A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli

A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli is described. Rabbit anti-human chorionic gonadotropin serum (0.2 ml) was digested with pepsin to convert IgG to F(ab')2 and applied to a column of human chorionic gonadotropin-Sepharose 4B, followed by elution at pH 2.5. The affinity-purified anti-human chorionic gonadotropin F(ab')2 was mixed with non-specific goat F(ab')2 (0.5 mg) as a carrier, reduced with 2-mercaptoethylamine to split F(ab')2 to Fab' and conjugated to beta-D-galactosidase using N,N'-o-phenylenedimaleimide. The affinity-purified rabbit anti-human chorionic gonadotropin Fab'-beta-D-galactosidase conjugate was separated from non-specific goat Fab'-beta-D-galactosidase conjugate and unconjugated beta-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B using 4 M urea. The amount of the affinity-purified conjugate obtained was 56-69 micrograms. The detection limit of human chorionic gonadotropin by a sandwich enzyme immunoassay technique was improved 30-fold by using the affinity-purified conjugate as compared with that before affinity-purification. This method is applicable to the conjugation with alkaline phosphatase from calf intestine and probably also other enzymes which are stable in 4 M urea.     

Improved procedure for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide.

The procedures for the conjugation of rabbit IgG and Fab' antibodies with beta-D-galactosidase from Escherichia coli using N,N'-o-phenylenedimaleimide were improved in several respects as compared with the previous methods (Eur. J. Biochem. 62, 285--292, 1976; J. Immunol. 116, 1554--1560, 1976). Maleimide residues were efficiently introduced into antibodies under an atmosphere of nitrogen; the average number of maleimide residues introduced into IgG and Fab' antibodies were 0.78 (0.65--0.86) and 0.86 (0.80--0.95) per molecule, respectively. The conjugation with the enzyme was performed at 4 degrees C at pH 6.5 for 15 or more hours. The conjugates were almost completely separated from unreacted IgG and Fab' by gel filtration. When the recoveries of IgG, Fab', and beta-D-galactosidase in the conjugates were 23-29, 35-44, and 99%, respectively, the average numbers of IgG and Fab' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8 per molecule, respectively. There was no significant impairment of beta-D-galactosidase activity or the activity of anti-human IgG antibody to bind to human IgG upon conjugation. However, the conjugate preparation was heterogeneous, and one-third of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab' antibody gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG antibody.     

Comparison of beta-D-galactosidase from Escherichia coli and horseradish peroxidase as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique

beta-D-Galactosidase from Escherichia coli and horseradish peroxidase were compared as labels of anti-human ferritin Fab' by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The anti-human ferritin Fab'-peroxidase conjugates gave lower nonspecific bindings and higher specific bindings than the corresponding Fab'-beta-D-galactosidase conjugates. As a result, the former provided more sensitive dose response curves for human ferritin than the latter. However, the peroxidase conjugates were required in a larger quantity, since peroxidase assay was much less sensitive than beta-D-galactosidase assay.     

Detection of one milliattomole of ferritin by novel and ultrasensitive enzyme immunoassay

A novel and ultrasensitive enzyme immunoassay (immune complex transfer two-site enzyme immunoassay) for ferritin is described. Ferritin was reacted simultaneously with affinity-purified dinitrophenyl biotinyl anti-ferritin IgG and affinity-purified anti-ferritin Fab'-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto affinity-purified (anti-dinitrophenyl group) IgG-coated polystyrene balls. After eliminating excess conjugate by washing, the complex was eluted from the polystyrene balls with an excess of epsilon N-dinitrophenyl-L-lysine and transferred to streptavidin-coated polystyrene balls. The beta-D-galactosidase activity bound to streptavidin-coated polystyrene balls was assayed by fluorometry. Nonspecifically bound beta-D-galactosidase activity was remarkably lowered but there was much less decrease in specifically bound beta-D-galactosidase activity. As a result, the detection limit of ferritin was lowered to 1 milliattomole (1 x 10(-21) mol, 600 molecules as calculated from Avogadro's number). This technique will be useful for measuring, for example, antigens in single cells.     

Use of rabbit antiboty IgG bound onto plain and aminoalkylsilyl glass surface for the enzyme-linked sandwich immunoassay.

Rabbit antibody IgG was bound onto aminoalkylsilyl or plain glass rods by simple adsorption. For comparison, rabbit antibody IgG was also bound onto glutaraldehyde-activated aminoalkylsilyl glass rods. These antibody-glass rods were tested by the sandwich procedure using Fab' fragments of rabbit antibody conjugated with beta-D-galactosidase from Escherichia coli. The glutaraldehyde-activated aminoalkylsilyl glass showed the largest capacity to bind antigen and the plain glass showed the smallest. However, the antibody-glass rods prepared by simple adsorption were as useful for the sandwich immunoassay of macromolecular antigens as those prepared with glutaraldehyde. With all the antibody-glass rods prepared, 0.1 to 10 fmol of ornithine delta-aminotransferase from rat liver and 2,4-dinitrophenyl human IgG were measurable. More than 10 fmol of the antigens may be measurable with larger amounts of the antibody-beta-D-galactosidase complexes, although the non-specific binding of the complexes to the solid phase increases to limit the sensitivity of the immunoassay.     

A more sensitive enzyme immunoassay of anti-insulin IgG in guinea pig serum with less non-specific binding of normal guinea pig IgG

An enzyme immunoassay of anti-insulin IgG in guinea pig serum was improved in sensitivity by reducing the non-specific binding of normal guinea pig IgG and enhancing the specific binding of anti-insulin IgG. Silicone rubber pieces or polystyrene balls were coated with normal rabbit IgG, followed by coupling of insulin using glutaraldehyde. The insulin-normal rabbit IgG-coated silicone rubber pieces or polystyrene balls were incubated with normal rabbit IgG and then with diluted guinea pig anti-insulin serum in the presence of normal rabbit IgG at a lower temperature (20 degrees C). Finally, the solid phases were incubated with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate to measure the amount of guinea pig IgG bound. The detection limit of anti-insulin IgG in guinea pig serum was improved 10 to 100-fold compared to that of enzyme immunoassay performed by incubating insulin-bovine serum albumin-coated solid phases with diluted guinea pig anti-insulin serum at 37 degrees C and then with rabbit (anti-guinea pig IgG) Fab' conjugated to beta-D-galactosidase from Escherichia coli, according to a previous report (Kato, K., et al. (1978) J. Biochem. 84, 93-102).     

A sensitive two-site enzyme immunoassay for human epidermal growth factor (urogastrone)

A sensitive enzyme immunoassay (EIA) was developed for human epidermal growth factor (hEGF) or urogastrone, which was isolated from human urine. Our EIA system is based on the sandwiching of an antigen between anti-hEGF IgG coated on a polystyrene tube and anti-hEGF antibody Fab'-linked beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). This method has the advantages that the procedures are simple and rapid and that the antibody Fab'-beta-D-galactosidase complex is more stable than radioisotope-labeled IgG. Purified hEGF is detectable at as low as 100 pg/ml, which is very sensitive compared to the radioimmuno-assays or radioreceptor assays already reported. Using this new EIA system, hEGF levels in human urine were examined. The values for normal males and females were 48.4 and 83.5 ng/mg creatinine, respectively, which shows that females excrete 1.7 times more hEGF than males.     

Specific protein-binding reactions monitored with ligand-ATP conjugates and firefly luciferase

A new method was developed to monitor specific protein binding reactions with an ATP-labeled ligand and firefly luciferase. The ligand, 2,4-dinitrobenzene, was covalently coupled to four ATP derivatives and three of these conjugates were measured quantitatively at nanomolar levels with firefly luciferase. Incubation of the conjugates with antibody to the 2,4-dinitrophenyl residue diminished the peak light intensities produced in the bioluminescent assay, whereas incubation with immunoglobulin from a nonimmunized rabbit did not affect light production. Therefore, the antibody-bound ligand-ATP conjugates were inactive in the bioluminescent assay and levels of unbound conjugate could be measured in the presence of the bound form. The firefly luciferase was used to monitor competitive binding reactions between the antibody, the conjugates, and N(2,4-dinitrophenyl)-β-alanine.     

Pretargeting for cancer radioimmunotherapy with bispecific antibodies: role of the bispecific antibody's valency for the tumor target antigen

The use of a divalent effector molecule improves bispecific antibody (bsMAb) pretargeting by enabling the cross-linking of monovalently bound bsMAb on the cell surface, thereby increasing the functional affinity of a bsMAb. In this work, it was determined if a bsMAb with divalency for the primary target antigen would improve bsMAb pretargeting of a divalent hapten. The pretargeting of a (99m)Tc-labeled divalent DTPA-peptide, IMP-192, using a bsMAb prepared by chemically coupling two Fab' fragments, one with monovalent specificity to the primary target antigen, carcinoembryonic antigen (CEA), and to indium-loaded DTPA [DTPA(In)], was compared to two other bsMAbs, both with divalency to CEA. One conjugate used the whole anti-CEA IgG, while the other used the anti-CEA F(ab')(2) fragment to make bsMAbs that had divalency to CEA, but with different molecular weights to affect their pharmacokinetic behavior. The rate of bsMAb blood clearance was a function of molecular weight (IgG x Fab' < F(ab')(2) x Fab' < Fab' x Fab' conjugate). The IgG x Fab' bsMAb conjugate had the highest uptake and longest retention in the tumor. However, when used for pretargeting, the F(ab')(2) x Fab' conjugate allowed for superior tumor accretion of the (99m)Tc-IMP-192 peptide, because its more rapid clearance from the blood enabled early intervention with the radiolabeled peptide when tumor uptake of the bsMAb was at its peak. Excellent peptide targeting was also seen with the Fab' x Fab' conjugate, albeit tumor uptake was lower than with the F(ab')(2) x Fab' conjugate. Because the IgG x Fab' bsMAb cleared from the blood so slowly, when the peptide was given at the time of its maximum tumor accretion, the peptide was captured predominantly by the bsMAb in the blood. Several strategies were explored to reduce the IgG x Fab' bsMAb remaining in the blood to take advantage of its 3-4-fold higher tumor accretion than the other bsMAb conjugates. A number of agents were tested, including those that could clear the bsMAb from the blood (e.g., galactosylated or nongalactosylated anti-id antibody) and those that could block the anti-DTPA(In) binding arm [e.g., DTPA(In), divalent-DTPA(In) peptide, and DTPA coupled to bovine serum albumin (BSA) or IgG]. When clearing agents were given 65 h after the IgG x Fab' conjugate (time of maximum tumor accretion for this bsMAb), (99m)Tc-IMP-192 levels in the blood were significantly reduced, but a majority of the peptide localized in the liver. Increasing the interval between the clearing agent and the time the peptide was given to allow for further processing of the bsMAb-clearing agent complex did not improve targeting. At the dose and level of substitution tested, galacosylated BSA-DTPA(In) was cleared too quickly to be an effective blocking agent, but BSA- and IgG-DTPA(In) conjugates were able to reduce the uptake of the (99m)Tc-IMP-192 in the blood and liver. Tumor/nontumor ratios compared favorably for the radiolabeled peptide using the IgG x Fab'/blocking agent combination and the F(ab')(2) x Fab' (no clearing/blocking agent), and peptide uptake 3 h after the blocking agent even exceeded that of the F(ab')(2) x Fab'. However, this higher level of peptide in the tumor was not sustained over 24 h, and actually decreased to levels lower than that seen with the F(ab')(2) x Fab' by this time. These results demonstrate that divalency of a bsMAb to its primary target antigen can lead to higher tumor accretion by a pretargeted divalent peptide, but that the pharmacokinetic behavior of the bsMAb also needs to be optimized to allow for its clearance from the blood. Otherwise, blocking agents will need to be developed to reduce unwanted peptide uptake in normal tissues.     

Characterization of a monoclonal antibody to hog thyroid peroxidase and its use for immunohistochemical localization of the peroxidase in the thyroid gland

A monoclonal antibody (30.1.2) to hog thyroid peroxidase was produced, purified, and characterized. The IgG of 30.1.2 formed an immune complex with the peroxidase in a 1:2 or 1:1 molar ratio depending on the IgG to antigen ratio in the incubation mixture. Immune complex formation did not inhibit the peroxidase activity, which was actually activated 2-fold in the 1:1 complex. Studies of the binding of the conjugate of the IgG or its Fab' with horseradish peroxidase to untreated and acetone-treated thyroid microsomes showed that the IgG conjugate could bind to only a very small portion of the total binding sites (thyroid peroxidase) present in untreated microsomes even after prolonged incubation. The binding of the Fab' conjugate to untreated microsomes, on the other hand, increased as the incubation time was increased, reaching 40% of the total sites after 20 h of incubation. These findings indicated that thyroid peroxidase is localized on the inner surface of the microsomal membranes and that the Fab' conjugate, but not the IgG conjugate, can slowly penetrate through the membrane barrier to reach the peroxidase. Immunohistochemical experiments using the Fab' conjugate as a probe revealed that most thyroid peroxidase in the thyroid gland is located in the endoplasmic reticulum and perinuclear cisternae of the follicular cell, although a small amount could occasionally be detected in the apical membrane including microvilli. In contrast to previous reports, no thyroid peroxidase could be found in other cellular structures such as Golgi apparatus and apical vesicles by the immunohistochemical technique employed.     

Interaction of a glutathione S-conjugate with glutathione reductase. Kinetic and X-ray crystallographic studies

S-Conjugates of glutathione influence the glutathione/glutathione disulfide (GSH/GSSG) status of hepatocytes in at least two ways, namely by inhibition of GSSG transport into the bile [Akerboom et al. (1982) FEBS Lett. 140, 73-76] and by inhibition of the enzyme GSSG reductase (EC 1.6.4.2). The interaction of GSSG reductase with a well-studied conjugate, namely S-(2,4-dinitrophenyl)-glutathione and its electrophilic precursor 1-chloro-2,4-dinitrobenzene are described. For short exposures both compounds are reversible inhibitors of the enzyme, the Ki values being 30 microM and 22 microM respectively. After prolonged incubation, 1-chloro-2,4-dinitrobenzene blocks GSSG reductase irreversibly, which emphasizes the need for rapid conjugate formation in situ. As shown by X-ray crystallography the major binding site of S-(2,4-dinitrophenyl)-glutathione in GSSG reductase overlaps the binding site of the substrate, glutathione disulfide. However, the glutathione moiety of the conjugate does not bind in the same manner as either of the glutathiones in the disulfide.     

Construction of DNA-probes and their immunochemical detection using nonradioactive hybridization tests

Simple and efficient chemical approaches to preparation of DNA probes carrying 2,4-dinitrophenyl, dansyl or biotin residues were developed. The residues were introduced using following DNA derivatization procedures: a) transamination of cytidine residues with O-(4-aminobutyl)hydroxylamine; b) mercuration of pyrimidine residues followed by beta-mercaptoethanol modification. It was shown that 2,4-dinitrophenyl-containing DNA probes can be used for nonradioactive hybridization detection of nucleic acids. DNP-DNA: DNA complexes were detected using mouse antibodies specific to 2,4-dinitrophenyl groups, which were developed with peroxidase-conjugated antimouse immunoglobulins. Peroxidase-catalyzed chemoluminescent reaction of luminol oxidation with hydrogen peroxide allowed to detect 10 picograms of the dinitrophenylated single-stranded DNA probe.     

Immune complex transfer enzyme immunoassays for anti-angiotensin I IgG in serum using angiotensin I conjugates prepared by two different methods

Anti-angiotensin I IgG in serum was measured by immune complex transfer enzyme immunoassays using angiotensin I conjugates prepared by two different methods. In the first method, angiotensin I was conjugated to dinitrophenyl bovine serum albumin and beta-D-galactosidase through covalent links. Anti-angiotensin I IgG in rabbit serum was reacted simultaneously with dinitrophenyl bovine serum albumin-angiotensin I conjugate and beta-D-galactosidase-angiotensin I conjugate, and the complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls. After washing, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to goat (anti-rabbit IgG) IgG-coated polystyrene balls. Beta-D-Galactosidase activity bound to (anti-rabbit IgG) IgG-coated polystyrene balls was assayed by fluorometry. In the second method, biotinylated angiotensin I was coupled with dinitrophenyl bovine serum albumin-avidin conjugate and Beta-D-galactosidase-avidin conjugate and substituted for the two conjugates in the first method. The detection limits of anti-angiotensin I IgG in serum were 10-30 ng/liter (0.2-0.6 pg/assay). These methods were 330 to 1,000-fold more sensitive and much less affected by serum effect than the conventional enzyme immunoassay, in which an angiotensin I-bovine serum albumin-coated polystyrene ball was incubated with anti-angiotensin I IgG in serum and, after washing, with (anti-rabbit IgG) Fab'-peroxidase conjugate. The first method was more sensitive than the second method, but the second method may be superior in applicability to the first method.     

Analysis of Fc receptors for IgG on guinea pig peritoneal macrophages by the use of a monoclonal antibody to one of the Fc receptors

The variety and properties of Fc receptors (FcR's) for homologous IgG on guinea pig peritoneal macrophages were investigated with the use of a mouse monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea pig macrophages with a mouse myeloma cell line. VIA2 IgG1 completely inhibited the formation of macrophage rosettes with IgG1 antibody-sensitized erythrocytes, but not that with IgG2 antibody-sensitized erythrocytes. The Fab' of VIA2 IgG1 also completely inhibited the bindings of both monomeric and ovalbumin-bound IgG1 antibodies to macrophages. On the other hand, the Fab' did not affect the binding of monomeric IgG2 antibody to macrophages, although it partially inhibited that of ovalbumin-bound IgG2 antibody. These results show that at least two distinct types of FcR are present on guinea pig macrophages; one (FcR1,2) binds monomeric IgG1 antibody and also antigen-bound IgG1 and IgG2 antibodies, and the other (FcR2) binds monomeric and antigen-bound IgG2 antibodies alone, and also that VIA2 IgG1 binds specifically to FcR1,2. When FcR1,2 was isolated by affinity chromatography on F(ab')2 of VIA2 IgG1 coupled to Sepharose, it gave a main band with a molecular weight of 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was indistinguishable from the main band isolated with the IgG1 immune complex. The number of FcR1,2 per macrophage cell was estimated to be 2 X 10(5) by measuring the binding of 125I-Fab' of VIA2 IgG1.     

The variability of nitro group--protein interaction in the 2,4-dinitrophenyl-binding antibodies M315, M460 and X25 investigated by resonance Raman spectroscopy.

Pre-resonance Raman spectroscopy was used to study the interactions of the nitro groups of dinitrophenyl haptens with three dinitrophenyl-binding antibody fragments: M315 Fv, M460 Fab' and X25 Fab'. The observed changes in frequency of modes associated with the nitro moieties are compared with solvent-induced changes for the model hapten 2,4-dinitroaniline. These comparisons demonstrate a specific interaction via the H2N--C--C--2-NO2 and 4-NO2 groups with the protein. The interaction with the 4-NO2 group appears to be absent for epsilon-N-2,4-dinitrophenyl-L-lysine bound to M315 Fv fragment in contrast with either 2,4-dinitrophenylaspartate or 2,4-dinitrophenylglycine bound to M315 Fv fragment, despite the much tighter binding of the lysine derivative. The implications of this for M315 Fv fragment in terms of the antibody specificity are discussed. Comparisons of the effect of binding to M460 Fab' and X25 Fab' fragments also revealed significant differences in the shifts of the nitro group vibrations of 2,4-dinitrophenyl-lysine and 2,4-dinitroaniline.     

Synthesis of S6-(2,4-dinitrophenyl)-6-thioguanosine phosphoramidite and its incorporation into oligoribonucleotides

The preparation of N(2)-phenoxylacetyl-S(6)-(2,4-dinitrophenyl)-6-thioguanosine phosphoramidite and its subsequent incorporation into oligoribonucleotides is described. The identity of the oligonucleotides was confirmed by UV spectrophotometry and nucleoside composition analysis.     

Modification of the viral immunoassay for determining ultramicro-quantities of antigens

A technique for ultramicro-quantitation of antigens is suggested. It is based on the antigen isolation by immunosorption and a subsequent assay using phage-antigen conjugate (modification of viral immunoassay). Antibody Fab' fragments were bound to both the immunosorbent and the conjugate through their terminal thio groups. The technique sensitivity was one-two orders higher than that of ELISA for the corresponding antigens.     

Targeting weak antigens to CD64 elicits potent humoral responses in human CD64 transgenic mice

Previous studies have documented that targeting foreign Ags to IgG FcgammaR leads to enhanced Ag-specific responses in vitro and in vivo. However, the ability to overcome immunologic nonresponsiveness by targeting poorly immunogenic Ags to FcgammaR has not been investigated. To address this question in a simple model, we immunized transgenic mice expressing human CD64 (FcgammaRI) and their nontransgenic littermates with Fab' derived from the murine anti-human CD64 mAb m22. The m22 Fab' served as both the targeting molecule and the Ag. We found that only CD64-expressing mice developed anti-Id titers to m22. Furthermore, chemically linked multimers of m22 Fab', which mediated efficient internalization of the human CD64, were significantly more potent than monomeric m22 F(ab')(2) at inducing anti-Id responses. In all cases, the humoral responses were specific for m22 Id and did not react with other murine IgG1 Fab' fragments. Chemical addition of a second murine Fab' (520C9 anti-human HER2/neu) to m22 Fab' multimers demonstrated that IgG1 and IgG2a anti-Id titers could be generated to 520C9 only in the CD64-expressing mice. These results show that targeting to CD64 can overcome immunological nonresponsiveness to a weak immunogen. Therefore, targeting to CD64 may be an effective method to enhance the activity of nonimmunogenic tumor vaccines.     

Characterisation of a highly hydrophobically modified lactate dehydrogenase

1. Lysine residues of porcine H4 lactate dehydrogenase (L-lactate:NAD+ oxidoreductase EC 1.1.1.27) were modified with methyl-epsilon-(N-2,4-dinitrophenyl)aminocaproimidate - HCl. With increasing incorporation of the reagent a linear decrease of enzymatic activity was noticed. No essential lysyl group with an extraordinary reactivity was modified. 2. The active forms of the modified enzyme with different incorporation values were separated from denatured material by fractional precipitation and gel chromatography. An epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase was obtained with an average incorporation of 38 groups per tetramer and a residual activity of 42%. This material proved to be homogenous in cellulose electrophoresis. 3. The epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase is soluble only in glycine buffer at pH 8 and can be stabilized as ternary complex with NAD+ and sodium sulfite. Gel chromatography and ORD measurements show no strong conformational change. 4. epsilon-(N-2,4-dinitrophenyl)aminocaproamidinate lactate dehydrogenase has similar Km values for pyruvate, NADH, lactate and NAD+ as the native enzyme, and shows a lower thermostability due to a diminished stabilization by the hydrate layer on the surface.     

Membrane anchoring and surface distribution of glycohydrolases of human erythrocyte membranes

The membrane anchoring of the following glycohydrolases of human erythrocyte plasma membranes was investigated: alpha- and beta-D-glucosidase, alpha- and beta-D-galactosidase, beta-D-glucuronidase, N-acetyl-beta-D-glucosaminidase, alpha-D-mannosidase, and alpha-L-fucosidase. Optimized fluorimetric methods for the assay of these enzymes were set up. Treatment of the ghost preparation with 1.0 mol/l (optimal concentration) NaCl caused release ranging from 4.2% of alpha-D-glucosidase to 70% of beta-D-galactosidase; treatment with 0.4% (optimal concentration) Triton X-100 liberated 5.1% of beta-D-galactosidase to 89% of alpha-D-glucosidase; treatment with 1.75% (optimal concentration) octylglucoside yielded solubilization from 6.3% of beta-D-galactosidase to 85% of alpha-D-glucosidase. Treatment with phosphoinositide-specific phospholipase C caused no liberation of any of the studied glycohydrolases. These results are consistent with the notion that the above glycohydrolases are differently anchored or associated with the erythrocyte plasma membrane, and provide the methodological basis for inspecting the occurrence of these enzymes in different membrane microdomains.