Isolation and characterization of mucopolysaccharides from rat liver mitochondria

Abstract

Mucopolysaccharides were isolated from rat liver mitochondria which had been labeled with 35S-sulfate. They were prepared from trichloroacetic acid (TCA)-insoluble and -soluble fractions of lipid-free mitochondria. These fractions were digested with pronase exhaustively, and the mucopolysaccharides were recovered in the void volume fractions of gel filtration of the pronase digests on Sephadex G-50, monitored by radioactivity determination. Identification of these mucopolysaccharides was based on electrophoresis on cellulose acetate film using three different media, enzymatic and chemical degradations specific to each type of mucopolysaccharide, using chondroitinases, heparitinase, and nitrous acid. From the TCA-insoluble fraction, chondroitin sulfate A and dermatan sulfate were obtained in a ratio of about 1 : 2, based on 35S-radioactivities, whereas the TCA-soluble fraction yielded chondroitin sulfates A/C, dermatan sulfate, and heparan sulfate in a ratio of about 1 : 3 : 12. The total amount of mitochondrial mucopolysaccharides was about 3 mg/g protein, distributed between the TCA-insoluble and -soluble fractions in a ratio of about 1 : 3.