Purification and Characterization of Myosin from Calf Brain

Abstract

Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.

Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells

“…7b). These activities were lower than those of gizzard and platelet myosin II, as has been previously reported by others [Hobbs and Frederiksen, 1980;Malik et al, 1983;Matsumura et al, 1988].…”

A calcium‐protease activator associated with brain microsomal‐insoluble elements

“…110 kDa, usually composed of 72-82 and 25-30 kDa subunits 151. The smaller subunit obtained from bovine brain shows an approximate Mr of 17000 and is required for maximum activity of the larger subunit [13,25]. An activator of calpain partially purified by DeMartino and Blumenthal 1201 from bovine brain cytosol is apparently similar to the activator described in this paper, but its amount in the cytosol is extremely small (20-30,ug/40 g brain) [20].…”

Multiple phosphorylated variants of the high molecular mass subunit of neurofilaments in axons of retinal cell neurons: characterization and evidence for their differential association with stationary and moving neurofilaments.

“…One or both of the 200-kD soluble polypeptides may correspond to brain myosin (48), which migrated to an apparent molecular mass in the 197-200-kD range (Lewis, S. E., and R. A. Nixon, unpublished observations). In addition, we have observed that several [35S]methionine-labeled axonal polypeptides with similar apparent molecular masses coassemble with microtubules, suggesting that one or both may be microtubule-associated proteins (Nixon, R. A., and I. Fischer, unpublished observations).…”