Cystathionase Deficiency: Evidence for Genetic Heterogeneity in Primary Cystathioninuria

Summary: Optimal conditions are described for measurement of cystathionase activity in long term lymphoid cell lines. In 21 control lines established from normal subjects, cystathionase (EC 4.4.1.1) specific activity was 25.8 ± 1.7 (mean ± SE) nmole cysteine/hr/mg protein. Extracts of three lymphoid lines (NB-36, NB-95, and NB-77) established from three vitamin B6-responsive patients with primary cystathioninuria had activity of 3.6–7.3; from a B6-unresponsive patient (NB-68) had no detectable activity; from five obligate heterozygotes for cystathioninuria had activity of 11.2–18.9. Two, or possibly three, different modifications of the cystathionase molecule could be demonstrated in cultured cells from the patients with primary cystathioninuria, based on the effects of added pyridoxal phosphate (PLP) in the enzyme assay, on the extent of reaction with rabbit antihuman hepatic cystathionase, and on the ability to compete with normal lymphoid cell line enzyme extract for the antibody combining sites. When PLP is not added to the assay system, the normal enzyme extract still had 89% of its activity in 1.0 mM PLP; on agar double diffusion analysis it gave a band of identity with normal human hepatic cystathionase; the precipitin band had cystathionase activity, but inhibition by antibody could be shown in solution. Lymphoid line extract from the B6-unresponsive patient had no detectable activity in the absence or presence of PLP, did not form a precipitin band, and did not compete with normal enzyme extract for the antibody combining sites. Thus, synthesis of apocystathionase is absent or significantly reduced; alternatively, the protein produced has lost its antigenic determinants as well as catalytic activity. Extracts of the three cell lines established from the three B6-responsive patients had no activity in the absence of added PLP, but progressively greater activity was found with increasing concentrations of PLP: at 1.0 mM PLP 37, 26, and 16% of the mean normal value, respectively was attained. Cell extract from each of the B6-responsive patients formed a band of identity with normal enzyme on agar double diffusion and cystathionase activity could be demonstrated on the precipitin band only if PLP was added to the extracts before immunodiffusion. Extract of the B6-responsive lymphoid cell line NB-36 combined with the antibody sites for cystathionase, thereby blocking inhibition of normal enzyme, whereas extract of the line NB-95 had only a slight blocking effect and extract of the line NB-77 had no blocking ability. Thus, the lymphoid cell lines from the three B6-responsive patients produce a cyctsthionase with an altered capacity to combine with PLP; the enzyme in lines NB-95 and NB-77 also may have been affected in a manner which changes as well the ability to compete for the antibody combining site. These altered cystathionases, both B6-responsive and B6-unresponsive, also may represent forms more readily degradable than the normal enzyme. Antigenic identity was shown for cystathionase from various organs within a given vertebrate species, but only partial identity was observed among cystathionases from different vertebrate species. Speculation: Primary cystathioninuria is caused by different mutations affecting the cystathionase molecule. One form of cystathioninuria, vitamin B6-unresponsive, appears to result from absence of synthesis of the enzyme protein. B6-responsive forms appear to result from production of cystathionase molecules altered in ability to combine with coenzyme, but with antigenic indentity on agar double diffusion analysis maintained; in some cases, there may be changes in antibody binding capacity as well, although immunologic cross-reactivity remains.