Soluble and Membrane-Associated Forms of Acid Phosphatase Associated With the Lysosomal Fraction of Rat Liver

Abstract

Portions of this work were submitted in partial fulfillment of the requirements for the Ph.D. degree. The work was supported by funds from an NIH training grant in the Department of Zoology, The University of Michigan, (NIH 5T 01GM00989). 514Sloat & Allen: Lysosomal Acid Phosphatase MATERIALS AND METHODS Preparation of Liver Homogenates 575Normal tissue. Normal tissue used in quantitative and electrophoretic studies was obtained from 300 to 400-gram albino male rats. Animals were fed a diet of Purina Lab Chow and water ad libitum. After a starvation period of 12 to 18 hours, animals were sacrificed by cervical dislocation. The liver was removed immediately, weighed, and an initial 20% (w/v) homogenate prepared by bringing 20 g of tissue to a final volume of 100 ml with 0.25 M sucrose (Merck, reagent grade). Tissue was homogenized at 0°C in a smooth glass homogenizer with a Teflon pestle using 5 to 8 up-down strokes at 1200 revlmin.Regenerated tissue. Animals were partially hepatectomized under Nembutal anesthesia (Merck, 20 mg/ kg body weight) according to the method of Higgins and Anderson (1931). At the time of partial hepatectomy, the median and left lateral lobes were ligated and removed. This tissue was homogenized as above and utilized as control tissue. During the first 24 hours after the operation, animals were provided with 20% dextrose (Mallinckrodt analytical) ad libitum. After this period, food and water were given as usual. After the required regeneration period, animals were sacrificed and homogenates prepared as previously described. Difleren tial CentrifugationNormal and regenerated whole tissue homogenates were subjected to differential centrifugation at 4 O C according to one of the following schemes. Centrifugation times and g values are designated exclusive of acceleration and deceleration times and are based on average r-values. Scheme 1 :Nuclear fraction. Whole homogenates were centrifuged at 480 X g for 10 minutes followed by two washes ( 5 up-down strokes each), resuspensions, and recentrifugations at 270 X g for 10 minutes each. The washed pellet was resuspended to starting volume in 0.25 M sucrose to yield a final 20% fraction. All other pellets were resuspended to starting volume in the same manner.Mitochondrial-lysosomal fraction. The combined supernatants from the nuclear step were centrifuged at 22,000 X g for 30 minutes to sediment mitochondria and lysosomes.Microsomal-soluble fraction. The supernatant remaining after the mitochondrial-lysosomal centrifugation was designated the microsomal-soluble fraction. All centrifugations were carried out in a Servall RC 2 refrigerated centrifuge equipped with an SS-34 rotor. Scheme 2 :Nuclear fraction. Whole homogenates were centrifuged at 270 X g for 10 minutes. Two resuspensions (homogenized with 8 up-down strokes at 1,200 rev/ min) and recentrifugations followed at the same speed and time. The washed pellet was brought to starting volume with 0.25 M sucrose.Mitochondria1 fraction. The combined supernatants from the nuclear sedimentation wer...

Studies on the structure-bound sedimentability of some rat liver lysosome hydrolases

“…This behaviour, at first sight contradictory, could be explained on the assumption that there exist at least two varieties of acid phosphatase associated with the L fraction from rat liver, the first easily and the othei not readily dissociable from structures. This interpretation fits with the observations made by Sloat & Allen (1969). Since the solubilized acid phosphatase added to homogenates is extracted from lysosomes by freezing and thawing, it could be the soluble variety that exhibits low adsorbability.…”

“…The action of detergents is more complex, since they not only disrupt particles, by dissociating structural constituents of the membranes, but they are also able to solubilize membrane-bound enzymes (see, e.g., Sloat & Allen, 1969;Soltysiak & Kaniuga, 1970). As suggested by Weissmann et al (1967), the distribution pattern of enzymes can be obscured or complicated by the presence within lysosomes ofsome organizational principle (stroma?, internal membranes?)…”

“…As de Duve (1969) points out, the functional significance of membrane-bound acid hydrolases in the lysosomes is unclear, since soluble enzymes can more easily attack the substrates sequestered in these particles. Sloat & Allen (1969) adduced evidence showing that lysosomes in regenerating liver contain a larger proportion of bound to soluble acid phosphatase than do particles from control animals; they suggested that the bound form of the enzyme derives either from the membrane of Golgi vesicles or from the membrane ofthe endoplasmic reticulum.…”

Tissue Fraction-Past and Present

“…As found by Tappel and his co- workers (23,122,166), after repeated freezing and thawing of the particles, some enzymes, for instance fl-glucosidase and acid lipase, remain largely bound to the sedimentable fraction, whereas many others are largely released in soluble form. Acid phosphatase is unequally distributed between the two fractions, apparently in the form of two distinct isozymes (161). The significance of these observations is not easily assessed, since some of the bound activities can be partly detached by simple washing with solutions, and cannot therefore be considered truly membrane bound.…”

Isozyme Variations in Human Cells Grown <i>in vitro</i>. VII. Subcellular Distribution of Ammo Acid Naphthylamidases

“…The sum of the activities of subfractions R and T had a recovery of 87% for amino acid naphthylami dase and 96% for acid phosphatase. The results of the gradient experiments are presented graphically according to Sloat and Allen [1969]. The recoveries were: amino acid naph thylamidase 88, acid phosphatase 96, glucose-6-phosphatase 108 and cytochrome oxidase 116.…”