Enzyme Activities in Human Liver Biopsies: Assay Methods and Activities of Some Lysosomal and Membrane-Bound Enzymes in Control Tissue and Serum

1. Highly sensitive techniques are described for the assay of plasma membrane (5′-nucleotidase, alkaline phosphatase), microsomal (neutral α-glucosidase, leucyl-2-naphthylamidase) and biliary canalicular (γ-glutamyltransferase) enzymes and for nine acid hydrolases (acid phosphatase, phosphodiesterase, β-glucosidase, α-glucosidase, α-galactosidase, β-galactosidase, α-mannosidase, N-acetyl-β-glucosaminidase, β-glucuronidase) in human liver. 2. Optimum and specific assay systems have been developed which give linear kinetics for all enzymes. 3. The range of enzyme activities in samples of human liver, obtained by closed needle biopsy, and in sera have been determined.

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Analytical subcellular fractionation of needle-biopsy specimens from human liver

Biochemical Journal ◽

10.1042/bj1740435 ◽

1978 ◽

Vol 174 (2) ◽

pp. 435-446 ◽

Cited By ~ 39

Author(s):

T J Peters ◽

C A Seymour

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Density Gradient ◽

Needle Biopsy ◽

Human Liver ◽

Resolving Power ◽

Equilibrium Density ◽

Marker Enzyme ◽

Acid Hydrolases ◽

Biopsy Specimens

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were resolved: plasma membrane (1.12-1.14; 5′-nucleotidase); lysosomes (1.15-1.20; N-acetyl-beta-glucosaminidase); mitochondria (1.20; malate dehydrogenase); endoplasmic reticulum (1.17-1.21; neutral alpha-glucosidase); peroxisomes (1.22-1.24; catalase). 2. The distribution of particulate alkaline phosphatase and, to a lesser degree, leucine 2-naphthylamidase followed that of 5′-nucleotidase. gamma-Glutamyltransferase was associated with membranes of significantly higher equilibrium density than was 5′-nucleotidase. 3. The distribution of 12 acid hydrolases was determined in the density-gradient fractions. beta-Glucosidase had a predominantly cytosolic localization, but the other enzymes showed a broad distribution of activity throughout the gradient. Evidence was presented for two populations of lysosomes with equilibrium densities of 1.15 and 1.20 g/ml, but containing differing amounts of each enzyme. Further evidence of lysosomal heterogeneity was demonstrated by studying the distribution of isoenzymes of hexosaminidase and of acid phosphatase. 4. The resolving power of the centrifugation procedure can be further enhanced with membrane perturbants. Digitonin (0.12 mM) selectively disrupted lysosomes, markedly increased the equilibrium density of plasma-membrane components and lowered the density of the endoplasmic reticulum, but did not affect the mitochondria or peroxisomes. Pyrophosphate (15 mM) selectively lowered the equilibrium density of the endoplasmic reticulum.

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