T. L. Oei

Quantitative radiochemical enzyme assays in single cells: Purine phosphoribosyl transferase activities in cultured fibroblasts

Abstract An ultra-microchemical method using radioactive substrates has been developed for enzyme activity measurements at the single cell level. In order to demonstrate the possibilities of this radiochemical microassay, activity measurements of hypoxanthine-guanine phosphoribosyl transferase (HG-PRT) and of adenine phosphoribosyl transferase (A-PRT) in isolated human fibroblasts are described. There was a linear relationship between the number of cells incubated and the enzyme activities found. It was observed that the HG-PRT activity in single, skin derived, fibroblasts did not differ from that in amniotic fluid derived fibroblasts, thus providing a new, quantitative assay for rapid prenatal diagnosis in the Lesch-Nyhan syndrome.

Purine Metabolism in Intact Erythrocytes from Controls and HG-PRT Deficient Individuals

The present paper reports on studies on the purine metabolism in normal and HG-PRT deficient human erythrocytes.

Characterisation of purine nucleoside phosphorylase from fibroblasts using ultra-microchemical methods.

A new technique to quantitate nucleoside phosphorylase (NP) activity in single or small numbers of counted visually selected cells is presented. Fibroblasts were cultivated on the plastic film bottom of culture dishes. After lyophilisation in situ, plastic film leaflets carrying a counted number of cells were cut out and tested for NP activity. Some properties of NP, including temperature stability, pH optimum and substrate affinity, have been studied. The data obtained suggest that Np might play a regulatory role in the purine interconversion pathway.

A new ultramicrochemical assay for purine nucleoside phosphorylase

Abstract An ultramicrochemical method using radioactive substrates has been developed for the quantitation of purine nucleoside phosphorylase (NP) activity in single cells. NP activity can reproducibly be measured in both directions of the enzyme reaction. A quantitative relationship between the number of cells incubated and the NP activity was found. Fibroblasts from a patient with impaired T-lymphocyte function showed a substantial reduction of NP activity (both directions). In contrast to previously available methods, the present ultramicro approach makes it possible to analyze normal and immunodeficient single B and T lymphocytes.

Molecular and tissue-specific heterogeneity in HPRT deficiency

In several patients with different degrees of HPRT deficiencies, residual activities have been determined in both lysed and intact erythrocytes. No close correlation could be found between the degree of HPRT deficiency and the severity of the clinical expression. Unless HPRT activity in both intact and lysed erythrocytes was below detection level, the residual activity in intact red blood cells was higher than in lysates. Tissue-specific heterogeneity was illustrated with a patient suffering from X-linked gout. Lysates from erythrocytes, leukocytes, and cultured fibroblasts showed 1%, 8%, and 100% of normal HPRT activity, respectively. Characterization of the erythrocyte and fibroblast HPRT from this patient showed no kinetic abnormalities. However, there was a decreased heat stability. It is concluded that for a better understanding of the pathophysiology in HPRT deficiency studies on nucleated cells from the different tissues are needed.

Incorporation of Purine Bases by Intact Red Blood Cells

Mature human erythrocytes do not possess the ability to perform purine synthesis de novo (1–3). These cells meet their requirements for purine nucleotides by re-utilisation of preformed bases that are derived from the diet and those released from other tissues into the blood circulation (1,4).

Development of a Micro HG-PRT Activity Assay: Preliminary Complementation Studies with Lesch — Nyhan Cell Strains

The main purpose of the present study was to look with the heteropolykaryontest (ref. 1,2) for genetic complementation between different HG-PRT deficient cell strains, which had been fused and cultured in Plastic Film Dishes (PFD’s ref, 3,4) A radiochemical HG-PRT microassay, based on the use of the Parafilm Micro Cuvette (PMC; ref. 3,4) was developed, which permits the quantification of the enzyme activity at the single cell level.

Heterozygote detection in Fabry's disease using mailed hair roots

Hair roots can successfully be employed as an easily obtainable biopsy material for heterozygote detection in inborn errors of metabolism (Gartler et al., 1971; de Bruyn, Oei & ter Haar, 1974; De Bruyne; ah, 1979). We have recently described an easy and rapid method for measuring a-galactosidase and acid phosphatase activities in individual human hair roots (Vermorken et al., 1978). It was shown that acid phosphatase is a suitable reference enzyme with respect to a-galactosidase since it has a comparable distribution in the hair follicle and it is equally stable on storage. In this study we report that mailing hair roots does not interfere with the possibility of measuring the enzyme activities and distinguishing between patients, heterozygotes and normal controls. Hair roots are plucked, preferably from the scalp, using a pincct such as nr. 805 or nr. 970 Medicon Instruments (Tuttlingen, F.G.R.). Twenty-five follicles, from each individual, with visible bulb and sheath (Fig. iA) are attached to

Fluctuating adenosine deaminase activities in cultured fibroblasts

Abstract A sensitive and reproducible ultramicroassay for adenosine deaminase in cultured human fibroblasts is presented. The assay is based upon the incubation of a counted number of lyophilised cells (5 or 10) with a mixture containing [8-14C]adenosine. The mean ADA activity of desiccated lyophilised fibroblasts from the same seeding remained constant for at least 1 week when stored at 18°C. The mean fibroblastic ADA activities from control strains displayed considerable fluctuations in different passages. Because culture conditions were kept identical, the fluctuations were attributed to biological rather than to methodological variations. It was concluded that even with a sensitive quantitative measurement of ADA activity in amniotic fluid-derived fibroblasts it might be very difficult to discriminate between normal, heterozygous, and ADA-deficient foetuses.

Metabolic cooperation studied by a quantitative enzyme assay of single cells

Abstract A new method making use of a radiochemical enzyme assay at the single cell level is presented to investigate metabolic cooperation, a widely studied form of cellular communication. In this case metabolic cooperation between normal human fibroblasts and fibroblasts derived from a patient deficient for the enzyme hypoxanthine-guanine phosphoribosyl transferase has been studied. A mixture of an equal number of both cell types was cultured in close physical contact and after trypsinisation, replating and culturing the cells for several hours in a high dilution, quantitative enzyme measurements with individual cells isolated from the mixture were carried out. From the distribution curve of the enzyme activities of the individual cells the conclusion could be drawn that a macromolecule, either the enzyme itself or DNA or mRNA, coding for that enzyme, is transferred from normal to mutant cells.