Ronald G. Davidson

Differential assay of arylsulfatase A and B activities: a sensitive method for cultured human cells.

Abstract Conventional colorimetric methods for determining arylsulfatase A and B activities are cumbersome and insufficiently sensitive for microassays. A more sensitive fluorogenic technique to determine activities of the two enzymes in human cultured fibroblasts without any prior treatment has been developed. In the assay mixture, lead ions (3 m m ) are used to remove endogenous inhibitors and silver ions (0.3 m m ) are used to inhibit specifically arylsulfatase A activity. This assay is optimal at pH 5.6 for both enzymes, linear for up to 2 h of incubation at 37° at protein concentrations of 3 to 10 μg for arylsulfatase A and 7 to 40 μg for arylsulfatase B. “Lysis buffer” is the best for extracting arylsulfatase A and 0.15 m sodium acetate for extracting arylsulfatase B. This technique is three- to fivefold more sensitive than the conventional method and applicable to assays of arylsulfatase A or B activity in fibroblasts from patients with various sulfatase deficiencies.

Biochemical variability of arylsulphatases-A,-B and-C in cultured fibroblasts from patients with multiple sulphatase deficiency

Multiple sulphatase deficiency (MSD) in man is inherited as an autosomal recessive trait and associated with deficient activities of various sulphohydrolases. Cultured fibroblasts from seven different patients were assayed for arylsulphatases-A,-B and-C activities. On the basis of the results, they may be classified into three groups: I, deficient in all three arylsulphatases: II, deficient only in arylsulphatases-A and-C with half or near-normal arylsulphatase-B; electrophoretically, arylsulphatase-A activity bands are undetectable as in metachromatic leukodystrophy; III, same as in II except electrophoretically, the residual arylsulphatase-A is detectable as faint activity bands similar to those in pseudo arylsulphatase-A deficiency. In addition to the variability among different strains, within the same strain of MSD or normal cells, each enzyme activity increased several fold with increasing time in culture. These sources of biochemical variability among and within different cell strains have not been recognized before in the study of this apparently monogenic trait with multiple enzyme deficiencies. They may account for some of the discrepancies reported in the literature on arylsulphatase activities among cultured cells from different multiple sulphatase deficient patients.

Action of surface-active agents on arylsulfatase-C of human cultured fibroblasts☆

Arylsulfatase-C is a microsomal membrane-bound enzyme with unusual biochemical and genetic properties. Whether it is a single enzyme hydrolyzing different sterol sulfates or a complex of enzymes, with each enzyme hydrolyzing a specific substrate, has not been resolved. Its locus has been mapped to the human X chromosome but appears to escape inactivation. As a first step to clarify its biochemical properties, a systematic search was undertaken for a suitable detergent that can release this enzyme from human cultured fibroblast membranes in a form that is biologically active and electrophoretically mobile. Four non ionic (Triton X-100, Nonidet P-40, Digitonin, and saponin) and four amphoteric (lysolecithin, Zwittergent, Miranol, and Chaps) detergents were studied. At 1% concentration, they released more than 80% of the activity into a low-speed supernatant fraction, except for Saponin which had no effect. With Triton X-100 and Miranol representing the two groups of detergents, significant release occurred only when the detergent concentrations exceeded their respective critical micelle concentrations, thus indicating that arylsulfatase-C is an integral membrane protein. The apparent molecular weight of the detergent-enzyme complex, ascertained by gel filtration, was 85,000 in the presence of Triton X-100 and 335,000 in the presence of Miranol. However, only the preparation solubilized by Miranol (and Chaps, to a lesser degree) permitted migration of the enzyme in nitrocellulose acetate during electrophoresis at pH 7.0, while the enzyme extracted with all other detergents remained at the origin. Therefore, the amphoteric detergent, Miranol, appears to fulfill the requirements for further characterization of the membrane-bound arylsulfatase-C in human cultured fibroblasts.

Gene dosage effects in human diploid and tetraploid fibroblasts

The effects of cell ploidy on the biochemical characteristics of cultured cells were compared using human diploid vs tetraploid fibroblasts isolated with a non-selective method. Their DNA replication was compared by thymidine incorporation, and DNA content by Feulgen staining and quantitative analysis. Their RNA and protein content, cell sizes and the specific activities of glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD) were assayed quantitatively. With the exception of RNA content, all other parameters demonstrated a 2-fold increase reflecting the increase in cell ploidy. These direct gene dosage effects on the genetic material and functional expression of the human genome were in contrast to previous observations in other species and validate the use of human intraspecific euploid hybrids for biochemical and genetic studies.

Perspectives in the teaching of human genetics

There is a growing sense of concern among geneticists with regard to the place occupied by their discipline in the curriculum throughout the entire educational system. Surveys of genetics teaching in North American medical schools, especially over the last 30 years, have shown dramatically increasing interest in the subject (Table I). For example, the proportion of schools with formal courses in genetics rose from 8.6% in 1953 (Herndon, 1954) to 74.7% in 1978 (Childs et al., 1981). Similar studies have revealed the importance of genetics in the education of dentists (Sanger, 1980; Farrington et al., 1982), nurses (Brantl and Esslinger, 1962; Feetham, 1984; Mertens et al.,1984; Monsen, 1984), nurse practitioners (J.K. Williams, 1983), and social workers (Mealer et al., 1981; Bishop, 1984). The last decade has even seen the emergence of an entirely new type of genetic practitioner, the genetic counselor or genetic associate (Kenen, 1984; Marks, 1982, 1984; Marks and Richter, 1976).

Somatic cell hybridization studies on the genetic regulation and allelic mutations in metachromatic leukodystrophy

SummaryMetachromatic leukodystrophy is a hereditary neurodegenerative disease associated with deficient arylsulfatase A activity. Clinical variants differ in onset times and severity of the disease but each breeds true within families. Somatic cell hybridization techniques were used to clarify the genetic relationship among these mutants. Hybrid clones isolated with a nonselective method from fusing fibroblasts of an infantile and a juvenile variant did not show complementation of arylsulfatase A activity. Hence, these clinical variants are allelic mutants.Previous somatic cell hybridization studies suggested that “arylsulfatase A-deficiency” is a dominant phenotype, in contrast to its apparent recessive mode of inheritance. To resolve this discrepancy, hybrid clones from fusing normal and arylsulfatase A-deficient fibroblasts were isolated nonselectively. They continued to express arylsulfatase A activity. Hence, even in vitro, “arylsulfatase A-deficiency” remains as a recessive phenotype.

The use of fluorogenic substrates to locate rapidly enzyme activity in chromatographic fractions

Abstract A rapid method of screening chromatographic fractions has been developed for enzymes which metabolize fluorogenic substrates. Samples of the eluted fractions are applied to cellulose acetate gels and then incubated with the specific fluorogenic substrate. Fractions which possess enzymatic activity are visible as fluorescent spots when the gels are examined under long-wave ultraviolet light.