Iodosulfanilic Acid Research Articles

Labeling of the proteins at the surface of the pancreatic zymogen granule using diazotized [ 125I]iodosulfanilic acid

Purified pig and rat pancreatic zymogen granules have been covalently labeled with the membrane impermeant agent diazotized [ 125I]iodosulfanilic acid. Following alkaline lysis, the radioactivity was almost entirely (92%) recovered in a dense protein pellet designated as the 1 M sucrose pellet. The rest (8%) of the label was recovered in the membrane fraction. The specificity of this procedure in labeling the cytoplasmic aspect of the granule is demonstrated by the absence of label from granule content proteins and by the removal of iodinatable proteins following protease treatment of intact granules. No characteristic integral membrane proteins were labeled. In the pig, four major protein bands were labeled in both subfractions at M r of 15 000, 33 000, 35 000 and 38 000. In the rat, a similar set of protein bands was labeled except for that of 15 000 M r which was poorly labeled. Due to their location, it is suggested that these proteins may play an important role in the recognition between the granule membrane and the cell membrane and thereby the control of the exocytosis process.

A membrane protein in LRM55 glial cells cross-reacts with antibody to the anion exchange carrier of human erythrocytes

Cl − HCO 3 − exchange transport in the glial plasma membrane is remarkably similar to anion exchange transport in erythrocytes. When LRM55 cells were pretreated with the extracellular labeling reagent [ 125I]iodosulfanilic acid, radioactivity appeared primarily in two bands on denaturing polyacrylamide gels at M r 70,000 and 30,000. Antibody to the human erythrocyte anion exchanger (Band 3) cross-reacted on immunoblots with an LRM55 band at M r 68,000. The LRM55 Cl − HCO 3 − exchanger may be a component of M r 68,000 to 70,000.

Biochemical and immunologic characterization of a major surface antigen of Dirofilaria immitis infective larvae.

A 35 kD major surface antigen of Dirofilaria immitis third-stage larvae was characterized biochemically and immunologically. Living larvae were iodinated by using Iodo-gen, iodosulfanilic acid, lactoperoxidase-glucose oxidase, and Bolton-Hunter reagents. Detergent extracts of larvae labeled by the first three methods showed one major 35 kD component and a number of smaller components of about 6 kD, as analyzed by one-dimensional SDS-PAGE. In contrast, extracts from larvae labeled with the Bolton-Hunter reagent showed multiple bands on gels. The 35kD molecule was shown to be exposed on the larval surface, insofar as it was accessible to trypsin-proteolysis on living radiolabeled larvae. Two-dimensional gel electrophoresis resolved the 35 kD band into two components: a major one with a pI of 3.8, and a minor one of pI 7.3. The lower m.w. bands were resolved into about 12 constituents with pI values from 3.5 to 8.0. Of all these surface molecules, the only one that was antigenic was the 35 kD component. It could be immunoprecipitated with sera from dogs carrying an occult experimental D. immitis infection or with sera from dogs immunized with irradiated third-stage larvae of this parasite. Similarly, sera from rabbits immunized repeatedly with normal unirradiated larvae also precipitated the 35 kD antigen. None of these sera, however, contained detectable antibodies to the surface-labeled low m.w. molecules. Sera from rabbits immunized with D. immitis adult worms and microfilariae precipitated the 35 kD antigen, which is therefore not stage specific. In contrast, sera from dogs experimentally infected with Toxocara canis and Ancylostoma caninum or with Uncinaria stenocephala (a canine hookworm) did not contain antibodies to the 35 kD antigen, but did cross-react with many other D. immitis adult and microfilarial antigens. This molecule may therefore be species specific. Evidence for glycosylation of the 35 kD molecule was not found: it did not bind to peanut, wheat germ, lentil, or Ulex europeus lectins, and its electrophoretic mobility was not altered after treatment with endoglycosidase-F or mild alkali solutions.

Turnover of the surface proteins of adult and third and fourth stage larval Brugia pahangi

The turnover of surface proteins in adults, fourth-stage and third-stage larvae of Brugia pahangi was measured using [ 125I]iodosulfanilic acid. Groups of worms ( n = 10 adult, 20 L 4, 50 L 3) were labelled and surgically implanted into the peritoneal cavity of naive jirds. The amount of radioactivity remaining on worms recovered over a 7–8 day period was determined. Adult females showed no significant loss of label during a 7 day period. The recovery of fourth-stage larvae was low but the counts per minute remaining on each group of larvae recovered over an 8 day period, encompassing the major part of the instar, did not fall below the limits of the standard deviation of the time 0 groups, indicating that no significant loss of surface label had occurred. Third-stage larvae showed a significant loss of 125I-labelled proteins prior to the third moult, although it was not confirmed that these proteins occur on the worm surface. Electrophoresis and autoradiography of labelled homogenates of adult, fourth and third stage larvae suggested that [ 125I]iodosulfanilic acid labels polypeptides of different molecular weights on each life cycle stage of B. pahangi.

Characterization and localization of CIV polypeptides

In order to detect the structural proteins linked with disulfide bonds, CIV was solubilized and electrophoresed under nonreducing conditions in the first dimension and then under reducing conditions in the second dimension. The viral polypeptides linked originally with disulfide bonds were separated into subunits. The complexes were trimers (P′50) or dimers (P60 and P10). The apparent molecular weights of P81, P53, and P49 changed significantly according to the composition of the lysis buffer used, suggesting that the differences in their molecular weights were due to conformational changes produced by reduction of their intramolecular disulfide bonds. Sulfhydryl-containing polypeptides (P′50-P50, P60, P100, and P33) were detected by N-[ 14C]ethylmaleimide, and the accessibility of these residues was analyzed after successive stripping of the CIV particle. Radioiodination of external polypeptides by [ 125I]iodosulfanilic acid shows only one intensively labeled spot corresponding to the P50 polypeptide, whereas P′50 was only slightly labeled. Six viral polypeptides P81, P60, P31, P17, P13, and P10 were revealed to possess high affinity for CIV DNA. A structural model of CIV is proposed and discussed.

An evaluation of different methods for labelling the surface of the filarial nematode Brugia pahangi with 125iodine

The specificity of a range of 125I labelling techniques (Chloramine T, Iodogen, Bolton and Hunter reagent, lactoperoxidase and iodosulfanilic acid) to the surface of the filarial nematode Brugia pahangi was evaluated by autoradiography of sections of labelled worms and of dried SDS-polyacrylamide gels following electrophoresis of homogenised worm extracts. It was concluded that Bolton and Hunter reagent was not surface specific but labelled proteins throughout the body of the worm. At the light microscope level autoradiography of worms labelled using Chloramine T, Iodogen, lactoperoxidase and iodosulfanilic acid demonstrated that the 125I labelling was restricted to the worm surface. Electrophoresis and autoradiography showed that each method produced a different pattern of labelled polypeptide. A polypeptide of molecular weight 30 kDa was labelled using each method except Bolton and Hunter reagent, and appears to be a major surface component.

Isolation and partial characterization of a carbohydrate-binding protein from a nematode-trapping fungus.

A developmentally regulated carbohydrate-binding protein from the capture organs of Arthrobotrys oligospora, not present on hyphae, was isolated and partially characterized. Surface structures of A. oligospora were radiolabeled with [125I]iodosulfanilic acid. The fungus was homogenized, and the homogenate was passed over an affinity column containing N-acetyl-D-galactosamine immobilized to Sepharose 6B. The bound radiolabeled protein was eluted from the affinity column with a glycine-hydrochloride buffer (pH 3.0), concentrated, and chromatographed on a metal chelate affinity gel containing Ca2+. EDTA was used as an eluant for the radiolabeled protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in combination with autoradiography revealed a molecular weight for the carbohydrate- and cation-binding polypeptide of ca. 20,000.

Isolation and characterization of plasma membranes from strains of Neurospora crassa with wild type morphology.

A variety of commercially available cell wall hydrolytic enzyme preparations were screened alone and in various combinations for their ability to degrade the cell wall of Neurospora crassa wild type strain 1A. A combination was found which causes complete conversion of the normally filamentous germinated conidia to spherical structures in about 1.5 h. Examination of these spheroplasts by scanning electron microscopy indicated that, although they are spherical, they retain a smooth coat that can only be removed upon prolonged incubation in the enzyme mixture (about 10 h). The 10-h incubation in the enzyme mixture appears to have no obvious detrimental effects on the integrity of the plasma membrane since the activity and regulatory properties of the glucose active transport system in 10-h spheroplasts are essentially unimpaired. Importantly, plasma membranes can be isolated from the 10-h spheroplasts by an adaptation of the concanavalin A method developed previously in this laboratory for cells of the cell wall-less sl strain, which is not the case for the 1.5-h spheroplasts. The yield of plasma membrane vesicles isolated by this procedure is 18-36% as indicated by surface labeling with diazotized [125I]iodosulfanilic acid, and the preparation is less than 1% contaminated with mitochondrial protein. The chemical composition of the wild type plasma membranes is similar to that previously reported for membranes of the sl strain of Neurospora. The isolated wild type plasma membrane vesicles also exhibit all of the functional properties that have previously been demonstrated for the sl plasma membrane vesicles. The wild type vesicles catalyze MgATP-dependent electrogenic proton translocation as indicated by the concentrative uptake of [14C]SCN- and [14C]imidazole under the appropriate conditions, which indicates that they contain the plasma membrane H+-ATPase previously shown to exist in the sl plasma membranes and that they possess permeability barrier function as well. The vesicles also contain a Ca2+/H+ antiporter as evidenced by their ability to catalyze protonophore-inhibited MgATP-dependent 45Ca2+ accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of the isolated vesicles indicate that the protein composition of the wild type vesicles is roughly similar to that of the sl plasma membranes with the H+-ATPase present as a major band of Mr approximately 105,000. The wild type plasma membrane ATPase forms a phosphorylated intermediate similar to that of the sl ATPase, and the specific activity of the H+-ATPase in both wild type and sl membranes is approximately 3 mumol of Pi released/mg of protein/min.(ABSTRACT TRUNCATED AT 400 WORDS)

Proteins and glycoproteins in plasma membranes and in the membrane lamellae produced by purified oligodendroglia in culture

Oligodendroglial plasma membranes are complex structures composed of a heterogeneous mixture of proteins and glycoproteins. The Coomassie stained gel patterns showed a maximum of 40 proteins with molecular weights ranging from > 200 000 to 12 500 . Autoradiography was used to detect binding of radioiodinated lectins to glycoproteins. With concanavalin A, 5 major glycoproteins were seen; with wheat germ agglutinin, 2 major glycoproteins with approximate molecular weights of 95 000 and 78 000 were found; with Ulex europaeus, 7 major glycoproteins were observed. Additional minor bands were also seen. The impermeant probe diazodi[ 125I]iodosulfanilic acid was used to radiolabel intact cells. It was found that 5 major proteins were radiolabeled in the plasma membranes. In all cases, the whorls of membrane lamellae produced in culture by oligodendroglia tend to have a somewhat less complicated pattern with fewer proteins and glycoproteins than the plasma membranes. However, the whorls of membrane lamellae have far more complicated protein patterns than myelin.

Characterization of the blood-brain barrier: protein composition of the capillary endothelial cell membrane.

Microvessels were isolated from canine cerebral cortex, and the composition of the endothelial cell membrane was investigated. Endothelial cell membranes were separated from the surrounding basement membrane, solubilized, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 12% gels. Staining with Coomassie Blue revealed a characteristic banding pattern of at least 12 major proteins with apparent molecular weights between 14,000 and 250,000. When proteins from red blood cell ghosts were run simultaneously, no similarities were observed, except for proteins at apparent molecular weights of 43,000 (band 3) and 35,000 (band 4). These two proteins migrated exactly to the positions of the erythrocyte proteins actin and glyceraldehyde 3-phosphate dehydrogenase, respectively. Membrane glycoproteins in gels were also examined by the use of fluorescent lectins. Of the fluorescein isothiocyanate-conjugated (FITC) lectins tested, only FITC-concanavalin A had an affinity for any membrane components. Diazotized [125I]iodosulfanilic acid, a membrane-impermeable reagent, was used to label the internal (lumen) cell surface and the external (antilumen) cell surface. Autoradiography and determination of radioactivity levels in gel slices showed that several proteins were specifically labeled, and that major differences in radioactivity of proteins existed in internal and external labeling experiments. It is concluded that the protein composition of the luminal membrane is different from that of the antiluminal membrane.

Sequential removal of outer bilayer and apical plasma membrane from the surface epithelial syncytium of Schistosoma mansoni

The outer and inner bilayers of the apical membrane complex of Schistosoma mansoni were sequentially stripped from adult worms by two incubations in 0.1% digitonin solutions. Membrane removal was evaluated by electron microscopy of worms and bilayer material, using Con A-ferritin as a marker for the outer bilayer. Amounts of Con A removed by the digests were measured with a tritiated Con A marker. To measure the purity of the fractions membrane markers were characterised and quantitated for both bilayers. In the absence of the usual enzymatic markers for plasma membrane diazotised [ 125I]-iodosulfanilic acid was used as a marker for the outer bilayer. Alkaline phosphatase and a Na +, Mg 2+-ATPase were localised to the inner bilayer. From these results we can deduce that the inner bilayer is analogous to the typical, apical plasma membrane of other animal epithelia. The outer bilayer does not share these enzymatic similarities. The integrity of the syncytium after removal of the outer bilayer and the increased levels of lactate dehydrogenase in the supernatant after removal of the inner bilayer suggests that the outer bilayer is secondary in maintaining the permeability barrier of the apical membrane complex, with respect to soluble proteins. The possible significance of these results in terms of the destructive action of complement on the parasite are discussed.

The effects of 20-hydroxyecdysone on the metabolic labeling of membrane proteins in Drosophila imaginal discs

20-Hydroxyecdysone induces evagination of imaginal discs of Drosophila melanogaster cultured in vitro. The possible involvement of cell-surface proteins in this process has prompted us to study the synthesis of membrane proteins in imaginal discs. A procedure is reported for the isolation of membrane vesicle fractions from discs that are enriched for the plasma membrane enzyme, Na +/K +-ATPase, and that label with the surface-labeling reagent [ 125I]iodosulfanilic acid. 20-Hydroxyecdysone alters the pattern of [ 35S]methionine incorporation into polypeptides in these membrane vesicle fractions. Increased and decreased incorporation as well as changes in migration on two-dimensional gels of specific polypeptides are caused by the hormone. These changes parallel in time the onset and the continuation of evagination.

Discoidin I-membrane interactions III. Interaction of discoidin I with living Dictyostelium discoideum cells

We have examined the interaction of exogenous 125I-discoidin I with living wild type (NC-4) Dictyostelium discoideum cells and the subcellular localization of endogenous discoidin I during development. The living cells bind 125I-discoidin I relatively rapidly, with an apparent steady state being reached within 40 min at 23°C. The rate of association is reduced to a similar extent by lowering the temperature to 4°C and by glutaraldehyde fixation of the cells. The level of steady-state binding is not developmentally regulated. Living cells do not degrade significant amounts of the 125I-discoidin I during incubations at 23°C. Differentiated cells bind the 125I-discoidin I via both the carbohydrate or C site receptors and the ionic or I site receptors, first identified on the corresponding glutaraldehyde-fixed cells (Bartles, J.R. and Frazier, W.A. (1982) Biochim. Biophys. Acta 687, 121–128). Depending on the concentration of 125I-discoidin I, 50% or more of the binding to the 8.5-h differentiated cells is via the I sites. Binding exhibits apparent positive cooperativity with respect to discoidin I. This causes the binding to appear nonsaturable, with a capacity for more than 10 7 discoidin I tetramers per cell. Independent verification of the nonsaturability of the interaction was obtained from experiments using diazotized [ 125I]iodosulfanilic acid to radiolabel the surfaces of cells treated with increasing concentrations of unlabeled discoidin I. The susceptibility of the surface radiolabeled discoidin I to various extractive treatments of the cellular particulate fraction suggests that as much as 10% is very tightly associated with cellular membranes. Dissociation experiments also suggest the existence of an irreversible component of binding, the magnitude of which increases with time of association. While the 8.5-h differentiated NC-4 cells exhibit maximal developmental cohesiveness and contain about 5·10 6 discoidin I tetramers per cell, cell surface labeling and radioimmunoassay indicate that they display only 1.3·10 3 tetramers per cell on their surface and only 6·10 3 tetramers per cell in their surrounding extracellular medium.

Survival of baboon platelets labeled with diazotized (125-1)-iodosulfanilic acid: No effect of drugs that modify platelet behavior

A baboon animal model has been developed to study the kinetics of platelets doubly labeled with 51-Cr, a cytoplasmic label, and diazotized 125-I-iodosulfanilic acid (D 125-ISA), which binds to glycoprotein components of the platelet membrane. This study was undertaken to evaluate the possible usefulness of this platelet label, and to extend to primates previous observations of D 125-ISA-labeled platelets in rabbits. D 125-ISA-platelet activity disappeared exponentially with a mean t 1 2 of 0.90 days ± 0.11. Labeling 51-Cr-platelets with D 125-ISA resulted in a modest but significant reduction in the baseline 51-Cr-platelet survival time (from 5.5 ± 0.2 days to 4.7 ± 0.4; p < 0.02). 51-Cr and D 125-ISA kinetics were not changed by the administration of aspirin-dipyridamole, sudoxicam, or high molecular weight dextran. These studies suggest that platelet membrane components are continually lost during circulation in vivo, but that this process is unaffected by pharmacologic inhibition of platelet function.

Labelling of intestinal brush border membrane proteins in vivo using diazotised [ 125I]iodosulfanilic acid

Membrane proteins of the intestinal brush border were labelled in vivo by intraluminal injection of diazotised [ 125I]iodosulfanilic acid, a highly polar molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of brush border membranes labelled in this manner showed 20 protein bands, 11 of which contained significant radioactivity. The most heavily labelled proteins had molecular weights greater than 150 000, indicating that they were the most exposed to the intestinal lumen. Little radioactivity was detected in proteins with molecular weights of less than 94 000. The majority of these smaller proteins were likely to have been brush border core proteins. The evidence that diazotised [ 125I]iodosulfanilic acid bound primarily to brush border membrane proteins when administered in this way, was: (a) the specific activity of brush border proteins was up to 3-fold greater than that of total cell particulate proteins (pelleted at 27 000 × g from mucosal homogenates); (b) principal peaks in the gel radioactivity profile of total cell particulate proteins corresponded to the most heavily labelled proteins of the isolated brush border membrane; and (c) brush border core proteins showed minimal radioactivity in vivo, but considerably higher radioactivity when brush border membranes were labelled in vitro. A small amount of label was absorbed across the intestinal mucosa. However, secondary labelling of brush border proteins by this absorbed label was minimal, since the specific activity of brush border proteins in jejunum adjacent to the labelled loop was only 0.22% of the level for those proteins in the labelled segment. Since this technique did not affect the cellular morphology, enzyme activity or biochemical integrity of the membrane, it should prove useful as a means of accurately studying in vivo turnover rates of brush border membrane proteins.

Localization of the dicarboxylate binding protein in the cell envelope of Escherichia coli K12.

Examination of the localization of the dicarboxylate binding protein (DBP) in the cell envelope of Escherichia coli K12 reveals that this protein is present on the cell surface, and also in the inner and outer regions of the periplasmic space. The cell surface DBP is release by treating the cells with EDTA. This protein can be surface labeled by lactoperoxidase radioiodination, and by diazo[125I]iodosulfanilic acid in whole cells. It also binds tightly, but not covalently, to lipopolysaccharide. The DBP located in the outer region of the periplasmic space is released when the outer membrane is dissociated by EDTA-osmotic shock treatment. The DBP located in the inner region of the periplasmic space is released only when the EDTA-osmotic shocked cells are subjected to lysozyme treatment. At the moment, it is not certain whether this protein is bound to or trapped by the peptidoglycan network. This protein cannot be surface labeled in whole cells or in EDTA-osmotic shock treated cells; and it is not associated with lipopolysaccharide. Analysis of transport mutants indicates that these DBP are coded by the same gene.

Labeling and Isolation of Plasma Membranes from Corn Leaf Protoplasts

A plasma membrane-enriched fraction has been isolated from corn leaf mesophyll protoplasts and its identity confirmed with the aid of an external label, diazotized [(125)I]iodosulfanilic acid. Gentle cell disruption enabled internal organelles to be maintained intact and thus facilitated separation from the plasma membrane. The plasma membrane-enriched fraction was devoid of chloroplast or mitochondrial markers, whereas markers for the endoplasmic reticulum and golgi indicated minimal contamination. The highly enriched plasma membrane fraction contained a Mg(2+)-dependent, K(+)-stimulated ATPase with a pH optimum near neutrality. The position of the membranes on sucrose density gradients indicates that the plasma membranes have characteristics similar to other plasma membrane fractions.

Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte's zona pellucida

The zona pellucida is an acellular coat which surrounds the plasma membrane of fully grown mammalian oocytes and which performs a variety of important functions during oogenesis, fertilization, and preimplantation development. In this investigation the proteins of the mouse oocyte's zona pellucida have been identified and characterized by using zonae pellucidae isolated individually from fully grown oocytes with mouth-operated micropipets. Various morphological and biochemical criteria were employed to assess the purity of the isolated zonae pellucidae and, in most cases, they were found to be virtually free of contamination by other oocyte proteins. It was determined that each zona pellucida contains 4.8 ng of protein, which represents 80% or more of the dry weight of the zona pellucida and about 17% of the oocyte's total protein. Electrophoretic analyses of as few as five isolated zonae pellucidae treated with diazotized [ 125I]iodosulfanilic acid revealed the presence of only three radiolabeled proteins, designated ZP1, ZP2, and ZP3. The same three proteins were identified by Coomassie blue staining when large numbers of isolated zonae pellucidae (approximately 750) were subjected to SDS-polyacrylamide gel electrophoresis. These three proteins migrate as broad bands on SDS-polyacrylamide gels, consistent with their being glycoproteins, with apparent molecular weights of 200,000 (ZP1), 120,000 (ZP2), and 83,000 (ZP3). The same proteins were radiolabeled when intact oocytes were treated with diazotized [ 125I]iodosulfanilic acid, a reagent which does not penetrate the oocyte's plasma membrane, or when isolated zonae pellucidae were treated with 3H-labeled 1-dimethylaminonaphthalene-5-sulfonyl chloride. Results of amino acid analysis and high-resolution two-dimensional electrophoresis of the individual proteins suggest that each protein represents a unique polypeptide chain. The proteins ZP1, ZP2, and ZP3 represent about 36, 47, and 17%, respectively, of the total protein of the zona pellucida. In the presence of reducing agents which cause dissolution of the zona pellucida, ZP1 is converted into a species which migrates with an apparent molecular weight of 130,000, suggesting that it exists as an oligomer, stabilized by disulfide bonds, in the unreduced state. The results of these experiments are discussed in terms of the properties of the zona pellucida before and after fertilization and are compared with results obtained using vitelline envelopes of eggs from nonmammalian animal species.

15] Radioiodination of cell surface lipids and proteins for use in immunological studies

This chapter describes the use of radioiodinated diazotized iodosulfanilic acid (ISA) and a lactoperoxidase-catalyzed iodination method for the labeling and examination of plasma membrane proteins and lipids in cells under humoral immune attack. There are currently available several reagents, including ISA and a NaI-lactoperoxidase (LPO) complex, that have been used to label the cell membranes radioisotopically, presumably by labeling exposed tyrosine and/or histidine residues on the plasma membrane proteins. The lactoperoxidase-catalyzed NaI reaction has been shown to label a variety of cell surface lipids, including phospholipids, triacylglyceddes, free fatty acids, and lysophosphatides. The labeling reaction is enzyme-dependent in its initial steps, in which an oxidizing agent (H2O2) is generated, with the subsequent oxidation of I– (from NaI) to I2. The final interaction of I2, with lipid molecules, does not involve a simple addition reaction of I to the unsaturated fatty acid constituents of lipids, because fully saturated fatty acids or saturated fatty acid-containing neutral lipids and phospholipids can also be labeled by this reaction. This suggests that I2 substitution or exchange reactions can also occur in this system.

Studies on the transverse localization of lysophospholipase II in bovine liver microsomes by immunological techniques

1. 1. Lysophospholipase activity solubilized from bovine liver microsomes could be precipitated for more than 80% by antibodies evoked in rabbits against the purified bovine liver lysophospholipase II. 2. 2. After solubilization of the microsomes in 1.5% sodium deoxycholate, an immunoprecipitate containing lysophospholipase II in enzymically active form could be isolated. 3. 3. Microsomal lysophospholipase activity was completely inhibited by [ 3H]-diisopropylphosphofluoridate. Enzyme labelled in this way was isolated by immunoprecipitation from control and chymotrypsin-treated microsomes. Sodium dodecyl sulfate disc gel electrophoresis of the immunoprecipitates showed that chymotrypsin treatment of intact microsomes had no influence on the molecular weight of the enzyme. 4. 4. Attempts to label the lysophospholipase II in microsomes by lactoperoxidase catalyzed iodination or by reaction with the diazonium salt of [ 125I]iodo-sulfanilic acid were negative, although both techniques labelled other microsomal proteins efficiently. 5. 5. Antibody absorption experiments gave no indication for the presence of lysophospholipase antigenic sites on the outside surface of microsomes. 6. 6. These experiments are interpreted to indicate that lysophospholipase II is exclusively located at the luminal side of the microsomal membrane.