High Strength Buffer Research Articles

A Method for Isolation of Total RNA from Fruit Tissues of Banana

We describe a rapid and efficient method for isolation of total RNA from banana fruit tissues. The RNA was extracted with a high ionic strength buffer at room temperature. The proteins, genomic DNA and secondary metabolites in the extract were then removed by precipitation with pre-cooled potassium acetate and repeated phenol/chloroform/isoamyl alcohol extractions. The RNA was recovered by ethanol precipitation. It was relatively free of ribonucleases and was suitable for RT-PCR and northern blot analysis. The procedure can be completed in less than 4 hours.

Expression and characterisation of the homodimeric E1 component of the Azotobacter vinelandii pyruvate dehydrogenase complex.

We have cloned and sequenced the gene encoding the homodimeric pyruvate dehydrogenase component (E1p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and expressed and purified the E1p component in Escherichia coli. Cloned E1p can be used to fully reconstitute complex activity. The enzyme was stable in high ionic strength buffers, but was irreversibly inactivated when incubated at high pH, which presumably was caused by its inability to redimerize correctly. This explains the previously found low stability of the wild-type E1p component after resolution from the complex at high pH. Cloned E1p showed a kinetic behaviour exactly like the wild-type complex-bound enzyme with respect to its substrate (pyruvate), its allosteric properties, and its effectors. These experiments show that acetyl coenzyme A acts as a feedback inhibitor by binding to the E1p component. Limited proteolysis experiments showed that the N-terminal region of E1p was easily removed. The resulting protein fragment was still active with artificial electron acceptors but had lost its ability to bind to the core component (E2p) and thus reconstitute complex activity. E1p was protected against proteolysis by E2p. The allosteric effector pyruvate changed E1p into a conformation that is more resistant to proteolysis.

Streptomycin binds to the decoding center of 16 S ribosomal RNA

Streptomycin, an error-inducing aminoglycoside antibiotic, binds to a single site on the small ribosomal subunit of bacteria, but this site has not yet been defined precisely. Here, we demonstrate that streptomycin binds to E. coli 16 S rRNA in the absence of ribosomal proteins, and protects a set of bases in the decoding region against dimethyl sulfate attack. The binding studies were performed in a high ionic strength buffer containing 20 mM Mg2+. The pattern of protection in the decoding region was similar to that observed when streptomycin binds to the 30 S subunit. However, streptomycin also protects the 915 region of 16 S rRNA within the 30 S subunit, whereas it did not protect the 915 region of the naked 16 S rRNA. The interaction of streptomycin with 16 S rRNA was further defined by using two fragments that correspond to the 3′ minor domain of 16 S rRNA and to the decoding analog, a portion of this domain encompassing the decoding center. In the presence of streptomycin, the pattern of protection against dimethyl sulfate attack for the two fragments was similar to that seen with the full-length 16 S rRNA. This indicates that the 3′ minor domain as well as the decoding analog contain the recognition signals for the binding of streptomycin. However, streptomycin could not bind to the decoding analog in the absence of Mg2+. This contrasts with neomycin, another error-inducing aminoglycoside antibiotic, that binds to the decoding analog in the absence of Mg2+, but not at 20 mM Mg2+. Our results suggest that both neomycin and streptomycin interact with the decoding center, but recognize alternative conformations of this region.

ISOLATION AND CHARACTERIZATION OF PECTINESTERASE FROM VALENCIA ORANGE

Thermolabile (TL) and thermostable (TS) pectinesterases (PE) were extracted from the pulp of fresh Valencia oranges by enzyme hydrolysis using a native pH and high ionic strength buffer system. Each form was separated individually by CM-Sephadex C-50, Phenyl Sepharose CL-4B and CM-Bio-Gel A chromatography. TLPE and TSPE exist as multiple forms which were shown to differ in their stability and effects on cloud loss in single strength orange juice.

Free radical induced oxidative depolymerisation of chondroitin sulphate and dermatan sulphate

The glycosaminoglycan (GAG) dermatan sulphate (DS) is a potentially useful therapeutic for antithrombotic applications. Natural DS is isolated as a polymer of a molecular weight of around 25000. The predominant disaccharide unit is IdoA-GalNAc4SO 3 but the antithrombotic activity is mediated by the binding of heparin cofactor II (HCII) to repeats of IdoA2SO 3-GalNAc4SO 3. We hoped, in parallel with other bioactive GAGs, such as heparin, to improve the phamacological properties of DS by lowering the molecular weight using oxidative free radical depolymerisation. We found indeed that the method gives a smooth and repeatable reduction in molecular weight using chondroitin sulphate as a model system but it appears to selectively attack the disaccharide unit IdoA2SO 3-GalNAc4SO 3 in DS, severely reducing its activity towards HCII. In the course of these investigations we found that, in contrast to prior work on heparin, uncharged polysaccharides cannot be used as standards for the determination of molecular weight of DS and chondroitin sulphate by high performance size exclusion chromatography with high ionic strength buffers.

Autolysis, Ca2+ Requirement, and Heterodimer Stability in m-Calpain

The roles of N-terminal autolysis of the large (80 kDa) and small (28 kDa) subunits in activation of rat m-calpain, in lowering its Ca2+ requirement, and in reducing its stability have been investigated with heterodimeric recombinant calpains containing modified subunits. Both autolysis and [Ca2+]0.5 were influenced by the ionic strength of the buffers, which accounts for the wide variations in previous reports. Autolysis of the small subunit (from 28 to 20 kDa) was complete within 1 min but did not alter either the Ca2+ requirement ([Ca2+]0.5) or the stability of the enzyme. Autolysis of the NHis10-80k large subunit at Ala9-Lys10 is visible on gels, was complete within 1 min, and caused a drop in [Ca2+]0.5 from 364 to 187 microM. The lower value of [Ca2+]0.5 is therefore a property of the Delta9-80k large subunit. Autolysis at Ala9-Lys10 of the unmodified 80-kDa large subunit is not detectable on gels but was assayed by means of the fall in [Ca2+]0.5. This autolysis was complete in 3.5 min and was inhibited by high [NaCl]. The autolysis product of these calpains, which is essentially identical to that of natural m-calpain, was unstable in buffers of high ionic strength. Calpain in which the large subunit autolysis site had been mutated was fully active but did not undergo a drop in [Ca2+]0.5, showing that m-calpain is active prior to autolysis. The main physiological importance of autolysis of calpain is probably to generate an active but unstable enzyme, thus limiting the in vivo duration of calpain activity.

High-efficiency entrapment of superoxide dismutase into cationic liposomes containing synthetic aminoglycolipid.

A monofatty acid ester of glucosamine (PGlcN) was synthesized to provide liposomal membranes with a positive charge, and the trapping efficiency of negatively charged substances (superoxide dismutases, SODs) into cationic liposomes containing PGlcN or stearylamine (SA) prepared by various methods was compared to find the most efficient trapping methods. We demonstrated that cationic liposomes, which were prepared in a buffer of low ionic strength containing sorbitol by a simple hydration method, could entrap a large amount of negatively charged SODs which retained their activity, as compared with cationic liposomes prepared in a buffer of high ionic strength. We also showed a reverse-phase evaporation method entrapped a large amount of SODs. However, SODs were inactivated during the preparation; therefore, this method was not suitable to entrap the enzyme. Freeze-thaw method induced the formation of cationic liposomes which were smaller than extruded liposomes and could entrap the SODs in a buffer of low ionic strength. Dehydration-rehydration method with a buffer of low ionic strength also entrapped a large amount of SODs, indicating that the integrity of liposomes was lost in the lipid bilayer after freeze-drying and the SODs were entrapped in the reconstruction of liposomes during rehydration. These findings showed that the hydration method based on electrostatic attraction with a buffer of low ionic strength was simple and the most effective for entrapping SODs without loss of their activity.

Common nuclear matrix proteins in rat tissues.

Nuclear matrix proteins have been defined as insoluble residual proteins resulting from treatment of isolated nuclei with nucleases, detergents and high ionic strength buffers. They are considered as in part representing the proteins constituting the three-dimensional framework of the interphase nucleus. Though cell-specific nuclear matrix proteins have been differentiated from ubiquitously occurring (common) nuclear matrix proteins, the number and types of common nuclear matrix proteins have not yet been unequivocally established. In the present study nuclear matrix proteins were prepared from isolated nuclei of rat kidney, liver, lung, spleen and testes. The matrix proteins were separated by two-dimensional (2-D) electrophoresis and silver stained. Then the spot patterns were compared by computer-assisted image analysis. Composite images were derived for nuclear matrix proteins of individual tissues. Finding between 396-483 spots per tissue, a total of 964 individual spots were registered. Of these, 102 were common nuclear matrix proteins, as appearing in each of the tissue-characteristic images. The apparent molecular mass and pI data may serve for further identification of these nuclear proteins.

The Effect of Concentration on the Structure of α-Crystallin

Several models have been proposed for arrangement of the subunits in α-crystallin. These include the contrasting proposals that subunits are arranged in three layers and that subunits assemble into micelle-like structures. The validity of the micelle model was investigated by examining the effects of variations in protein concentration on the surface tension, conductivity, molecular weight and conformation of α-crystallin. The data were compared with those obtained for bovine serum albumin (BSA) and sodium dodecyl sulphate (SDS). Measurements of surface tension were conducted in the range, 10μgml -1to 130mgml -1, in low and high ionic strength buffers. An apparent point of inflection, independent of ionic strength, was seen in α-crystallin's surface tension at around 1.9mgml -1(95μ m). The surface tension did not plateau beyond this point, as is the case with surfactants, but continued to decrease up to 130mgml -1. BSA exhibited similar surface tension properties with an apparent inflection at 0.9mgml -1(13μ m). The conductivity of α-crystallin and BSA solutions increased smoothly with no sign of any transition up to 96mgml -1and 60mgml -1, respectively. In contrast, SDS showed a clear transition in this property at the concentration corresponding to its CMC. The aggregation state of the α-crystallin aggregates was examined by comparing molecular masses and Stokes radii. The size of the protein remained uniform over a wide concentration range and was unaffected by variations in ionic strength. Protein conformation, which was monitored by examining the microenvironment of tryptophan residues, was also found to be independent of protein concentration. It is concluded that over the concentration range that was investigated, α-crystallin does not exhibit any of the properties associated with classical micelles formed from small amphiphilic molecules.

Interaction of recombinant human bone morphogenetic protein-2 with poly(d,l lactide-co-glycolide) microspheres.

The combination of recombinant human bone morphogenetic protein-2 (rhBMP-2) with poly(d,l lactide-co-glycolide) (PLGA) porous microspheres provided for "sustained release" of the protein from the microspheres. Soaking 50:50 PLGA microspheres in a buffered rhBMP-2 solution for a sufficient period of time to permit equilibrium binding enabled quantification of "free" and "bound" protein. "Free" protein is defined as protein present within the porous matrix of the microspheres, whereas "bound" refers to protein adsorbed to PLGA surfaces. Kinetics of the rhBMP-2-microsphere association revealed that equilibrium was attained within 8 hr for two buffer systems (arginine/histidine, pH 6.50; and glutamic acid/sodium glutamate, pH 4.50). Increasing the concentration of the rhBMP-2 stock solution used for the interaction studies from 0.025 to 1.0 mg/ml increased the amount of rhBMP-2 adsorbed and the concentration of free rhBMP-2. Beyond a 1.0 mg/mL concentration, only free rhBMP-2 levels increased. Linearized Langmuir treatment of the adsorption data yielded values corresponding to monolayer coverage of the microspheres (Cm) and the equilibrium adsorption constant (K) of 0.17 microgram/cm2 and 7.57 ml/mg, respectively. Studies performed to determine the effect of ionic strength revealed that increasing NaCl and buffer concentration decreased the amount of protein adsorbed. rhBMP-2 release studies, conducted in an isotonic phosphate buffered saline, pH 7.4 vehicle, revealed that free rhBMP-2 was released during an initial period of 72-96 hr. Following this period, there was no discernible release of rhBMP-2 from the microspheres for up to 7 days, suggesting that the bound protein would remain at a defect site and release slowly upon erosion of the polymer. Mass balances performed by using an extraction buffer of high ionic strength confirmed this prediction.

Large scale procedure for fractionation of albumins and globulins from pea seeds.

The buffer extractable proteins of pea, albumins and globulins, were successively extracted in a large amount at a pilot scale. In order to preserve as much as possible the native structure of proteins, a selective solubilization step procedure was performed. Firstly, albumins were extracted with acetate buffer and secondly globulins with phosphate buffer of high ionic strength. Each extract was desalted by diafiltration without protein precipitation. From 15 kg of flour, 380 g of albumin fraction and 1000 g of globulin fraction were obtained with a protein content of 86.0% and 90.7% of dry matter respectively. The characterisation of albumin and globulin fractions by electrophoresis, ultracentrifugation and anion-exchange chromatography, showed that the cross-contamination of these two fractions was minimal.

Purification and characterisation of soluble invertases from leaves of Arabidopsis thaliana

Multiple isoforms of beta-fructofuranosidase (invertase, EC 3.2.1.26) were identified in mature green leaves of the cruciferous plant Arabidopsis thaliana (L.) Heynh. There were four major and one minor isoforms of soluble acid invertase and an additional activity which could be released from the cell wall by buffers of high ionic strength. This study reports the separation and characterisation of three soluble isoforms following ammonium sulphate and polyethylene glycol 6000 precipitations, Concanavalin A, MonoQ ion exchange, Superose 12 size-exclusion chromatography and chromatofocusing. These isoforms, designated INV1, INV2 and INV3, had isoelectric points of 4.75, 4.70 and 4.65 and a Km for sucrose of 5, 12 and 5 mM, respectively. Each had a pH optimum of 5.5, exhibited optimal activity at 45 degrees C and used sucrose as the preferred substrate. All fractions containing these isoforms contained a 52-kDa polypeptide which was specifically detected by immunoblotting with an antibody raised against deglycosylated wheat invertase. The N-terminal amino-acid sequence of this polypeptide was homologous to acid invertases isolated from other plant species. The possible origin of isoforms of soluble acid invertase is discussed.

Mg(2+)-dependent conformational changes in the hammerhead ribozyme.

Conformational changes of the hammerhead ribozyme were examined by fluorescence changes of 2-aminopurine riboside incorporated either in the substrate or in the ribozyme. Fluorescence changes could be observed for both the substituted substrate and ribozyme upon complex formation, indicating a different environment for the 2-aminopurine in the complex. Ribozyme-substrate constructs for ciscleavage containing 2-aminopurine at various sites were used for the determination of binding constants of Mg2+ and Ca2+. Depending upon the site of 2-aminopurine substitutions, the fluorescence intensity upon addition of Mg2+ or Ca2+ was reduced by 0-50%. The measurements were performed in high ionic strength buffers such that base pairing in the helical regions is expected to be complete. With three of the ribozymes, the dependence of the fluorescence emission as a function of Mg2+ concentration could be fitted by single binding processes, whereas for the two remaining ribozymes a second binding process needed to be included. The binding constants range from 7600 M-1 down to 12 M-1 in 75 mM Tris-HCl (pH 7.5) and indicate the presence of multiple binding sites in the ribozymes with varying degrees of affinity toward the metal ions. Mg2+ binding constants determined in the same buffer from the Mg2+ dependence of the cleavage rate are of the order of 100 M-1; thus, Mg2+ sites directly involved in catalysis are of intermediate affinity. The ribozyme containing 2-aminopurine in loop III demonstrated the highest binding constant whereas the ribozyme with a 2-aminopurine next to a 2'-deoxy-2'-aminocytosine at the cleavage site exhibited only low metal ion affinity. The data obtained for Ca2+ are very similar to those found for Mg2+. This approach provides a first set of data describing a Mg2+ binding topography to hammerhead RNA molecules and should be useful for the analysis of other RNA molecules.

Identification and localization of proteins in gregarines that are immunologically related to smooth muscle α-actinin

Summary The immunological cross-reactivity of proteins from gregarine cell extracts with polyclonal and monoclonal antibodies against smooth muscle α-actinin was demonstrated by immunoblotting in one- and two dimensional gel electrophoresis and by immunofluorescence microscopy. In immunoblotting, two proteins of Gregarina blaberae with Mr = 90,000 and Mr = 66,000 and with isoelectric points of 5.5 and 5.9, respectively, showed cross-reactivity with anti-α-actinin antibodies. Electrophoretic analysis showed that the two proteins were associated with ghosts and a cytoplasmic extract of trophozoite and sexual stages. The Mr = 90,000 and the Mr = 66,000 proteins were extracted from G. blaberae ghosts by 6 M urea in high ionic strength buffer. Indirect immunofluorescence antibody staining showed these polypeptides to be localized in the cell cortex, within longitudinal lines underlying the folds of G. blaberae (Polycystidae) and Lecudina pellucida (Monocystidae), and with lines separating the large folds of Selenidium pendula (Selenidiidae). The presence of two distinct forms (Mr = 90,000 and Mr = 66,000) of α-actinin is discussed with regard to the motility of gregarines.

THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. II. ISOLATION OF A BINDING PROTEIN WHICH PARTIALLY ACTIVATES EGGS1

ABSTRACT An in vitro binding assay involving egg plasma membrane vesicles (PMVs) of Fucus serratus L. and proteins contained in a KCl extract of sperm has been used to identify a sperm protein involved in egg binding. High‐performance gel filtration (HPGF) separated the sperm KCl extract into several major fractions, and a protein (apparent M, 60 kDa) was identified as being involved in binding to the egg PMVs. This protein ran on denaturing sodium dodecyl sulfate (SDS)gels with an apparent molecular weight of 27 kDa. This suggests that either the native form of the protein is a dimer or the molecular weight on HPGF is an artifact caused by high ionic strength buffer promoting hydrophobic interactions. When KCl‐sol‐uble proteins were separated by SDS‐polyacrylamide gel electrophoresis (PAGE), blotted onto nitrocellulose, and incubated with biotinylated egg PMVs, these bound to a band at 27 kDa, confirming the role of this protein. Addition of the Fucus sperm extract or HPGF fractions containing the binding protein to eggs in the absence of sperm induced the release of polysaccharides onto the egg cell surface. This labeling was patchy, in contrast to the uniform release of polysaccharides observed when sperm were added to eggs. The monoclonal antibody (MAb) FS17 was raised against the 27‐kDa sperm protein. It labeled the sperm body and both flagella by immunofluorescence, though the sperm had to he permeabilized to observe labeling, suggesting that the epitope recognized is not exposed at the cell surface. Addition of FS17 to the KCl extract in the binding assay reduced subsequent binding of egg PMVs. Removal of the 27‐kDa protein recognized by FS17 from the sperm extract prevented the binding of egg PMVs in the binding assay and the triggering of the patchy release of polysaccharides when added to eggs. Overall the results suggest that the 27‐kDa sperm protein is involved in binding to the egg plasma membrane and can trigger partial activation of the egg.

Immunization of a rabbit with beta 2-glycoprotein I induces charge-dependent crossreactive antibodies that bind anionic phospholipids and have similar reactivity as autoimmune anti-phospholipid antibodies.

A rabbit immunized with beta 2-glycoprotein I (beta 2-GPI) produced Abs that bind to negatively charged phospholipids and to beta 2-GPI. After affinity purification of the Abs to beta 2-GPI, the dual reactivity could still be detected. Adsorption studies with a phosphatidylserine affinity column depleted phospholipid-reactive Abs, but beta 2-GPI reactivity was retained. The same pattern of reactivity was found with culture supernatants from rabbit anti-beta 2-GPI splenocytes fused with an immortalized rabbit cell line. The reactivity to negatively charged phospholipids is likely to involve ionic interactions, as high ionic strength buffers eliminated binding to anionic phospholipids, but not to beta 2-GPI. Affinity-purified anti-phospholipid (aPL) Abs from four of seven autoimmune patients bound anionic phospholipids in the absence of beta 2-GPI. However, in high ionic strength buffer, this binding was abolished in three patients and significantly reduced in the fourth. In contrast, affinity-purified aPL Abs from seven autoimmune patients bound to beta 2-GPI-coated plates, and binding in high ionic strength buffer was reduced only moderately in three patients. Therefore, autoimmune-type aPL Abs display anti-beta 2-GPI reactivity and charge-dependent binding to anionic phospholipids similar to affinity-purified rabbit anti-beta 2-GPI Abs.

The Nuclear Pore Complex Protein p62 Is One of Several Sialic Acid-containing Proteins of the Nuclear Envelope

While investigating the glycosylation of nuclear envelope proteins of neuroblastoma cells, we found several proteins that bound the sialic acid-specific Sambucus nigra agglutinin. The strongest signals were obtained for proteins with apparent molecular masses of 66 and 180 kDa. The specificity of the lectin binding was checked by acylneuraminyl hydrolase treatment of nuclear envelope proteins, which prohibited S. nigra agglutinin binding. Digestion of nuclear envelope proteins with the N-glycosidase F revealed that sialic acid was N-glycosidically linked to the 180-kDa protein and very probably O-glycosidically linked to the 66-kDa protein. Upon extraction, the latter behaved like the nucleoporin p62 in that it was partly extracted by high ionic strength buffers, could not be solubilized by nonionic detergent, and was completely removed from the nuclear envelope with urea. Two-dimensional gel electrophoretic comparison showed that the S. nigra agglutinin-binding protein and p62 have an identical isoelectric point of about 5.0 and an identical apparent molecular mass of 66 kDa. This, together with the binding of the anti-nucleoporin antibody, demonstrated the identity of the 66-kDa sialoprotein and p62. S. nigra agglutinin inhibits nuclear protein transport in neuroblastoma cells, strongly suggesting a functional significance of sialylation of p62.

High molecular weight polyarginine as a capillary coating for separation of cationic proteins by capillary electrophoresis

Fused silica capillaries treated with dilute solutions of high molecular weight polyarginine (PA) provide capillary electrophoresis separation efficiencies of more than 2 million theoretical plates per meter and improved migration time reproducibility for the analysis of protein mixtures. The non-covalently bound PA coating was stable for several days and could be easily regenerated by exposing the capillary to a dilute solution of PA for several minutes. A comparison of PA and polybrene (PB) treated capillaries indicates that the PA capillary is capable of providing higher separation efficiencies, but that PB provides a more stable coating when used under acidic conditions and with high ionic strength buffers.

Colocalization of a high molecular mass phosphoprotein of the nuclear matrix (p255) with spliceosomes.

It was previously demonstrated that monoclonal antibody CC-3 binds to a phosphorylation dependent epitope present on a 255 kDa nuclear protein (p255). We show here that in interphase cells, p255 distributes to typical nuclear speckles that correspond to the localization of spliceosome components as revealed by antibodies to the m3G cap of snRNAs or to the non-snRNP splicing factor SC-35. Immunofluorescence and immunoblot studies indicated that p255 is resistant to extraction with non-ionic detergents, nucleases and high ionic strength buffers and may thus be defined biochemically as a nuclear matrix phosphoprotein. To determine the nature of the association of p255 with the nuclear structure, its distribution was studied at different stages of the cell cycle and after the cells were treated with nucleases or heat shocked. We found that the antigen diffused into the cytoplasm during metaphase but was reorganized into cytoplasmic speckles during anaphase-telophase transition, where it colocalized with SC-35. Nuclear matrix preparations that were digested with DNases and RNases showed that interphasic p255 still localized to nuclear speckles even though snRNA and snRNP antigens were removed. Heat-shocked cells labelled with monoclonal antibody CC-3 exhibited more rounded and less interconnected speckles, identical to those decorated by anti-SC-35 antibody under such conditions. These results indicate that p255 and SC-35 are present in the same nuclear structures, to which they are more tightly bound than the snRNP antigens. They further suggest that both proteins are implicated in spliceosome assembly or attachment.

Activity of cell wall‐associated enzymes in ripening olive fruit

Enzymatically active cell wall isolaled from olive (Olea europaea) fruit was employed Hi investigate some hydrolytic enzymes bound to the cell wall and the changes in these during ripening. Seven glycosidases. β‐glucosidase (EC 3.2.1.21) α‐galactosidase (EC 3.2.1.22). β‐galactosidase (EC 3.2.1.23). α‐arabinosidase (EC 3.2.1.55), α‐mannosidase (EC 3.2.1,24). β‐xylosidase (EC 3.2.1.37) and β‐N‐acetylglucosamidase (EC 3.2.1.30). as well as Cx‐cellulase (EC 3.2.1.4) and endo‐polygalacturonase (EC 3.2.1.15). were identified in the cell wall preparation, at four stages of ripeness (mature green. changing colour, black and black‐ripe). Activities of all these cell wall‐associated enzymes fionicallv and covalently linked) were determined either by cell wall incubation with artificial substrate or after extraction from the cell wall with buffers of high salt concentration (Cx‐cellulase). and were compared to those of forms solubilized from acetone powders with 500 nM citrate buffer (cytoplasmic and/or apoplastic plus ionically hound to cell wall) In general, the activities of low ionic strength buffer‐soluble enzymes were found to be much higher than those of the bound enzymes. The bound enzymes are present in the fruit at the green colour stage, whereas the activities of the soluble enzymes only increased from the changing colour stage onwards. The tenacity of binding of enzymes to the wall was investigated by treating the walls with high salt and measuring residual activity. The nature of the ionic and covalent binding and the changes during ripening were also established for wall‐hound glycosidase During ripening there was a marked change in the percentages of covalently‐ and tonically linked activities of β‐glucosidase and β‐galaclosidase: al the changing colour stages about 75–80% of the bound active in was present in high ionic strength buffer while al the black‐ripe stage it was only 15–20. A possible role for these cell wall degradative enzymes in olive softening is discussed.