Lysosomal Marker Enzyme Research Articles

Phagocytosis and onset of human labor

An in vitro experimental model has been developed which allows the study of amnion lysosomal enzyme release under controlled conditions. Briefly, a layer of human amnion membrane mounted on a specially designed reaction vessel serves as the reaction surface. We have noted that the addition of particulate material to these membranes incubating in pseudoamniotic fluid results in an increased release of the lysosomal marker enzyme N-acetylglucosaminidase when compared to the release in the absence of particles. This release is completely inhibited by iodoacetate and slightly by azide. A similar increased release is also noted with the use of term amniotic fluid as incubation medium when compared to centrifuged (30,000 g/20 min) amniotic fluid. Lecithin and lysolecithin were effective in releasing increasing amounts of enzyme. This increased release was noted only from membranes of placentas collected from subjects who had undergone cesarean section prior to labor. Membranes collected from vaginal deliveries after labor showed a baseline increased release but no further stimulation upon the addition of any of the substances. These results suggest that the release of lysosomal enzymes from amnion membranes is brought about by substance(s) present in amniotic fluid. Very probably, these are surfactants. The interaction of these substances with amnion cells would eventually result in an exponential burst of prostaglandin synthesis, which would result in labor.

Effect of temperature and pH on release of enzymes from lysosomes of the bovine retinal pigment epithelium in vitro

The effects of buffer concentrations, pH, and temperatures on the release of enzymes from lysosomes of the bovine retinal pigment epithelium were studied in vitro. Cathepsin D, arylsulfatase, and acid phosphatase were used as lysosomal marker enzymes. Elevation of temperature caused a marked increase in the release of cathepsin D and arylsulfatase from lysosomes, but little changes in the release of acid phosphatase. Acidic conditions accelerated the release of arylsulfatase and acid phosphatase. Different buffer concentrations had little effect on the release of these enzymes from lysosomes.

Sequestration of cytoplasmic enzymes in an autophagic vacuole-lysosomal system induced by injection of leupeptin.

Administration of leupeptin to rats induces the accumulation of numerous autophagic vacuoles in the liver. Furuno et al. (Furuno, K., Ishikawa, T., and Kato, K. (1982) J. Biochem. (Tokyo) 91, 1485-1494) have recently devised a method for Percoll density gradient equilibrium fractionation of crude lysosomal fractions to isolate a highly enriched preparation of autophagic vacuoles. This system was used to determine whether cytoplasmic enzymes are normally sequestered into autophagic vacuoles in fed animals. Within 30 min following the administration of leupeptin to fed rats, several cytoplasmic enzymes could be demonstrated in vacuolar fractions heavier than mitochondria and normal lyosomes. The activities of tyrosine aminotransferase and lactic dehydrogenase as well as antigens of fructose-bisphosphate aldolase were detectable in fractions with densities of 1.115 to 1.15 g/ml containing cathepsins and acid phosphatase. The cytoplasmic enzymes in these fractions exhibited latency and were sequestered within membranous organelles. Six hours after the administration of leupeptin, the autophagic vacuoles gradually disappeared from these fractions concurrently with the loss of both cytoplasmic and lysosomal marker enzymes. For 6 h after injection of leupeptin the activities of cathepsin D and acid phosphatase increased in autophagic vacuoles and decreased in the postvacuolar lysosomal fraction. Administration of dexamethasone, which induces the synthesis of tyrosine aminotransferase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Cycloheximide administered simultaneously with leupeptin rapidly inhibited formation of autophagic vacuoles and the sequestrations of both cytoplasmic and lysosomal enzymes. However, when cycloheximide was administered 1 h after leupeptin, the formation of autophagosomes and the sequestration of cytoplasmic enzymes were inhibited but the vacuolar uptake of acid phosphatase and cathepsin D continued to increase for several hours. When cycloheximide was injected 1 h after leupeptin, losses of lactic dehydrogenase and aldolase proteins were observed in autophagic vacuoles isolated 1 and 2 h later.

Characterization of molluscan phagocyte subpopulations based on lysosomal enzyme markers

AbstractThis study quantitatively analyzed the distribution and abundance of the lysosomal enzyme markers, acid phosphatase (AP), nonspecific esterase (NE), and peroxidase (PO) within and between the phagocytes (hemocytes) of two strains of the snail, Biomphalaria. Glabrata, vector host for the human blood fluke, Schistosoma mansoni. Discrete subpopulations of AP‐, NE‐, and PO‐staining cells were found in the glass‐adherent hemocyte population of both schistosome‐resistant (10‐R2) and susceptible (PR albino) strains of the snail. Furthermore, there were significant differences in the distribution and abundance of the different enzymes defining the subpopulations within and between the snail strains. Generally, 10‐R2 hemocytes have a higher enzymatic activity than PR albino cells, which, in part, can be attributed to larger NE‐only, AP‐only, and AP + NE‐staining hemocytes in the 10‐R2 strain, and a larger subpopulation of negative cells in PR albino snails. Moreover, these differences in hemocyte enzyme content are further augmented by the fact that the 10‐R2 strain has nearly twice as many circulating, glass‐adherent blood cells when compared to PR albino snails. These findings may be of additional significance, since previous studies have implicated hemocyte lysosomal enzymes in the killing of schistosome larvae in naturally resistant 10‐R2 snails, although accurate measurements of strain‐specific enzyme levels in these cells have not been conducted. The present study provides direct evidence that 10‐R2 snails naturally possess a much greater lysosomal enzyme content than do those of the PR albino strain, which, in turn, may be related to the observed resistance or susceptibility in these two molluscan strains.

Jejunal mucosal enzymes in untreated and treated coeliac disease.

A series of marker enzymes for brush borders, basolateral membrane, and lysosomes were assayed in mucosal biopsy specimens from patients with untreated and treated coeliac disease and from controls. The brush border enzymes lactase, sucrase, neutral alpha-glucosidase, alkaline phosphatase, and leucyl-beta-naphthylamidase showed reduced activities in the untreated state and complete or partial normalization during treatment. The lysosomal marker enzyme acid phosphatase increased in activity in untreated coeliac disease and was normalized by treatment. The brush border enzyme gamma-glutamyl transferase was nearly normal in untreated patients and slightly increased in treated patients. The basolateral membrane marker, 5'-nucleotidase, was reduced both in untreated and treated patients, whereas the lysosomal marker N-acetyl-beta-glucosaminidase was normal in the untreated state and decreased during treatment. The possible pathogenetic role of the three latter enzymes in coeliac disease is discussed. The patterns of the other enzymes are suggested to be attributable to the morphologic changes in the mucosa.

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of 125I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.

Remarks on the differentiation of lysosomes from cultured human fibroblasts by silica gradient centrifugation

Two bands of lysosomal marker enzyme activity from cultured skin fibroblasts can be obtained by silica gradient centrifugation (Rome, L H, Garvin, L H, Allietta, M M & Neufeld, E F, Cell 17 (1979) 143) [3]. It is shown that the distribution of lysosomes into the bands in the upper and lower part of the density gradient depends on the starting density and the shape of the self-forming gradient. A plot of marker enzyme activity vs density reveals that the two bands stem from a lysosomal population with unimodal density distribution.

Properties and subcellular localization of acid phosphatase activity in cultured arterial smooth muscle cells.

Smooth muscle cells were cultured from pig aortas and their acid phosphatase activity toward 4‐methylumbelliferyl phosphate was studied. Cell homogenates hydrolysed this substrate with a pH optimum of 5.0, with very little activity at alkaline pH. The apparent Km of the activity was approximately 20 μM at pH 5.0, and the activity was directly proportional to both the incubation time and to the concentration of homogenate. Acid phosphatase activity was inhibited by p‐chloromercuribenzoic acid, by fluoride ions, and by chloroquine.Postnuclear supernatants of the cells were subjected to analytical subcellular fractionation on sucrose density gradients. Acid phosphatase activity had a modal equilibrium density of 1.13 g/ml, which was similar to that of 5′‐nucleotidase and galactosyltransferase, marker enzymes for the plasma membrane and Golgi complex, respectively. The distribution of acid phosphatase activity also had a shoulder of equilibrium density about 1.16 g/ml, which may have corresponded to activity in the lysosomes. The activity of N‐acetyl‐β‐glucosaminidase, a lysosomal marker enzyme, had a very broad distribution with much of its activity equilibrating at low densities.When the cells were homogenized in sucrose medium containing digitonin, a selective membrane perturbant, the N‐acetyl‐β‐glucosaminidase activity remained almost entirely in the sample layer, showing that the lysosomes had been disrupted. The modal equilibrium density of 5′‐nucleotidase activity was increased to 1.19 g/ml, whereas the modal equilibrium density of galactosyltransferase activity was not significantly affected. Acid phosphatase activity showed a similar increase in modal equilibrium density to 5′‐nucleotidase, but had a shoulder on the least dense side of its peak, which may have corresponded to activity in the Golgi complex. To confirm the plasma membrane localization of a large proportion of the acid phosphatase activity, intact cells were treated with digitonin and were then washed and homogenized in sucrose medium without digitonin. The modal equilibrium densities of both 5′‐nucleotidase and acid phosphatase activity were increased to similar extents, whereas the equilibrium densities of the marker enzymes for the other organelles were not significantly affected.Thus, although acid phosphatase is a widely used marker enzyme for the lysosomes in subcellular fractionation studies, its activity towards 4‐methylumbelliferyl phosphate cannot be used as a marker for these organelles in pig arterial smooth muscle cells in culture.

Extended chain analogs of [arginine8]vasopressin as model prohormones: investigation of precursor-processing enzymes in extracts of the rat hypothalamus and neural lobe.

Analogs of [arginine8]vasopressin (AVP) in which the peptide chain was elongated from the N-terminus by the addition of Ala-Arg-Arg-, Ala-Ala-Phe-, Pro-Arg-Val-, Pro-Ala-Arg-Arg, and Pro-Ala-Ala-Phe-, and from the C-terminus by the addition of -Ala-Met-Ala-NH2 and -Gly-Arg-Arg-Ala-NH2 were synthesized by the solid phase method and purified by Sephadex G-15 chromatography. At the final step of the synthesis, the extent of formation of the intramolecular disulfide bond was found to be sequence dependent. These peptides were incubated with extracts of the rat hypothalamus (supraoptic region) and neural lobe and with isolated neurosecretory granules from the neural lobe, and the release of vasopressin was measured by the rat pressor assay. All peptides resisted conversion to the hormone in the presence of tissue extracts, except (Ala-Ala-Phe)-AVP which was converted to AVP in the presence of all three tissue extracts at pH 4.7 but not at pH 8.0. When these peptides were treated with trypsin, chymotrypsin, or leucine aminopeptidase at pH 8.0, only the action of chymotrypsin on [Ala-Ala-Phe]AVP resulted in AVP formation. Evidence obtained using lysosomal enzyme markers suggested that the converting enzyme activity in neurosecretory granule preparations was not of lysosomal origin.

Morphogenesis of the lamellar body in fetal lung tissue in vitro

By use of both biochemical and morphological techniques it has been reported that lysosomal enzymes are localized in the lung lamellar body. In the present investigation, the association between lysosomal enzymes and lamellar body morphogenesis in fetal lung was studied. We employed an organ culture system in which the epithelium of immature human fetal lung explants differentiates in vitro into an epithelium of type II pneumonocytes. The specific activities of two lysosomal marker enzymes, viz., N- acetyl-β- d- glucosaminidase and β-galactosidase, as well as phosphatidate phosphohydrolase were increased 2-fold in homogenates of lung explants that had been maintained in organ culture compared to the specific activities of the enzymes in homogenates of fetal lung tissues before culture. The increase in total activity of the lysosomal enzymes during culture was greatest in the heavy mitochondrial fraction (5 000 × g pellet). The greatest increase in the specific activity of these enzymes during the culture period was also in the heavy mitochondrial fraction. We subfractionated the mitochondrial fractions by use of a step gradient (20–60% sucrose). By electron microscopy we found that lamellar bodies were localized in the fraction with the lowest density, whereas lamellar body precursors (i.e., multivesicular bodies) were present principally in the next two fractions. Mitochondria were identified in high-density fractions of the sucrose step gradient. All of the lysosomal enzymes of starting lung tissue were enriched in the gradient fractions at high densities of the step gradient. However, after the tissue differentiated, the enzyme activities were enriched in the less dense fractions that contained lamellar and multivesicular bodies. The NADPH-cytochrome c reductase (a microsomal marker enzyme) activity increased after culture in the low-density fractions that contained lamellar and multivesicular bodies. Thus, an increase in the total and specific activities as well as a change in the subcellular localization of lysosomal enzymes takes place during type II cell differentiation. These data are suggestive that several lysosomal enzymes, as well as phosphatidate phosphohydrolase and a microsomal enzyme, NADPH-cytochrome c reductase, are associated with the morphogenesis of the mature lamellar body and its precursor i.e., the multivesicular body.

A Ca 2+-activated ATPase specifically released by Ca 2+ shock from Paramecium tetraurelia

Deciliation of Paramecium tetraurelia by a Ca 2+ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca 2+-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca 2+, Sr 2+, or Ba 2+, but not by Mg 2+ or by monovalent cations. The crude enzyme has a specific activity of 2–3 μmol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50°C, and which has a sedimentation coefficient of 8–10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.

Brush border and lysosomal marker enzyme profiles in duodenal mucosa from coeliac patients before and after organ culture.

Five brush border and 2 lysosomal enzymes were measured in duodenal tissue explants from 21 children and young adults (16 coeliac and 5 non-coeliac) before and after organ culture. Reduced activity of brush border enzymes and increased activity of lysosomal enzymes were recorded in flat mucosas from coeliac patients compared with remission coeliac explants and biopsy specimens from non-coeliac controls. Slightly increased activity of alkaline phosphate and sucrase was recorded during culture (24 h) of coeliac explants. Coeliac specimens in the exacerbation state showed increased activity of acid phosphatase after culture in the presence of gluten, whereas gluten did not provoke detectable alterations in brush border enzyme activities during culture unless the wet weight of material was 1.5 mg or more. In such explants lower activity of brush border enzymes was measured after in vitro gluten exposure than after culture on gluten-free medium. Mucus removed from the specimen surface after culture contained considerable amounts of brush border enzymes and reflected the variations in the tissue homogenates. Culture media contained smaller quantities of enzymes.

Subcellular Fractionation of Bone Marrow-Derived Macrophages: Localization of Phospholipase A1 and A2 and Acyl-CoA:1-Acylglycero-3-phosphorylcholine-0-acyltransferase

Analysis of the subcellular distribution of lipid-metabolizing enzymes was carried out in bone marrow-derived macrophages with special respect to a comparison of the subcellular localisation of phospholipase A1 and A2 and to acyl-CoA:1-1-acylglycero-3-phosphorylcholine-0-acetyltransferase. After cell disruption differential centrifugation was followed by additional sucrose gradient purification of three main fractions. Satisfactory enrichment factors were obtained by this method for the following marker enzymes. The plasma-membrane enzyme alkaline phosphodiesterase I was enriched up to 25-fold and the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase was enriched up to 30-fold. The marker enzyme for the endoplasmic reticulum, NADPH-cytochrome c reductase showed a similar enrichment and distribution as the acyltransferase. Therefore it was concluded that the acyl-CoA:1-acylglycero-3-phosphorylcholine-0-acyltransferase of bone marrow-derived macrophages is mainly located in the endoplasmic reticulum. Phospholipase A1 and A2 occurred in a high proportion together with the lysosomal marker enzyme N-acetyl-beta-glucosaminidase in the soluble supernatant and in the gradient fractions. In the endoplasmic reticulum phospholipase A2 occurred only in trace activities whereas phospholipase A1 was maximally enriched in this subcellular fraction. No subcellular fraction could be obtained where phospholipase A2 was enriched exclusively. However, it can be concluded that the two enzymes which are responsible for the balance of fatty acid liberation and re-acylation are located in two different cellular compartments. Furthermore it can be claimed that in the cell there has to exist an exchange of substrates and products between these compartments to achieve a complete metabolic cycle of the de- and re-acylation reaction of phospholipids in bone marrow-derived macrophages.

Properties of acid and neutral cholesterol ester hydrolases in rat and pigeon aortas

The activity of cholesterol ester hydrolase was measured in subcellular fractions from rat and pigeon aortas using a glycerol-dispersed cholesterol oleate substrate preparation. The specific activity of acid cholesterol ester hydrolase (assayed at pH 5) in adventitia tissue fractions was 40–50 fold greater than in media-intima fractions from rat aorta. Soluble and particulate subcellular fractions from rat aorta (media-intima) were observed to have cholesterol ester hydrolase activity with both an acid (pH 4.5–5) and a neutral (pH 7.5) pH optimum. A comparison of the subcellular distribution of acid cholesterol ester hydrolase with the lysosomal marker enzyme, N-acetylglucosaminidase, suggests that the acid hydrolase activity originated in aortic lysosomes; the neutral cholesterol ester hydrolase was predominantly soluble. Acid and neutral cholesterol ester hydrolases could also be distinguished on the basis of the effects of MgCl 2 and NaCl on hydrolase activity and on rates of thermal denaturation. Both acid and neutral hydrolases from rat aorta (media-intima) were inhibited by chloroquine (half-maximal at 2–4 mM), and both hydrolases were characterized as having the same apparent affinity for the glycerol-dispersed cholesterol oleate substrate. Acid and neutral cholesterol ester hydrolases were also observed in preparations from pigeon aortas. The specific activity for both acid and neutral hydrolases was higher in atherosclerosis-susceptible White Carneau pigeon aortas in comparison to Show Racer pigeon aortas.

Effects of trichloroethylene inhalation on acid phosphatase in rodent brain

Rats, mice and gerbils were continuously exposed to 150 ppm trichloroethylene (TCE) for 30 days. In all three species, there was a marked increase in liver weight. In mice the weight increased more (86%) than in rats and gerbils (20%). After exposure the activity of acid phosphatase, a lysosomal marker enzyme, was tested in different brain areas, using a system which had a limit of detection of ± 10–15%. In most areas no significant influence was found. However, in the brain stem of mice and gerbils the phosphatase activity increased by approx. 10%.

Subcellular localization of O2- generating enzyme in guinea pig polymorphonuclear leukocytes; fractionation of subcellular particles by using a Percoll density gradient.

An iso-osmotic Percoll density gradient was applied to determine the subcellular localization of the O2- generating enzyme, NADPH oxidase, in guinea pig polymorphonuclear leukocytes (PMN). [14C]Myristate (MA) was employed as a metabolic stimulant in order to clarify whether the myristate binding site on PMN membrane was identical with the O2- generating site. The distribution pattern of the O2- generating activity of MA-activated PMN was compared with that of unactivated PMN in parallel experiments to find fractions showing an enhanced O2- generating activity (i.e., above the background values due to O2-generation by other electron-transport systems). We observed very high O2- generating activity concentrated in a single peak with MA-activated PMN but little activity was seen with unactivated PMN in the Percoll density gradient. The O2- generating activity of MA-activated PMN was consistently associated with 5'-nucleotidase activity as a membrane marker enzyme, but was not associated with lysosomal marker enzymes such as myeloper-oxidase and lysozyme. O2- generating and 5'-nucleotidase activities in the peak fraction of MA-activated PMN were increased to about six and four times those of whole cells in terms of specific activity, respectively. These results indicate that the O2- generating enzyme is located on PMN plasma membrane. Furthermore, [14C]myristate-binding activity was mainly found in the peak fraction containing O2 - generating enzyme. This suggests that [14C]myristate binds to plasma membrane, and the O2 - generating enzyme may thus be activated.

Effects of new antiinflammatory steroids derived from prednisolone on pituitary‐adrenal axis and lysosomal stabilization

AbstractThe pituitary‐adrenal axis suppression and lysosomal membrane stabilization of methyl steroid‐21‐oates, synthesized by modifying the 17β‐ketol side chain of prednisolone, were investigated by measuring plasma corticosterone and by determining abilities of the drugs to decrease extrusion of three liver lysosomal marker enzymes (acid phosphatase, beta‐glucuronidase, and aryl sulfatase) during incubation in severely hypo‐osmotic buffer. Animals were injected with 10 mg/kg drug suspensions i.m. at 20 hr and 2.5 hr prior to sacrifice. The parent compound prednisolone significantly suppressed pituitary adrenal function indicated by decreased plasma corticosterone levels. In contrast, the derivatives did not alter plasma corticosterone levels. When compared to controls, prednisolone, methyl 20‐dihydroprednisolonate, 1‐dehydro‐11β‐hydroxyandrostenedione, and to a lesser extent methyl prednisolonate retained significant lysosomal stabilization capacities, indicative of antiinflammatory activity. These data suggest that the new ester derivatives of prednisolone retained beneficial antiinflammatory activity but were devoid of significant adverse effects upon the pituitary‐adrenal axis.

Kallikrein and prekallikrein on the basolateral membrane of rat kidney tubules.

Basolateral membrane (BLM) enriched fraction was isolated from homogenized rat kidney cortex by differential centrifugation. We also obtained a fraction enriched in plasma membrane (PM). The morphology of the isolated BLM fragments was studied by transmission and freeze fracture electron microscopy. The relative specific activity of Na+-K+-ATPase was enriched 7-fold, while that of marker enzymes for PM, endoplasmic reticulum, and lysosomes was lower than in the crude homogenate. There was a 10-fold difference in the ratios of activities of Na+-k+-ATPase to Mg2+-ATPase in the BLM and in the PM enriched fractions. Kallikrein activity was determined with S-2266 substrate and by radioimmunoassay of kinin released. It was low in the BLM fraction prior to adding detergent, but Triton X-100 increased the activity 12 to 16-fold. Both free trypsin and Sepharose 4B-bound insoluble trypsin increased kallikrein activity 2- to 3-fold in both the membrane-bound and soluble fractions, probably by activating a prekallikrein. The results were interpreted that the kallikrein studied originated from the distal tubular BLM.

Analytical subcellular fractionation of rat pituitary homogenates, with special reference to prolactin proteolysis by lysosomes

Prolactin proteolysis by rat pituitary homogenates was assayed by measuring the release of trichloroacetic acid-soluble peptides from 125I-labelled rat prolactin. There was a distinct optimum at pH 4.3, with only trace amounts of activity at neutral and alkaline pH. Rat pituitary homogenates were subjected to analytical subcellular fractionation by sucrose density gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their respective marker enzymes, including: cytosol (lactate dehydrogenase); plasma membrane (5′-nucleotides); lysosomes ( N-acetyl- β-glucosaminidase, β-glucuronidase); mitochondria (particulate malate dehydrogenase); endoplasmic reticulum (neutral α-glucosidase); prolactin granules (radioimmunoassayable prolactin). Acid prolactin protease had a similar distribution to the lysosomal marker enzymes. A localisation of the activity to lysosomes was confirmed by subcellular fractionation experiments in which the lysosomes were selectively disrupted with low concentrations of the membrane pertubant, digitonin. Experiments with specific inhibitors of the lysosomal cathepsins indicate that both cathepsins B and D are implicated in pituitary prolactin proteolysis.

Phospholipase A 2 activity of lysosomal origin secreted by polymorphonuclear leucocytes during phagocytosis or on treatment with calcium

1. Peritoneal neutrophil leucocytes, derived from the rabbit, release phospholipase A (EC 3.1.1.4) activity during phagocytosis of complement-coated zymosan particles, or during treatment with Ca 2+. This enzyme is able to release [1- 14C]oleate from the membrane phospholipids of Escherichia coli. 2. The release of phospholipase A paralleled that of the known lysosomal marker enzymes β-glucuronidase and lysozyme. The phospholipase A would thus appear to be derived from the ly sosomal granules of the cells. 3. The released enzyme is of A 2 specificity, has an absolute requirement for divalent cations, and is active over a broad pH range (pH 6–9).