BRAIN LYSOSOMES AND LYSOSOMAL ENZYMES

Summary Biochemical, enzymatic and metabolic studies of purified lysosomal fractions from rat kidney and liver have shown that lysosomes contain two types of glycoproteins, enzyme glycoproteins, and soluble acidic lipoglycoproteins (SALGP) without catalytic properties. The SALGP comprise about 50% of the lysosol protein, are readily labelled by precursors of phospholipids, aminosugars, sialic acid and peptides, have a molecular weight of about 15,000 and a pI of about 4. Most or all of the acid hydrolases are glycoenzymes that are synthesized in a restricted portion of the rough endoplasmic reticulum in the form of basic glycoproteins containing N-acetylglucosamine and mannose. N-Acetylneuraminic acid (NANA) and additional N-acetylglucosamine are attached to the nascent glycoenzymes in the Golgi apparatus. The lysosomal enzymes are packaged in lysosomes exclusively as acidic sialoglycoproteins with pIs between 3.5 and 4.9. The basic forms of the lysosomal hydrolases apparently originate from the corresponding acidic forms during biodegradation through a partial autolytic cleavage of NANA, carbohydrate and (glyco)peptide. Similar changes occur during incubation of lysosomal extracts in vitro at an acid pH. The molecular heterogeneity of lysosomal enzymes has been investigated in submitochondrial and submicrosomal fractions of rat brain. The solubility varies with the enzyme species and increases with increasing pH and Triton X-100 concentration of the medium. The hydrolases in the nerve ending (NE) fraction are generally less soluble than those in the mitochondrial-lysosomal (M-L) fraction, but are quantitatively solubilized in alkaline buffer containing 0.5% (v/v) Triton X-100. Polyacrylamide gel electrophoresis in an alkaline system (pH 8.8) resolves multiple components of acid phosphatase, organophosphorus-resistant acid esterase, β-glucuronidase, arylsulfatase and β-N-acetylhexosaminidase. The patterns vary with the subcellular fraction. The acidic forms of the enzymes are labile and are readily converted to more basic forms, probably owing to a partial autolytic cleavage of NANA, sugar and peptide residues. Gel electrophoretograms of a Triton X-100 extract of the lysosome-enriched, dense M-L and d fractions show distinctive patterns for the various enzymes. On the other hand, gel electropherograms of the lighter, membrane-rich fractions, including the myelin and NE fractions, reveal poor penetration of protein and enzymes, and the enzymes, and the enzyme patterns are single and stereotyped, usually consisting of a major cathodic and a minor anodic component. These enzyme components also stain for protein, carbohydrate, lipid and acidic groups, indicating that they are associated with acidic glycolipoglycoproteins of membranous origin as stable multienzyme-glycolipoglycoprotein complexes. Similar complexes have been identified by ultracentrifugal flotation of Triton X-100 extracts of membrane-rich fractions. Polyacrylamide gel electrophoresis in an acidic system (pH 4) permits adequate penetration of protein and enzyme into gels and yields uniform patterns of the hydrolases for the various subcellular fractions, while excluding the acidic glycolipoglycoproteins from the gels. However, the latter system suffers from the handicap that the acidic isoenzymes are labile at this low pH. Pretreatment of the NE fraction with cold acetone markedly increases the solubilization of protein and lysosomal hydrolases in detergent-free alkaline buffer, and sharply modifies the electrophoretic patterns of soluble acidic glycolipoglycoproteins and the hydrolases in Triton X-100 extracts. Thus, the partial binding of lysosmal hydrolases to membrane glycolipoglycoproteins may alter several properties of these enzymes including their solubility, buoyant density and electrophoretic mobilities in polyacrylamide gels. Solubilization studies involving purified liver and kidney lysosomal fractions have indicated that the “membrane-bound” acid hydrolases are, in fact, complexed with phospholipid-rich, SALGP aggregates present in the lysosomal matrix, and are not bound to the limiting membrane of lysosomes as commonly supposed. Further, lysosomal SALGP, prelabeled in vivo in the phospholipid moieties, form complexes with acid hydrolases that tend to persist during gel filtration in a Sephadex G-200 column but can be split by dissociating agents such as 6 N urea or 1 M KCl. Finally, an improved method is described for the partial purification of lysosomes from brain homogenates. This method involves sequential isopycnic centrifugations of a crude mitochondrial fraction on metrizamide and sucrose density gradients.