Changes in electronegativity of lysosomal hydrolases during intracellular transport: An isoelectric-focusing study in subcellular fractions of rat kidney

Abstract

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, β-glucuronidase, β-galactosidase and β-N- acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electrofocusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4°C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1% p- nitrophenyloxamic acid) and adding p- nitrophenyloxamic acid (0.1%), an inhibitor of lysosomal neuraminidase and cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, β-glucuronidase and β-N- acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and β-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4.1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1–2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1–2.5 units, indicating the presence of some N- acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the rough endoplasmic reticulum as a bound, basic glycoenzyme containing a few N- acetylneuraminic acid residues. In the Golgi complex additional glycosylations occur involving the attachment of sugar sequences containing numerous N- acetylneuraminic acid residues. The nascent glycoenzymes are thereby converted into soluble, acidic isoenzymes with isoelectric points close to 4 and are subsequently packaged in lysosomes in this form.