We investigated the peroxisomal ,8-oxidation system in liver from a patient with clinical features similar to those in the cerebrohepatorenal (Zeliweger) syndrome and with elevated levels in body fluids of very-long-chain fatty acids and intermediates in

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1. A two‐step procedure is described for the isolation of lysosomal α‐glucosidase from human urine. In the second step, affinity chromatography on Sephadex G‐100, two fractions with acid α‐glucosidase activity were obtained. 2. Fraction I contained α‐glucosidase of Mr 109000, whereas fraction II contained components of Mr 76000 and 70000. 3. α‐Glucosidase in fraction I had an Mr similar to that of the precursor of α‐glucosidase detected in the medium of fibroblasts after labelling with [14C]leucine. The components in fraction II had Mr identical to those of the mature forms of α‐glucosidase found in placenta or cultured human skin fibroblasts. 4. α‐Glucosidase in fraction I contained mannose 6‐phosphate (3.5 mol/mol polypeptide). No mannose 6‐phosphate was present in the components in fraction II. 5. Fraction I, but not fraction II, was avidly endocytosed by α‐glucosidase‐deficient cultured human skin fibroblasts. Endocytosis of fraction I was inhibited by mannose 6‐phosphate. 6. The pH optimum and Km values for p‐nitrophenyl α‐glucoside, maltose and glycogen of fractions I and II α‐glucosidase were almost identical. However, the activity with glycogen relative to that of either p‐nitrophenyl α‐glucoside or maltose was lower in fraction I than in fraction II. 7. It is concluded that fraction I consists of the precursor form of α‐glucosidase and fraction II of the mature forms of the enzyme. 8. The importance of urine as a source of precursors of lysosomal enzymes is discussed.

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The maturation of lysosomal a-glucosidase in cultured human skin fibroblasts was studied using a monoclonal antibody that distinguishes between the precursor and mature forms of the enzyme.1. Monoclonal antibodies against a-glucosidase isolated from placenta were produced by the hybridoma technique [Hilkens et al. (1981) Biochim. Biophys. Acta 678, 7-11]. One of these monoclonal antibodies, that synthesized by clone 4368, reacts with the mature forms, but not with the precursor form of a-glucosidase isolated from urine.2. By means of pulse-labelling studies, it could be shown that monoclonal antibody 4368 does not react with either the intracellular or the secreted precursor of a-glucosidase from cultured human skin fibroblasts. However, the antibody does react with the intermediate and mature forms of a-glucosidase.3. Endocytosis of the precursor of a-glucosidase from urine by fibroblasts is followed by its conversion to a form with lower molecular mass. 4. After endocytosis urinary precursor a-glucosidase is converted to a form that binds to monoclonal antibody 4368. The t'Iz for this conversion is 2 h. The conversion is inhibited by addition of leupeptin to the culture medium. 5.It is concluded that a thiol proteinase is involved in the maturation of a-glucosidase in fibroblasts and the appearance of the antigenic determinant for 4368.Lysosomal a-glucosidase, like all other lysosomal enzymes of which the 'life-cycle' has been studied [l], is synthesized as a large precursor that is processed to mature forms of lower molecular mass [2]. Although very little is known about this proteolytic process, it is thought to take place in the lysosomes Two factors have led us to select a-glucosidase as a convenient model for studying the proteolytic processing of lysosomal enzymes. The first is the availability of substrate amounts of a precursor form of a-glucosidase; its isolation from human urine is described in the preceding paper [4]. The second is the availability of a monoclonal antibody that enables us to distinguish between different forms of a-glucosidase.Using the hybridoma technique [5], we have produced a number of monoclonal antibodies against placental a-glucosidase [6]. In the present paper we show that one of the antibodies, that produced by clone 4368, does not react with the precursor of a-glucosidase, whereas it does react with the intermediate and mature forms of the enzyme. We describe the use of this antibody in studying the processing of the precursor form of a-glucosidase from urine after endocytosis by cultured human skin fibroblasts and, in addition, the processing of labelled endogenous precursor. PI.Abbreviations. SDS, sodium dodecyl sulphate; PAGE, polyacrylEnzyme. a-Glucosidase (EC 3.2.1.20). amide gel electrophoresis. MATERIALS A N D METHODS Partial purification of monoclonal antibodiesMonoclonal antibodies were partially purified from the culture medium of the hybridoma cell lines by 50 % (NH,),SO, precipitation at 4 "C. The precipitate was dissolved in water, dialysed against phosphate-b...