Tryptic digestion and localization of calcium uptake and ATPase activity in fragments of sarcoplasmic reticulum

Abstract

Fractions sedimented at 25,000 g for 50 minutes from homogenates of mouse hind leg muscles were prepared according to the procedure of Von Korff originally designed to obtain intact cardiac mitochondria (41). Negatively stained preparations of this fraction (fragmented sarcoplasmic reticulum) appear to be essentially free of contamination with mitochondrial fragments and myofilaments. Vesicles consisting of a globular head with a diameter of 0.1–0.2 μ and one or more tail-like portions with a roughly constant width of 350 and a varying length of 0.05–1 μ (most frequently 0.2 μ) predominate. The surface of the vesicle, both of the head and tail portion, is covered with particles approximately 40 in diameter and spaced 50–80 from each other, the center being 40–60 from the surface of the vesicular membrane. Ca deposits, in the presence of oxalate, were localized in the globular portion. ATPase activity, as judged electron microscopically from the deposition of lead phosphate, was also localized in the globular portion, very frequently at the junction with the tail. Digestion of vesicles with trypsin caused changes in both the Ca uptake and ATPase activity. With a trypsin-vesicular protein ratio of 1:25 to 1:100 a rapid drop in Ca uptake was accompanied by an increase in ATPase activity. With even lower (1:300) ratios, a rapid initial drop in Ca uptake was eventually followed by a slower phase of decrease; the ATPase in this case increased monotonically with an inflection point subsequent to the rapid drop in Ca2+ uptale. The digestion caused structural disruption of the vesicle, first in the tail, and eventually led to swelling of the globular portion and the appearance of areas devoid of regularly arranged particles on the membrane.