Preparation and Properties of Vertebrate Smooth‐Muscle Myofibrils and Actomyosin

Abstract

A new technique for obtaining a myofibril‐like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate‐gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin‐like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+ ‐sensitivity in tropomyosin‐free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.

Mechanism of myosin and actomyosin ATPase in chicken Gizzard smooth muscle

“…Chicken gizzard myoiibrils and myosin were prepared as in [6] . Subfragment-l was prepared by direct digestion of the myofibrils with papain by a modifica.…”

Comparison of purified anti-actin and fluorescent-heavy meromyosin staining patterns in dividing cells.

“…Myofibrils were obtained from fresh chicken gizzards by a slight modification from the method of Sobieszek and Bremel (24). Fresh gizzards were chilled on ice and cleaned.…”

Purification and Characterization of a Kinase-associated, Myofibrillar Smooth Muscle Myosin Light Chain Phosphatase Possessing a Calmodulin-targeting Subunit

“…In addition, a homogenous preparation of the PC catalytic subunit was obtained when the targeting PT subunit was degraded during purification. The phosphatase must be considered as being myofibrillar in origin because the starting material (myofibrils) was obtained by extensive fragmentation and washing of gizzard muscle, as originally described by Sobieszek and Bremel (32). The myofibrils are practically depleted of cytosolic proteins, and only tightly bound enzymes (including the phosphatase considered here) are extracted with the AA buffer containing 25 mM MgCl 2 .…”

“…32 P labeling of the isolated ReLC, used as the substrate, was carried out by adding 25 nM of purified MLCKase and 35 nM of CaM to the light chain at a 500 M concentration. After 90 min, the phosphorylation reaction was terminated by placing the tube in boiling water for 6 min, and the radioactive ATP was removed by exhaustive dialysis against our AA buffer.…”

“…Formation of a complex between the holoenzyme and MLCKase is addressed in the accompanying report (15). 32 P]ATP was purchased from DuPont NEN and diluted with cold ATP of special grade (catalog number 519 987; Boehringer Mannheim) to the required specific activity and concentration (16). Q-Sepharose CL-6B, Sephacryl S-100 HR, and CNBr-activated Sepharose 4B-CL were obtained from Pharmacia Fine * These studies were supported by grants from the Austrian Science Foundation and from the East-West Program of the Austrian Ministry for Science and Research.…”