scholarly journals A new method to precipitate myosin V from rat brain soluble fraction.

2007 ◽  
Vol 54 (3) ◽  
pp. 575-581 ◽  
Author(s):  
Hugo Christiano Soares Melo ◽  
Milton Vieira Coelho
Keyword(s):  
Atpase Activity ◽  
Rat Brain ◽  
Soluble Fraction ◽  
New Method ◽  
Myosin V ◽  
Sds Page ◽  
Assay Conditions

Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg(2+)-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg(2+)-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg(2+)-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.

Effect of mineralocorticoids on rat brain Na, K-ATPase activity

1986 ◽  
Vol 102 (4) ◽  
pp. 1380-1382
Author(s):  
V. P. Komissarenko ◽  
E. K. Efimova
Keyword(s):  
Atpase Activity ◽  
Rat Brain

scholarly journals Catecholamine Binding Macromolecule in Soluble Fraction of Rat Brain

1977 ◽  
Vol 27 (2) ◽  
pp. 213-225 ◽  
Author(s):  
Minoru Inaba ◽  
Kunie Kamata
Keyword(s):  
Rat Brain ◽  
Soluble Fraction

Local ATP regeneration is important for sarcoplasmic reticulum Ca2+ pump function

1994 ◽  
Vol 267 (2) ◽  
pp. C357-C366 ◽  
Author(s):  
P. Korge ◽  
K. B. Campbell
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Atpase Activity ◽  
Muscle Relaxation ◽  
Creatine Phosphate ◽  
Coupling Ratio ◽  
Pump Function ◽  
Membrane Bound ◽  
Low Efficiency ◽  
Atp Regeneration ◽  
Assay Conditions

Ca2+ pump function of skeletal muscle sarcoplasmic reticulum (SR) vesicles was measured by monitoring Ca2+ uptake and efflux with a Ca(2+)-sensitive minielectrode and adenosinetriphosphatase (ATPase) activity of the same preparation under the same conditions. The efficiency of Ca2+ transport into SR vesicles, defined by the amount of Ca2+ transported per ATP hydrolyzed (coupling ratio), varied significantly depending on assay conditions. Coupling ratio increased in parallel with increase in precipitating anion concentration, which is supposed to decrease accumulation of free Ca2+ inside vesicles and its subsequent efflux. Membrane-bound creatine kinase-creatine phosphate (CK-CP) system, acting as a ADP sensor and local ATP regenerator, significantly improved Ca2+ pump function when the pump worked with low efficiency (coupling ratio < 1). The effect of CK-CP system on Ca2+ pump function was also dependent on extravesicular Ca2+ concentration ([Ca2+]o), the effect being most significant at high initial [Ca2+]o. Under conditions in which SR vesicles were allowed to decrease [Ca2+]o, as occurs also during muscle relaxation, plateau values of Ca(2+)-ATPase activity were reached at significantly higher [Ca2+]o (54 +/- 5.7, n = 6), compared with leaky vesicles or the condition in which [Ca2+]o was maintained. By preventing local accumulation of ADP, generated in ATPase reactions, CK-CP system also inhibited Ca2+ efflux under conditions in which this efflux was stimulated by the increase of free Ca2+ inside vesicles. This effect was at least partially responsible for the CK-CP-supported increase in Ca2+ uptake and coupling ratios that were more expressed at low precipitating anion concentration. We hypothesize that local ATP regeneration by CK-CP system is one mechanism the cell can use to improve Ca2+ uptake by SR in emergency conditions, where excessive increase in cytoplasmic [Ca2+] may have deleterious effects.

Separation of large mammalian ventricular myosin differing in ATPase activityThis paper is one of a selection of papers published in this Special Issue, entitled The Cellular and Molecular Basis of Cardiovascular Dysfunction, Dhalla 70th Birthday Tribute.

2007 ◽  
Vol 85 (3-4) ◽  
pp. 326-331 ◽  
Author(s):  
Heinz Rupp ◽  
Bernhard Maisch
Keyword(s):  
Atpase Activity ◽  
Roe Deer ◽  
Diastolic Pressure ◽  
70Th Birthday ◽  
Aortic Pressure ◽  
Myosin V ◽  
Norepinephrine Concentration ◽  
Ventricular Myosin ◽  
Selection Of ◽  
Wild Pig

To investigate a possible heterogeneity of human ventricular myosin, papillary muscles of patients with valvular dysfunction were examined using a modified native gel electrophoresis. Myosin was separated into 2 components termed VA and VB, whereby the VA to VB proportion appeared to depend on the ventricular load. The proportion of the faster migrating band VA was correlated (P < 0.05) with end-diastolic pressure and the aortic pressure-cardiac index product. The regression based on these variables accounted for 67% of the variation in VA (R2 = 0.67). The VA proportion was, however, not significantly correlated with cardiac norepinephrine concentration. The ATPase activity of the 2 components of myosin was assessed from the Ca3(PO4)2 precipitation by incubating the gel in the presence of ATP and CaCl2. The ATPase activity of VA was 60% of that of VB. The VA and VB forms were observed also in the cat (31.4% VA), dog (32.1% VA), pig (28.5% VA), wild pig (33.7% VA), and roe deer (30.5% VA). VA and VB were not detected in the rat exhibiting the 3 isoforms V1, V2, and V3, rabbit (100% V3), and hare (86% V1). The data demonstrate a heterogeneity of large mammalian ventricular myosin, whereby an increased cardiac load appeared to be associated with a higher myosin VA proportion that exhibited a reduced ATPase activity.

scholarly journals The Mg2+-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme

1991 ◽  
Vol 278 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T L Kirley
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Rabbit Skeletal Muscle ◽  
Cross Linking ◽  
Low Concentrations ◽  
Sds Page ◽  
Molecular Masses ◽  
Relationship Of ◽  
Peptide Core

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.

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