Cystathionine β-Synthase Deficiency: Differences in Thermostability between Normal and Abnormal Enzyme from Cultured Human Cells

Abstract

Summaryhepatic synthase activity obsewed in some patients receiving megavitamin therapy may not be mediated by a protective orThe thennostability of cystathionine 8-synthase and the effect stabilizing effect of the coenzyme, of pyridoxal phosphate (PLP) and other B, vitamers on this thennostability were studied in extracts of cultured skin fibroblasts from normal subjects, from heteroz~gotes for s~nthase The precise nature of the mutation(s) responsible for cystadeficiency, and from patients with homocystinuria due to Syn-thionine psynthaX deficiency is not known. However, we thaw deficiency. recently demonstrated a qualitative difference in a physical Incubation of crude extracts of normal fibroblasts at 55" property of hepatic synthase from affected individuals, i . e . , the reincu cub at ion) for short periods prior to assay consistently response to heat, which is corrected by vitamin B, therapy (10). resulted in an increase of cystathionine synthase activity (acti-Extracts of liver from normal individuals had increased cystavation) that reached a maximum at 2 min (Fig. 1). Further thionine synthase activity after brief incubation at 55" before preincubation resulted in inactivation. Addition of 0.4 mM PLP assay (preincubation). ~~~t -i n d u c e d activation was observed to the ~mincubation mixture resulted in greater activation that also in extracts of liver obtained from heterozygotes for synthase reached a maximum at 3-5 min. The subsequent inactivation deficiency, but occurred in extracts from patients only during phase proceeded at a slower rate than had 0ccurred in the vitamin B, therapy. These findings suggested that in patients absence of PLP, resulting in a doubling of the half-life of with homocystinuria due to cystathionine synthase deficiency thermal inactivation at 55"-PLP was the only one of the Be this enzyme is structurally altered. Whether or not the mechavitamers to have a protective effect.nism of action of the vitamin involved a direct effect on the No significant activation of synthase was observed when synthase molecule remained unanswered. extracts of fibroblasts from s~nthase-deficient patients were These results led us to investigate further the thermostability preincubated at 55"-Cells from eight patients were studied: of cystathionine synthase and the effects of the in vitro addition extracts from those of four patients demonstrated activity too of PLP, the coenzymatically active form of vitamin B,, using low to permit measurement of thennostability; extracts of cells cultured skin fibroblasts from normal subjects, heterozygotes, from two patients were less thermostable than those from and patients with cystathionine synthase deficiency. control subjects and showed a protective effect of PLP; extracts of cells from two other patients (sibs) showed very high heat stability in the absence of PLP, but significant inactivation of MATERIALS AND METHODS synthase in the presence of the coenzyme (Fig. 2, A and B ) . Three patterns of response to heating were observed with Skin from the in...

Homocystinuria: Studies on cystathionine β‐synthase,S‐adenosylmethionine synthetase and cystathionase activities in skin fibroblasts

“…Differences between the cell culture and enzyme assay systems may be at least partially responsible for this (Griffiths and Tudball, 1976;Uhlendorf et al, 1973). However, the widely reported degree of genetic heterogeneity associated with the condition is probably also a major contributory factor (Fleisher et al, 1978) and may explain the apparent link between the degree of enzyme defect and the severity of the pre-treatment symptoms, a relationship which appears to be independent of pyridoxine response. The nature of the genetic defect in the case of the pyridoxine responsive patient (Mi.L.)…”

Recent advances in the mechanism of pyridoxine‐responsive disorders

“…These include measurement of residual CS activity in cultured fibroblasts; effects of PLP addition on residual CS activity in fibroblasts (Seashore et al, 1972;Kim and Rosenberg, 1974;Fowler et al, 1978) and in liver (Mudd et al, 1970;Gaull et al, 1974); studies of CS activity in cells grown in pyridoxinedepleted medium (Lipson et al, 1980a); heat stability of mutant CS and the effects of PLP on this (Longhi et al, 1977;Fowler et al, 1978;Fleisher et al 1978); and immunologic studies (Skovby et al, 1982). There have been three main studies of residual CS activity in cultured fibroblasts from homocystinuric patients, based on the reasonable assumption that fibroblasts reflect findings in the liver.…”

Vitamin B-6 and Sulfur Amino Acid Metabolism

“…For the reasons previously listed, this research has been pursued in cultured skin fibroblasts (57,58). The heat-induced activation of the enzyme is retained by normal skin fibroblasts, although pyridoxal 5' -phosphate does not have such a comRlete protective effect on the subsequent inactivation of the enzyme from this source as it did on the normal liver enzyme (Fig.…”