Homocystinuria: Studies in Tissue Culture

Bw, Uhlendorf; Eb, Conerly; Sh, Mudd

doi:10.1203/00006450-197307000-00008

Cited by 58 publications

(35 citation statements)

References 29 publications

(79 reference statements)

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“…Furthermore, it is known that heterozygotes for synthase deficiency demonstrate only 20-25% of the mean control activity (2, 3 , 5, 15), rather than the 50% expected on the basis of the simple gene-dose relationship observed with monomeric enzymes. The present results lend further support to the suggestion (3,15) that in the heterozygote the normal polypeptide chains are influenced by the abnormal polypeptide chains via combination into multimeric enzyme units. With such multimeric enzymes, whether they represent the products of multiple alleles or multiple loci for synthase, a broad spectrum of response to heat might be anticipated.…”

Section: Discussionsupporting

confidence: 79%

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

et al. 1978

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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...

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Section: Discussionsupporting

confidence: 79%

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

et al. 1978

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“…In contrast, cells from five of seven nonresponsive patients had no detectable residual synthase activity. However, as in the study of Uhlendorf et al (15), there are exceptions to the thesis that in vivo pyridoxine responsiveness can be simply correlated with the presence of residual MUTANTS synthase activity in cultured cells. Cells from one responsive patient (mutant 6) had no residual activity even when 0.7 mg of protein was included in the assay, and cells from two nonresponsive patients (mutants 3 13 and 14) contained measurable activity in all batches of cells studied.…”

Section: Methodsmentioning

confidence: 51%

Homocystinuria

Fowler¹,

Kraus²,

Packman³

et al. 1978

J. Clin. Invest.

103

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A B S T R A C T We have compared in vivo pyridoxineresponsiveness with in vitro cystathionine /8-synthase activity in extracts of confluent fibroblasts from 14 synthase-deficient patients. Enzyme activity was measured with and without addition of its cofactor, pyridoxal-5'-phosphate, using a radioisotopic assay which detects as little as 0.25% of control activity. Six of seven lines from responsive patients had measurable activity without the added cofactor (0.6-15% of mean control). Two of these lines showed a five-and sevenfold stimulation of cystathionine ,B-synthase activity with added pyridoxal-5'-phosphate; in the other four, the cofactor addition increased activity only modestly, as in controls. Two of seven lines from nonresponsive patients had measurable activity (each 3% of mean control) which increased two-and fivefold with the added cofactor. Cystathionine ,8-synthase activity was undetectable in one line from a responsive patient and in five lines from nonresponsive ones. To characterize control and mutant synthase further, dissociation constants for pyridoxal-5'-phosphate were estimated and thermostability (54°C) was studied in two control and five mutant lines. In one mutant, both parameters were normal; in the others, the affinity for the cofactor was reduced 3-to 11-fold and thermostability was much impaired. We conclude that at least three general classes of cystathionine 83-synthase mutants exist: those with no residual activity; those with reduced activity and normal affinity for pyridoxal-5'phosphate; and those with reduced activity and a reduced affinity for the cofactor. Pyridoxine responsiveness in vivo cannot be correlated simply with the presence or absence of residual synthase activDr.

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“…Some patients respond to large doses of B6 with decreases in plasma and urinary homocystine and methionine, whereas other, unrelated patients show no such response (16,17,19). These responses to B6 are thought to be genetically determined manifestations of heterogeneity in cystathionine synthase-deficient patients (18). Therefore, each patient was classified with respect to his pyridoxine responsiveness (Table I), and the experiments were designed so that each patient would be in his basal state during the present studies, rather than in pyridoxine-induced response.…”

Section: Resultsmentioning

confidence: 99%

“…The control subj ects were normal volunteers aged [18][19][20][21][22][23][24] yr, in good health as indicated by medical histories, physical examinations, and laboratory tests, including urinary amino acid analyses.…”

Section: Methodsmentioning

confidence: 99%

“…Recent indications of genetic heterogeneity in cystathionine synthase-deficient patients (18), in conjunction with an increasing realization of the possible functional significance of small residual activities of cystathionine synthase in deficient patients (19), led us to question whether the generally accepted opinion of the essential nature of cyst (e) ine for such patients were not an oversimplification. As explained more fully in the Discussion, it seemed possible that patients with as little as 1-2% of the normal activity of cystathionine synthase might not require cyst(e)ine.…”

Section: Introductionmentioning

confidence: 99%

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