gel blue chromatography and hydroxyapatite chromatography. The molecular weight of the enzyme determined by Ultrogel AcA44 column was 39,000. When the enzyme was subjected to polyacrylamide gel electrophoresis in SDS. a 38.000 MW species was found to be the major protein band. The Km values for sepiapterin and NADPH were determined to he 75.4 µM. and 14 µM, respectively. The optimal temperature and pH for the reaction were 30°C and pH 5.7-6.7. The half-life of the activity was 30 minutes when treated at 48°C The enzyme was markedly inhibited by tri-and tetravalent cations. Fe3+ . Sn4+ and divalent cation. Cd2+ . It was found that pyrimidodiazcpine (a homopterin) and 2.4-diamino-6.7-diisopropyl caused the reduction of sepiapterin reductase activity by about 50% at the concentration of 0.1 mM. Among the neurotransmitters and their precursors tested. 3 mM concentrations of melatonin and N-acetylserotonin inhibited the enzyme activity completely. In addition to sepiapterin. the enzyme uses rather broad spectrum of carbonyl compounds as substrate including menadione. p-nitrohenzaldehyde, and various dicarhonyl compounds

Immunocytochemical localization of 155 kDa myosin light chain kinase and myosin heavy chain in bovine brain

1995 ◽  
Vol 682 (1-2) ◽  
pp. 212-214 ◽  
Author(s):  
Hiroyuki Shimada ◽  
Teruo Shimizu ◽  
Hideto Kuwayama ◽  
Masashi Suzuki ◽  
Ryozo Nagai ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Bovine Brain ◽  
Immunocytochemical Localization

The effect of ageing cooled milk on the composition of the fat globule membrane

1972 ◽  
Vol 39 (1) ◽  
pp. 95-105 ◽  
Author(s):  
M. Anderson ◽  
G. C. Cheeseman ◽  
Dorothy J. Knight ◽  
W. F. Shipe
Keyword(s):  
Gel Electrophoresis ◽  
Milk Fat ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Protein Band ◽  
Major Protein ◽  
Fresh Milk ◽  
Major Protein Band ◽  
Fat Globule

SummaryThe effect of ageing fresh milk for 24 h at 4°C on the composition of 4 fractions of milk fat globule membrane (FGM) – microsomal pellet membrane (MPM), deoxycholate soluble membrane (DOCM), high density membrane (HDM) and low density membrane (LDM) – prepared from washed cream treated with sodium deoxycholate (DOC), was studied for milk of individual cows. Total FGM and its fractions were solubilized by treatment with sodium dodecyl sulphate (SDS), EDTA and β-mercaptoethanol, and the dissociated FGM proteins were separated by polyacrylamide gel electrophoresis in the presence of SDS.Ageing resulted in a greater loss of phospholipid during cream preparation than was found with fresh milk, and also in a reduction in the total amount of FGM isolated. Loss of FGM material was confined to MPM, DOCM and LDM and was accompanied by compositional changes in DOCM and LDM, quantitatively the most significant fractions. Ageing also produced changes in the gel electrophoresis patterns of DOCM, where the band of greatest mobility (mol. wt about 16000) was partially lost, and of LDM, where there was an increase observed in the major protein band.The implication of the results on the proposed models of FGM structure is discussed.

scholarly journals Thrombokinase of the Blood as Trypsin-Like Enzyme

1962 ◽  
Vol 45 (4) ◽  
pp. 103-113 ◽  
Author(s):  
J. H. Milstone
Keyword(s):  
Methyl Ester ◽  
Trypsin Inhibitor ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Protein Band ◽  
Soybean Trypsin Inhibitor ◽  
Major Protein ◽  
Bovine Plasma ◽  
Major Protein Band

Thrombokinase of the blood, while resembling enterokinase in its role of activator, is more closely analogous to trypsin in its intrinsic origin. It probably arises from a plasma precursor; but it is different from plasmin (fibrinolysin). Like trypsin, thrombokinase can activate prothrombin without the aid of other factors; however, it is potentiated by platelets plus calcium. Unlike certain tissue "thromboplastins," it does not sediment appreciably in 2 hours at 85,000 g. Like trypsin, it hydrolyzes p-toluenesulfonylarginine methyl ester (TAMe). Chromatography on DEAE-cellulose separated thrombin from thrombokinase. The TAMe esterase associated with the thrombokinase fractions was largely suppressed by soybean trypsin inhibitor, while that associated with the thrombin fractions was not. Highly purified thrombokinase was used as starting material; and thrombokinase was eluted in the last major protein band. Under these conditions stepwise elution was as effective as gradient in leading to further purification. The product of 199 liters of bovine plasma was chromatographed in 1 day; and the specific activity was comparable to that attained previously by repeated electrophoretic fractionations. The assembled data suggest that the thrombokinase protein may be approaching homogeneity.

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