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1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5′-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together.

The structure-linked latency of acid phosphatase and beta-galactosidase was studied in rat liver lobes made ischaemic for 1 or 2 h and then recirculated with blood for increasing periods. Free activity of acid phosphatase and unsedimentable activity of beta-galactosidase are increased in homogenates of ischaemic livers. When ischaemia had been maintained for 1 h, the recovery of normal latency for both enzymes was observed 1 h after re-establishment of the blood flow. After a 2 h period of ischaemia, unmasked activity markedly decreases during the first 1 h after restoration of blood flow; after that, a large and irreversible secondary rise takes place. Chlorpromazine, injected 30 min before or just after induction of ischaemia, extensively prevents the latency decrease occurring during restoration of blood flow. Modifications of the hydrolase distribution pattern obtained after differential centrifugation are in agreement with the latency changes. These results suggest that a 2 h ischaemia causes an alteration of the liver lysosomes that is largely reversible and that restoration of blood flow induces an irreversible alteration of these organelles. Chlorpromazine treatment prevents the irreversible lesion from taking place.

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.

The release by glycyl-L-phenylalanine 2-naphthylamide (Gly-L-Phe-2-NNap) of endocytosed invertase associated with the MLP fraction (sum of the M, L and P fractions [de Duve, Pressman, Gianetto, Wattiaux & Appelmans (1955) Biochem. J. 63, 604-617]) of rat liver was investigated and compared with the release of cathepsin C. The percentage of invertase released increases with time after the enzyme injection, whereas the release of cathepsin C is not influenced by this treatment and corresponds to 85-90% of the total activity of the enzyme. It takes about 2h to attain a similar release of both enzymes. The quantity of invertase releasable or not by Gly-L-Phe-2-NNap was plotted against the time after the injection. Results agree well with the hypothesis that unreleasable invertase is associated with a pre-lysosomal compartment, whereas releasable invertase is present in lysosomes. A kinetic analysis indicates that invertase enters the pre-lysosomal compartment with a zero-order rate constant of 0.48 unit/min per g fresh wt., and leaves this compartment with a first-order rate constant of 0.042 min-1.

Glycyl-L-phenylalanine 2-naphthylamide (Gly-L-Phe-2-NNap), a cathepsin C substrate, induces an increase of the free and unsedimentable activities of this enzyme when incubated with a total mitochondrial fraction of rat liver. 1 mM-ZnSO4 considerably inhibits the cathepsin C total activity, measured with Gly-L-Phe-2-NNap as the substrate, in the presence of Triton X-100. The inhibition is markedly less pronounced when the free activity is determined; a high activity remains that depends on the integrity of the lysosomes; it decreases as the free activity of N-acetylglucosaminidase increases when lysosomes are subjected to treatments able to disrupt their membrane. Cathepsin C activity is reduced when thioethylamine hydrochloride is omitted from the incubation medium. Under these conditions at 37 degrees C, the free activity equals the total activity, although the lysosomes are intact, as indicated by the low free activity of N-acetylglucosaminidase. 1 mM-ZnSO4 strikingly inhibits the total activity, whereas more than 80% of the free activity remains. These observations are presented as evidence that Gly-L-Phe-2-NNap can possibly cause a disruption of the lysosomes as a result of its hydrolysis inside these organelles. In the presence of ZnSO4, intralysosomal hydrolysis becomes apparent, owing to a preferential inhibition by Zn2+ of extralysosomal hydrolysis; in the absence of thioethylamine hydrochloride, it is measurable because the disruption of lysosomes by Gly-L-Phe-2-NNap is delayed as a result of a slow-down of the reaction. The usefulness of Gly-L-Phe-2-NNap and related dipeptidyl naphthylamides in lysosomal-membrane-permeability studies is emphasized.

We have investigated the effect on the osmotic activation of rat liver lysosomes, by glucose penetration, of different substances known to inhibit the glucose transport through the plasma membrane. Diethylstilbestrol is the most efficient, particularly when purified lysosomes are used. It has no effect on osmotic activation induced by hypo-osmotic sucrose or by iso-osmotic KCl. It is proposed that diethylstilbestrol reacts with specific sites involved in the glucose translocation through the lysosomal membrane. These sites could not be identified by binding experiments, presumably owing to the considerable unspecific binding of the compound to the membrane.