A Phosphorylase in Calcifying Cartilage. ∗

Abstract

It has been shown by histochemical 1 , 2 , 3 and chemical 3 , 4 methods that cartilage cells preparatory to calcification accumulate large stores of glycogen, which disappear abruptly just prior to or during the early course of calcium deposition. The processes of glycogen depletion and of calcification appear to be closely coürdinated, suggesting some inter-relation; 1 , 2 for example, phosphoric esters formed during glycogen breakdown might serve as substrates for bone phosphatase. 2 , 3 , 4 , 5 This inherently plausible hypothesis, as yet unsupported by direct evidence, has been largely ignored in current speculation concerning the mechanisms of calcification.

In liver and muscle, according to recent studies by Cori, 6 Parnas 7 and others, the major pathway of glycogen degradation involves phosphorolysis and the formation of intermediate phosphoric esters. The glycogenolytic cycle is initiated by the reaction

Glycogen + inorganic phosphate glucose-1-phosphate

which is catalyzed by an enzyme, phosphorylase, shown to be present in liver, muscle, and certain other tissues. Evidence is offered here that a similar phosphorylating enzyme system, associating glyco-genolysis with calcification, occurs in calcifying cartilage.

Methods. The proximal and distal ends of the femora, tibiae and humeri of young rabbits (500-800 g) or rats (20-25 days) were used, after careful removal of all muscle and most shaft bone. The tissue was rapidly weighed, minced and ground, then transferred to glass-stoppered centrifuge tubes. These, in experiments with tissue pulp, contained a 1:1 proportion of digest mixture prepared as indicated in Table I (1.0 cc was used where the tissue weighed less than 1.0 g), with which the pulp was quickly stirred. After brief centrifugation, duplicate control samples were removed from the supernatant with a Lang-Levy micropipette. The digest was then mixed and allowed to stand, samples being taken at intervals after preliminary centrifugation.