A series of substituted salicylates and other structurally unrelated compounds having the common property of metal-ion complexation have been examined for the ability to inhibit the incorporation of sulfate into mucopolysaccharide. Several very potent inhibitors have been found among the mercaptoamines. It may be stated that the course of mucopolysaccharide sulfation is definitely affected by metal-binding agents, generally by inhibition. A close correlation between per cent inhibition of sulfation and metal-binding strength was observed, which establishes that the mode of inhibition is one of metal binding of the metalloenzymes involved i n mucopolysaccharide sulfation.
ALICYLATES have been shown to inhibit the S incorporation of "S into cartilage mucopolysaccharide (1). Whitehouse and Bostrom (2) have postulated that salicylate exerts its inhibitory effect on this reaction either by acting as an alternative sulfate acceptor, being itself sulfated, or by complexation of the metalloenzymes involved in mucopolysaccharide sulfation (2, 3). The results reported here permit a more definite conclusion to be made.
A series of substituted salicylates and other structurally unrelated compounds having the common property of metal-ion complexation were tested for the ability to inhibit the incorporation of into veal costal cartilage, according to the procedures of Rubin and Howard (4) and Whitehouse and Bos- trom (2). It may be stated that metal complexation agents definitely affect the sulfation, generally by inhibition. A correlation between metal-binding strength and degree of inhibition is apparent, although agents with comparatively high or low metalbinding constants were found t o stimulate the incorporation of sulfate.
Methods
Incubation Procedure.-Incubations were carried out a t 37" in air with continuous slow shaking. Costal cartilage from a newly killed calf was sliced to 0.5 mm. thickness and in each determination five slices were preincubated in 10 ml. of Krebs-Ringer solution with the inhibitory agent ( 5 X lod3 M ) . After 15 min., sodium sulfate 36S (5 X lo6 counts/ min.) was added, and incubation was continued for 3 hr. The medium was then decanted, and the slices were washed three times with chilled (4') Krebs-Ringer solution, blotted, and stored in the moist state at 4" until taken for counting.
Measurement of Incorporated Radioactivity.-Approximate (Direct) A ssuy.-The intact, moist, circular cartilage slices (dry weight approximately 40 mg.) were placed in tared planchets and dehydrated by standing overnight in the refrigerator and drying for 10 min. at 100". The planchets were weighed, and the radioactivity of the slices was