Pl Values Research Articles

Actin-related proteins in Anabaena spp. and Escherichia coli

Actin has been described in all eukaryotic cells as the major microfilament cytoskeletal protein. Although prokaryotic cells do not have a cytoskeleton, proteins related to the latter have been found in different prokaryotic species. We have found prokaryotic actin-related proteins in the enterobacterium Escherichia coli and in the cyanobacteria Anabaena cylindrica and Anabaena variabilis. They were identified by the following criteria: (1) by cross-reaction with a fluorescent conjugated anti-actin (rat-brain) mAb by Western blot analysis (in total cellular extracts); (2) specific binding of acetone powder and soluble cellular extracts to DNase I; and (3) specific binding of cells and total cellular extracts to phalloidin. In E coli, specific binding of phalloidin labelled with rhodamine to cells was detected by spectrofluorometry. In total cellular extracts, three bands of 60, 43 and 35 kDa were weakly recognized by the mAb by Western blot analysis; this recognition increased when phalloidin was added to the extracts. Furthermore, three polypeptides of kDa were isolated by binding to DNase I, showing pI values of 6.7, 6.65 and 6.6, less acidic than all reported actin pI values. In A. cylindrica and A. variabilis, specific binding of phalloidin labelled with rhodamine to cells was also detected by spectrofluorometry. In total and soluble cellular extracts, the mAb recognized two bands of 45 and 40 kDa by Western blot analysis, but only the first was purified by binding to DNase I, and it showed three isoforms of pI values 6.8, 6.5 and 6.4. These results suggest the presence, in prokaryotes, of proteins with similar biochemical characteristics to eukaryotic actin.

Isolation and characterization of two fatty acid binding proteins from mouse brain.

Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross-reacted with antisera to recombinant liver L-FABP. One protein, designated brain H-FABP, migrated on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H-FABP bound NBD-stearic acid and cis-parinaric acid with K D values near 0.02 and 0.5 microM, respectively. Brain H-FABP cross-reacted with affinity-purified antisera to recombinant heart H-FABP. The second protein, mouse brain B-FABP, migrated on tricine SDS-PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B-FABP bound NBD-stearic and cis-parinaric acid with K D values near 0.01 and 0.7 microM, respectively. The brain B-FABP doublet was immunoreactive with affinity-purified antibodies against recombinant mouse brain B-FABP, but not with affinity-purified antibodies against heart H-FABP. (3H)Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B-FABP and brain H-FABP represented 0.1% and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H-FABP and brain B-FABP.

Observations On the Specificity of Globodera Pallida and G. Rostochiensis Diagnostic Proteins To Amphids of Globodera Species

Globodera pallida and G. rostochiensis proteins (Gp-5.7 and Gr-5.9, of pl 5.7 and 5.9 respectively) were purified from acrylamide gel bands excised after isoelectric focusing. A panel of monoclonal antibodies (MAbs) and a monospecific polyclonal antibody (PC 320) were produced to Gp-5.7 and Gr-5.9, respectively. The polyclonal antibody reacted equally, but some of the MAbs reacted differentially with the two species of potato cyst nematodes (PCN). MAb IACR Gp-5.7.1 recognises a proteic epitope and preliminary results indicated that the molecule is N-terminally blocked. This MAb bound only to antigens derived from cysts containing live eggs. Proteins related to Gp-5.7 and Gr-5.9 were identified in all pathotypes of potato cyst nematodes and in other Globodera species, but not in the Heterodera species tested. The antigen has the same molecular weight in all the species tested, but the isoelectric points varied between the different species. The pl values were 5.9 for G. rostochiensis, 5.7 for G. pallida and 5.77 for G. tabacum tabacum, G. t. solanacearum, G. t. virginiae and G. mexicana. This might indicate that the Globodera species from Mexico and the USA are more closely related to each other, than they are to G. pallida or G. rostochiensis. Immunofluorescence studies demonstrated that the MAb bound to the amphids of the Globodera species tested; the genus-specificity of this molecule might account for very specialized functions in the amphids of these nematodes. The differences in isoelectric points and antigenicity observed probably indicate that the Globodera species have different isoforms of the same molecule which might be related to specific functions in the nematode amphids.

Purification of a new ribosome-inactivating protein from the seeds of Cinnamomum porrectum and characterization of the RNA N-glycosidase activity of the toxic protein.

Porrectin, a new type II ribosome-inactivating protein (RIP), was purified from the seeds of the camphor tree (Cinnamomum porrectum) by affinity chromatography on acid-treated Sepharose 4B. Porrectin is a glycoprotein (M(r)64,500, sugar content 2.5%) consisting of an A-chain (M(r)30,500) and a B-chain (M(r)33,500) linked by the disulfide bond. The terminal sugar of glycan in porrectin B-chain is determined to be mannose. By non-denaturing polyacrylamide gel electrophoresis, porrectin displayed three isoforms that have different pl values with the same molecular weight. Porrectin is a potent inhibitor of eukaryotic protein synthesis in the rabbit reticulocyte lysate system. The molecular mechanism of action of porrectin on rat liver ribosomes is demonstrated to be specific for RNA N-glycosidase. The cleavage site is the adenosine at position 4324 (rat liver 28S rRNA) embedded in the highly conserved ricin/alpha-sarcin ('R/S') domain.

Molecular biology and biochemistry of human recombinant follicle stimulating hormone (Puregon).

Follicle stimulating hormone (FSH) is a heterodimeric glycoprotein hormone produced in the anterior pituitary gland. The hormone is essential in the regulation of reproductive processes, such as follicular development and ovulation. It is clinically used for treatment of anovulation and in assisted reproduction technologies such as in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Until recently, the only source for human FSH has been the urine from post-menopausal women. Such a natural source implies limited availability and potential product variability. Thus, we have cloned the genes encoding the alpha- and beta-subunits of human FSH and transfected these into Chinese hamster ovary (CHO) cells. A CHO-clone was isolated capable of secreting intact glycosylated FSH with identical amino acid sequences to natural FSH. This cell line was grown in perfusion culture and enabled us to isolate highly pure FSH (> 99%). The complexity of the charge distribution of human recombinant FSH was demonstrated by Isoelectric focusing. The observed microheterogeneity is caused by the large number of carbohydrate chain structures which are added to the four potential glycosylation sites in the alpha beta-dimer. Furthermore, the carbohydrates show a variation in their degree of sialylation which reflects the different pl values of the individual isohormones. Despite the complexity of post-translational modification, the isoform distribution of recombinant FSH produced in a CHO-cell line and grown in perfusion culture is surprisingly similar to that observed with pituitary FSH and urinary FSH. In conclusion, we have shown that FSH-gene transfected CHO-cells are capable of stable serum-free production of recombinant FSH. A process has been developed which assures the consistent and reproducible production of highly-purified recombinant FSH.

Fluorophore‐labeled carbohydrate analysis of immunoglobulin fusion proteins: Correlation of oligosaccharide content with in vivo clearance profile

CTLA4 is a membrane receptor on cytotoxic T cells whose interaction with the B7 counterreceptor on B cells is important in alloantigen responses. Soluble recombinant human and murine CTLA4 were produced using either Chinese hamster ovary or NS-0 cell lines. Expression vectors were constructed containing the gene coding for the extracellular domain of CTLA4 fused to either human lgG1 hinge, CH2, and CH3 domains or murine lgG2a hinge, CH2, and CH3 domain genes. These glycoproteins were produced in hollow-fiber or packed-bed-type bioreactors and purified from conditioned media by protein A affinity chromatography. Batches of purified CTLA4lg were analyzed for size, composition, and isoelectric point (pl) patterns by standard protein methods; oligosaccharide and monosaccharide profiles using several carbohydrate specific techniques; and in vivo clearance profiles using a murine model. Significant differences were observed between lots in their pl, clearance, and crbohydrate profiles. Higher overall pl values correlated with accelerated alpha-phase clearance and changes in oligosaccharide composition as determined by lectin binding analysis and electrophoresis of fluorophore-conjugated carbohydrates. Preparations exhibiting slower clearance profiles had oligosaccharides with higher quantities of N-acetylneuraminic acid and were predominantly of an N-linked biantennary complex-type. Conversely, batches with accelerated clearance profiles had less detectable N-acetylneuraminic acid. Oligosaccharides from murine CTLA4lg produced in NS-0 cells had terminal N-glycolylneuraminic acid but no detectable N-acetylneuraminic acid and had concomitant accelerated clearance. These data suggest that the presence and quantity of N-acetylneuraminic acid is an important component in predicting CTLA4lg plasma clearance rates and that production lots can be analyzed for oligosaccharide heterogeneity and sialic acid content by electrophoresis of fluorophore-conjugated carbohydrates. (c) 1995 John Wiley & Sons, Inc.

Sperm-activating proteins from unfertilized eggs of the Pacific herring, Clupea pallasii.

Ripe unfertilized eggs of the Pacific herring, Clupea pallasii, release sperm-activating proteins into seawater at the time of fertilization. Five species of herring sperm-activating proteins (HSAP) with different pl values (4.8, 4.9, 5.0, 5.1 and 5.4) were purified from the egg-conditioned medium by gel filtration and isoelectric focusing. Molecular mass of the HSAP (pl = 5.1), the major species of the five HSAP, was determined to be 8.1 kDa by mass spectrometry. Molecular weights of all of the HSAP were estimated to be 7700 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The first 20 amino acid sequences from N-terminal ends of three HSAP (pl = 4.9, 5.0 and 5.1) were almost identical, suggesting that the HSAP have similar structures.

Hemicellulolytic enzymes in P- and S-strains of Heterobasidion annosum

Summary: The secretion of several hemicellulolytic enzymes and endoglucanase was studied in S- and P-group strains of a wood-rotting pathogen, Heterobasidio annosum, using enzyme activity assays and isoelectric focusing. In liquid cultures supplemented with birch sawdust or pure xylan, both strains of the fungus produced xylanases, mannanases, endoglucanases and α-galactosidases. Low activities were detected in cultures containing only glucose as a carbon source. When S-strain cultures were provided with a cart matrix, protein secretion to the culture medium started earlier and was significantly increased compared to free cell cultures. This was not the case with the P-strain. In both strains of H. annosum the pH of the birch sawdust culture medium fell within 12 d to below 3.5, and then started to increase towards the end of the cultivation period. The increase in pH appeared to corresprelate with the formation of hemicellulolytic and endoglucanase activitie. The isoenzyme patterns of xylanase, mannanase and endoglucanase were typical for P- and S-strains. In P-strain cultures containing xylan, a xylanase with an alkaline pl value was induced which, however, was absent when the strain was grown with the birch sawdust substrate. In the P-strain the mannanase pattern at the early stage of growth was comparable to that of t S-strain at the later stages of growth on birch sawdust medium. Later in the growth of the P-strain, this pattern was replaced by mannanases with more acid pl values. The relative intensities of the individual endoglucanases varie between the P- and S-strains.

Induction of Newly Synthesized Proteins in Astroglial Cells Exposed to Lead

Although astroglial cells accumulate lead (Pb), they appear to be less sensitive to its overt deleterious effects than are neurons. One possible mechanism in which as rocytes adapt to Pb may be by upregulating the expression of particular genes. This study evaluated the effect of exogenous Pb on proteins synthesized in primary rat astroglial cultures. Following incubations with 0-50 microM Pb acetate cells were radiolabeled with leucine for various time intervals. Proteins were subsequently analyzed using SDS-PAGE followed by fluorography. Changes observed within the first 24 hr resembled a classical stress response and included increased synthesis of proteins with apparent molecular weights of 90, 70, 32-35, and 23 kDa. The most notable change was the enhanced synthesis and abundance of the 23-kDa protein (p23) which persisted throughout a 14-day treatment period, whereas the synthesis of the other proteins declined. This protein did not appear to be induced in rat lung fibroblasts treated with similar concentrations of Pb. Subcellular fractionation indicated that p23 was localized to the cytosol. Treatment with actinomycin D or cycloheximide prior to the addition of Pb precluded induction of p23. Pulse labeling of cells following a 24-hr Pb exposure revealed that p23 continued to be synthesized for 12 but not 24 hr following removal of Pb. Pulse-chase experiments indicated that the protein was stable for at least 18 hr following the removal of Pb. Two-dimensional gel electrophoresis revealed that the 23-kDa Pb-induced protein consisted of multiple-charged species with pl values ranging from 4.3 to 7.8.

Detection of Embryogenesis- and Organogenesis-Specific Glycoproteins in Rice Calli.

Differential expression of glycoproteins during somatic embryogenesis and organogenesis (formation of adventitious shoots) in rice calli was investigated by lectin staining after twodimensional polyacrylamide gel electrophoresis (2D-PAGE). Several embryogenesis-specific and organogenesis-specific glycoproteins were detected by staining with Concanavalin A (Con A). Affinity chromatography on Con Aagarose was performed to concentrate embryogenesis-specific glycoproteins, and 18 glycoproteins specific to embryogenesis were detected. Based on their pl values and MWs, 13 glycoproteins were newly detected after concentration by affinfty chromatography on Con Aagarose. It is suggested that affinity chromatography on a lectin-conjugated support enables to concentrate regeneration-related glycoproteins that are present at low levels.

Electrofiltration of solutions of amino acids or peptides

Summary - Enhanced transfer according to the isoelectric point (pl) of amino acids and peptides (molecular mass 130-6 000 9 mol-1) was achieved using laboratory filtration in the presence of an electric field. The permeate was enriched with arginine and lysine of negative electrophoretic mobility, while the retentate was enriched with the amino acid characterized by the lowest pl value, aspartic acid. A model which assumes no retention of amino acids by ultrafiltration (UF) membranes describes reasonably weil their transmission to the permeate. The peptide mixture permeate was enriched with peptides which were either positively or negatively charged according to the electric field direction. The effect was significant even under low electric field. The model did not quantily satisfactorily the experimental transmission probably because of significant retention of sorne peptides by the ultrafiltration membranes. The present study shows that electrofiltration (EF) may be a useful and efficient process for achieving selective separation of charged biological molecules provided that further work is aimed at a better understanding of which mechanisms rule the retention in EF and of the effect of process variables (flux, electric field, conductivity, charge of the molecules). electrofiltration 1amino acid 1 peptide Nomenclature: Cp, Cp amino acid concentration of permeate, retentate, 9 1-1; E, electric field, Y m-'; J, permeation flux, m3 m-2 s-'; Mp molecular mass; MMCO, molecular mass eut-off; N, Avogadro number (6.023 x 1023); pl, isoelectric pH; Rm, membrane hydraulic resistance, m-'; Rf, hydraulic resistance of fouling layer, m-1; Tr, transmission rate; TP, transmembrane pressure, Pa; r, molecule radius, m; U, electrophoretic mobility, m2 S-1 y-1; Z, number of charges; E, charge of electron, Cb; <p, sieving coefficient; Il, dynamic viscosity of feed, Pa s; IlP, dynamic viscosity of permeate, Pa s; p, density, kg m~.

Pectin methylesterase from Botrytis cinerea: physiological, biochemical and immunochemical studies

Pectin methylesterase (PME) was purified from the supernatant of Botrytis cinereastrain Bd90. SDS-PAGE showed a single band at 42 kDa, but this band corresponded to two distinct isoforms observed by IEF-PAGE at pl values of 7.0 and 7.4. PME was produced during the exponential phase of fungal growth and independently of the carbon source. Unlike other pectinases of B. cinerea, which are polymorphic, no differences were observed between the PME profiles of 25 strains of different origins. Polyclonal antibodies were raised against purified PME from B. cinerea, and immunochemical comparisons with PMEs from Erwinia chrysanthemi, Vigna radiataand Glycine maxshowed the presence of common epitopes between these different enzymes.

Isoenzymes of manganese-dependent peroxidase and laccase produced by the lignin-degrading basidiomycete Ceriporiopsis subvermispora

The white-rot basidiomycete Ceriporiopsis subvermispora produces two families of ligninolytic enzymes, namely manganese-dependent peroxidases (MnPs) and laccases, when growing in liquid cultures of defined composition. In medium containing 11 p.p.m. of Mn(II), up to seven isoenzymes of MnP and four isoenzymes of laccase were resolved by isoelectrofocusing (IEF), with pI values in the range 4.10-4.60 and 3.45-3.65, respectively. Occasionally, a fifth laccase isoform of pI 4.70 was also detected. In cultures with 25 and 40 p.p.m. of Mn(II), mainly the MnPs with higher pI values are produced. The isoenzyme pattern of MnP is not altered throughout the growth period of the fungus. MnP and laccase are also produced by C. subvermispora when growing on wood chips of Pinus radiata. Highest levels of both enzymes were obtained during the first week of incubation. A second peak of MnP activity was observed during the fourth week, whereas very low levels of laccase were extracted from the chips after the second week of growth. IEF analysis showed that the pI values of these laccases are similar to those of laccases produced in liquid cultures, being in the range 3.45-3.65. In contrast, four isoforms of MnP were resolved during the first week of incubation on wood chips, with pI values of 4.40, 4.17, 4.04 and 3.53. This profile underwent a transition during the second week of growth, at the end of which isoforms of MnP with pI values of 3.53, 3.40, 3.30 and 3.20 were resolved by IEF.(ABSTRACT TRUNCATED AT 250 WORDS)

Critical Concepts for Column Testing

Concepts critical for the evaluation of the miscible transport parameters from laboratory soil columns are presented. Failure to recognize a distinction between solute concentrations within the soil and solute concentrations collected in an effluent reservoir may result in errors in the evaluation of the column Peclet number, PL, and the retardation factor, Rd. An evaluation of three analytical models commonly used to describe miscible transport through soil columns indicates that only one model is capable of predicting the diffusive dominated transport observed in laboratory and field studies for transport at low flow rates. The common assumption that the retardation factor (Rd) is equal to the pore volumes of flow (T) at which the relative effluent concentration [(ce-ci)/(c0-ci)] equals 0.5 is shown to be invalid at low column Peclet numbers (PL≤10) based on mass balance constraints for finite soil columns. A more correct assessment of Rd is obtained by considering the area above the effluent breakthrough curve at equilibrium conditions [i.e., at (ce-ci)/(c0-ci)=1]. Finally, when effluent is allowed to collect in a reservoir over regular sampling intervals, the measured concentrations represent average, incremental concentrations. Regression analyses indicate that errors in regressed values of both Rd and PL in terms of an analytical model based on instantaneous concentrations generally are ≤10% when the sampling interval, ΔT, ≤ 0.25 and the average, incremental concentrations are plotted at the middle of the sample interval, and the errors tend to decrease as PL, Rd, and ΔT decrease.

Pectin Methylesterase Isoforms in Tomato (Lycopersicon esculentum) Tissues (Effects of Expression of a Pectin Methylesterase Antisense Gene).

We have identified two major groups of pectin methylesterase (PME, EC 3.1.1.11) isoforms in various tissues of tomatoes (Lycopersicon esculentum). These two groups exhibited differential immuno-cross-reactivity with polyclonal antibodies raised against tomato fruit PME or flax callus PME and differences in their accumulation patterns in tissues of wild-type and transgenic tomato plants expressing a PME antisense gene. The group I isoforms with isoelectric points (pls) of 8.2, 8.4, and 8.5 are specific to fruit tissue, where they are the major forms of PME activity. The group II PME isoforms, with pl values of 9 and above, are observed in both vegetative and fruit tissues. The group I isoforms cross-react with polyclonal antibodies raised to a PME isoform purified from fruit, whereas the group II isoforms cross-react with antibodies to a PME purified from flax callus. Expression of a fruit-specific PME anti-sense gene impairs accumulation of the group I PME isoforms, with no apparent effect on the accumulation of the group II PME isoforms. The absence of any noticeable effects on growth and development of transgenic plants suggests that the group I PME isoforms are not involved in plant growth and development and may play a role under special circumstances such as cell separation during fruit ripening.

Epitope analysis of isoforms of the major allergen Phl p V by fingerprinting and microsequencing.

The major allergen of timothy grass pollen (Phleum pratense), designated as Phl p V, consists of isoallergenic components of 38 and 32 kDa with pl values of 5.2-7.5 and 4.8-5.9, respectively. The different-sized proteins reveal similarities in IgE reactivity, N-terminal sequence and protein staining. For epitope analysis of these allergens a combination of enzymatic cleavage of electrophoretically separated proteins and immunoblotting techniques with subsequent N-terminal sequencing was performed. After isolation of the components from two-dimensional PAGE gels, proteins were enzymatically cleaved and separated by SDS-PAGE. By endoproteinase Glu-C cleavage six IgE-reactive fragments of each 32 kDa protein and three of each 38 kDa allergen were obtained. Microsequencing of the fragments revealed internal sequences that did not show any similarities between the different-sized allergens. Therefore, we assume only slight structural variations among allergens of similar sizes, whereas the 32 and 38 kDa proteins reveal great differences.

Purification and characterization of human meningioma M2-type pyruvate kinase

The M2-type pyruvate kinase was purified from human meningioma by ammonium sulfate precipitation, followed by ion exchange and affinicty chromatography. The specific activity of the purified enzyme was 33.4 U/mg with a yield of 6.5% The enzyme gave a single band with 63,000 ± 2000 Da upon SDS polyacrylamide gel electrophoresis. On cellulose acetate electrophoresis zymograms, the purified enzyme (M2) showed a single band, while crude extracts gave two broad bands corresponding to pyruvate kinase isozymes. The pl value of purified enzyme was found to be 6.9 With phosphoenol pyruvate as substrate the purified enzyme showed sigmoidal kinetics, while in the presence of 0.6 m M fructose 1,6-diphosphate as modulator it gave a hyperbolic saturation curve with a Km value of 0.58 m M.

Skin Proteasomes (High-Molecular-Weight Protease): Purification, Enzymologic Properties, Gross Structure, and Tissue Distribution

Proteasomes (high-molecular-weight protease) were purified from rat skin, and their enzymologic properties, gross structure, and tissue distribution were investigated. Skin proteasomes were purified by successive (NH4)2SO4 fractionation and by phenyl Sepharose CL-4B and HPLC gel filtration chromatography. The molecular weights of the proteasomes were estimated from gel filtration to be 750 kD. On sodium dodecylsulfate-polyacrylamide gel electrophoresis, the purified enzymes dissociated into several bands, the majority falling into the range of 36-20 kD. Two-dimensional electrophoretic analysis demonstrated approximately 10-15 separate protein spots with pl values varying between 3 and 10. As analyzed by electron microscopy, the gross structure of the enzymes showed an almost symmetrical ring-shaped particle with a small hole in the center. Succinyl-leucyl-leucyl-valyl-tyrosine-4-methylcoumaryl-7-amide, a fluorogenic substrate for serine proteinases, demonstrated the highest activity in terms of substrate specificity. Sodium dodecylsulfate, Ca++, and some free fatty acids activated enzyme activity. Activity was inhibited by diisopropylfluorophosphate, leupeptin, N-ethylmaleimide, iodoacetamide, and chymostatin. These results show that both serine and cysteine residues are related to the enzyme activity of proteasomes. Total and specific enzyme activities in the epidermis were, respectively, 10 and 20 times higher than in the dermis. Immunohistochemical studies utilizing the avidin-biotin complex method with monoclonal antibody revealed that the enzyme is distributed throughout the epidermis. These findings indicate the epidermal localization of proteasomes.

Purification of staphylococcal enterotoxin E.

Staphylococcal enterotoxin E has been purified by a combination of Red-A ligand chromatography and Mono-S (cation-exchange) chromatography. Commencing with 30 litres of fermenter-grown culture the method has been used to purify 17 mg of toxin, giving a purification yield of around 40%. The purity of the toxin was over 98% when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, but demonstrated three isoelectric forms by isoelectric focusing, with pl values of 7.6, 7.4, and 7.0. This method adds to the versatility of the Red-A matrix system which can also be used for the purification of staphylococcal enterotoxins A, B, and C2.

Degradation and solubilization of pectin by β‐galactosidases purified from avocado mesocarp

Three β‐galactosidase (EC 3. 2. 1. 23) isozymes were purified from the mesocarp of ripe fruit of Persea americana Mill. cv. Lula. and their effects on pectin derived from mature‐green tomato fruit were investigated. The β‐galactosidases had pl values of 5. 0,5. 1 and 5. 2. and molecular weights of 41. 49 and 54 KDa. respectively. There was a partial degradation of pectin resulting in the release of monomeric galactose upon treatment with avocado β‐galactosidase. This degradation resulted in increased pectin solubility and decreased apparent average molecular size as determined by microfiltration and gel permeation by high‐performance liquid chromatography. The increase in solubility was due. in part. to an apparent decrease in the ability of pectin molecules to aggregate together.