Immunochemical and immunohistochemical studies on the 27 S iodoprotein of dog thyroid with reference to thyroglobulin-like reaction of the parafollicular cells.

Our earlier finding that the thyroglobulin-like material responsible for the immunoreaction of parafollicular cells obtained in peak I fraction of Bio-Gel A-5m was followed up in the present study by an investigation of the immunochemical and immunohistochemical reactions of 27 S iodoprotein which was the most prominent material in the peak I fraction. The antibody was raised against completely purified 27 S iodoprotein which was obtained as follows: Thyroglobulin was extracted from dog thyroids and chromatographed initially on Bio-Gel A-5m and then on Bio-Gel A-50m. The area of 27 S migrated as a single bank on polyacrylamide gel slab electrophoresis. This was cut and eluted. Anti-27 S antiserum showed the same immunochemical patterns to 27 S and 19 S as anti-19 S antiserum with three different immunochemical methods: double diffusion test, one dimensional and two dimensional immunoelectrophoresis. The immunoperoxidase reactions of the anti-27 S antiserum and anti-19 S antiserum were restricted to follicular cells and luminal colloids. No reaction of the parafollicular cells was obtained by these antisera. Thus, 27 S iodoprotein shared common immunochemical and immunohistochemical properties with 19 S thyroglobulin. It was concluded that 27 S iodoprotein was not responsible for the thyroglobulin-like reaction of the parafollicular cells.     

Immunohistochemical study of a large molecular fragment of thyroglobulin in parafollicular cells

Thyroglobulin-like immunoreactivity of the parafollicular cells was studied by an immunoperoxidase bridge technique using antisera against dog thyroglobulin fragments. 1. The dog parafollicular cells were specifically stained by anti-peak I (27S and larger components fraction) antiserum absorbed with peak II (19S fraction). By this method, they were easily distinguishable from the non-reactive follicular cells and colloid droplets. More sensitive staining of the parafollicular cells was possible with anti-peak I' (larger components fraction) antiserum. The staining reactions indicated that the antigenic material responsible for immunoreactivity of the parafollicular cells was due to larger molecular components of thyroglobulin corresponding to 32S, 37S or greater than 37S, and was not due to either the 19S thyroglobulin or to the 27S iodoprotein. 2. A conspicuous decrease of the immunoreactive material in the parafollicular cells occurred in the dog after both chronically induced hypercalcemia and antithyroid drug treatment. This coincided with movement of secretory granules containing calcitonin as shown by staining with silver impregnation, HCl-basic dye, and lead-hematoxylin. 3. The antisera against larger molecular components of dog thyroglobulin showed a high degree of cross-reactivity to the parafollicular cells of most of the mammalian species investigated; rats, rabbits, hamsters, mice, cats, lions, goats, cows, and human.     

Development and cytodifferentiation of C cell complexes in dog fetal thyroids

Summary The development of C-cell complexes was investigated in dog fetuses by an immunoperoxidase method with three specific antisera: anti-calcitonin, anti-C-thyroglobulin (C-Tg), and anti-19S thyroglobulin. Ultimobranchial bodies joined with the thyroid anlage and then dispersed into the parenchyma to form large C cell groups. Sparse reaction products of C-Tg initially appeared in C cells with small amounts of cytoplasm. Later at about day 39 of gestation, when the immunoreactivity of calcitonin and 19S thyroglobulin appeared weakly in C cells and follicular cells, C-cell complexes were identified as large cell masses containing numerous undifferentiated cells without no immunoreactivity for any of the antisera. As development proceeded, the undifferentiated cells developed progressively the morphology of C cells. In addition, the undifferentiated cells developed 19S thyroglobulin immunoreactivity, that is, within some of the complexes small clusters of cells filled with material immunoreactive for 19S thyroglobulin. They were not organized into follicles during the fetal period, and were very slow in development. Depending on the degree of development of the undifferentiated cells, several features of the complexes were noted. The present study indicates that not only C cells but also follicular thyroid cells appear to be derived from the ultimobranchial bodies.     

Occurrence of glucose polymer in undifferentiated PC12 cells

A large glucose polymer was found, following pronase digestion, in PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was excluded from a Bio-Gel A-0.5 m column and adsorbed by immobilized concanavalin A-Sepharose from which it was eluted with 10 mM alpha-methylmannoside. Glucose was found to be the sole component monosaccharide. Except for those capable of degrading glycogen, no exo- or endo-glycosidases cleaved the polymer. This is the first report on the occurrence of a glucose polymer in undifferentiated PC12 cells.     

Extraction of Mullerian inhibiting substance from newborn calf testis.

Using a dissociative solvent and a protease inhibitor, Mullerian inhibiting substance, a testicular substance responsible for regression of the Mullerian ducts in the mammalian male embryo, has been extracted from newborn calf testis. Data are presented which demonstrate that fractions with biological activity for Mullerian inhibiting substance can be extracted from whole tissue and that activity can be blocked by antisera raised to extracted testes components. Following extraction in guanidine hydrochloride the extract was fractionated by density gradient sedimentation, gel filtration chromatography, and ion-exchange chromatography. Fractions were subjected to amino acid and carbohydrate analyses and peptide constituents were determined by SDS gel electrophoresis. Fractions were dialyzed, concentrated, filtered, and added to an organ culture assay to detect Mullerian inhibiting substance activity, which was found (1) in the guanidine extract, (2) in a protein fraction of the cesium chloride gradient, (3) in constituents eluted with K_av values between 0.19 and 0.38 on gel filtration chromatography using a Bio-Gel A-0.5 M column, and (4) in constituents eluted between 0.15 and 0.20 M NaCl on ion-exchange chromatography using a DEAE Bio-Gel A-50 ion exchanger. Sequentially this scheme effected a 30-fold purification of a fraction with Mullerian inhibiting substance activity. Biological activity was lost when positive extracts were absorbed with antiserum raised to guanidine extract. The strong dissociative conditions employed in the gradient and extraction procedures make it likely that the distribution of activity obtained in the density gradient procedure was due to a macromolecule, and not to an interaction between an active low molecular weight component and an inactive macromolecule acting as a carrier. Further fractionation on the Bio-Gel column using a dissociative solvent also indicated that the active component was a macromolecule. Amino acid and carbohydrate analyses indicate that the active fractions are composed of proteins and glycoproteins.     

Studies on the function of cell surface glycoproteins. I. Use of antisera to surface membranes in the identification of membrane components relevant to cell-substrate adhesion

An antiserum prepared against purified surface membranes of transformed BHK21/C13 cells (C13/B4) reversibly rounded and detached hamster cells from plastic tissue culture plates but did not affect cells of other species. Antiserum treatment did not alter the growth rate of C13/B4 or BHK21/C13 cells; however, NIL-8 cells exposed to the antiserum detached from the substrate and stopped growing, but remained viable for up to 72 h in the presence of the antiserum. Rounding and detachment were not inhibited by DNP or cycloheximide. Antiserum-detached cells did not reattach in the presence of these inhibitors. F(ab)' fragments also induced rounding, thus ruling out the involvement of complement and ligand-induced rearrangement of surface antigens in rounding and detachment. Three different surface-reactive immunoglobulin preparations were used in indirect immunoprecipitation studies in an attempt to identify cell surface antigens involved in regulating adhesion and morphology. Antiserum against surface membranes (anti-M) and against material shed by the cells into serum-free medium (anti-SFM) caused rounding and detachment, but a third antiserum (anti-LIS) prepared against a partially purified glycoprotein did not. All three immunoglobulin preparations precipitated glycoproteins with an apparent mol wt of 120,000 daltons from a crude membrane preparation solubilized by Nonidet NP-40. The two immunoglobulin preparations that caused rounding precipitated an additional glycoprotein peak of 140,000 daltons. Extensive preabsorption of the extract with anti-LIS immunoglobulin enriched the anti-membrane and antiserum-free medium precipitates for the 140,000-dalton peak. Anti-M immunoglobulin eluted from intact cells and subsequently used to precipitate NP-40 solubilized membrane constituents also reacted with a group of glycoproteins of approximately 140,000 mol wt. Therefore, this group of glycoproteins was considered most likely to be the glycoproteins involved in substrate adhesion and maintenance of cellular morphology.     

High Molecular Weight Protease in Rumen Ciliate Protozoa

Mixed rumen ciliate protozoa (mainly Entodiniinae) from goats have two kinds of protease; one has a pH optimum of 3.0, the other is active at neutral or alkaline pH. The protease active at neutral or alkaline pH was partially purified from the supernatant after centrifugation of sonicated mixed rumen ciliate protozoa. The supernatant was chromatographed on Bio-Gel A-1.5m and a partially purified protease was obtained. This protease had a molecular weight of more than 400,000. When the sonicated protozoa were heated at 55°C for 15min, the active peak from the Bio-Gel A-1.5m column was shifted to a lower molecular weight, 27,000. The high molecular weight protease was strongly activated by high temprature and SDS, and inhibited by E-64 c. The protease degraded many proteins including those found in rumen bacteria. These findings suggest that rumen ciliate protozoa have high molecular weight protease that plays a role in the digestion of feed and bacterial protein.     

Purification from goat antiserum of immunoglobulins against G6PD from rabbits]

DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.     

Detection of nine chitinase species in germinating cells ofMucor rouxii

Chitinolytic activity in aerobically germinating cells ofMucor rouxii was studied. When an ammonium sulfate fraction (40% saturation) of cytosol was subjected to column chromatography in Bio-Gel P-100, two peaks of activity were separated: A and B, which showed molecular weights of 53 KDa and 28 KDa, respectively. After further purification by ion exchange chromatography in a column of DEAE Bio-Gel A, the lytic activity from peaks A and B resolved into four and five species, respectively. Species from peak A eluted with peaks at 0.075, 0.125, 0.160, and 0.225M NaCl, whereas those from peak B did so at 0.050, 0.125, 0.190, 0.240. and 0.300M NaCl. Like other chitinases from the same fungus, the ones detected here are of the exochitinolytic type, releasing diacetylchitobiose units from nascent chitin chains. The complex chitinolytic system in these cells is discussed and compared with that exhibited by exponentially growing cells of the same fungus.     

Rat heparins. A study of the relative sizes and antithrombin-binding characteristics of heparin proteoglycans, chains and depolymerization products from rat adipose tissue, heart, lungs, peritoneal cavity and skin.

35S-labelled heparins were recovered from adipose tissue, hearts, lungs, peritoneal cavities and skins of rats given H2(35)SO4. Their purification involved incubation with Pronase, precipitation with cetylpyridinium chloride in 1.0 M-NaCl, gradient elution from DEAE-Sephacel and incubation with chondroitinase ABC. Each product was divided into proteoglycan and "depolymerization products' fractions by gel filtration on Bio-Gel A-15m. Heparin chains were released from a portion of each proteoglycan fraction by beta-elimination with NaOH. Proteoglycans, chains and depolymerization products were separated by gradient elution from a column of antithrombin-agarose into fractions with no affinity, low affinity and high affinity for antithrombin. The relative sizes of the products were determined by gel filtration on columns of Bio-Gel A-50m, A-15m, A-1.5m and A-0.5m. Skin was the major source of heparin and contained the largest proteoglycans and the lowest proportion of depolymerization products. Lungs contained the smallest proteoglycans, the smallest depolymerization products and the highest proportion of depolymerization products. The highest proportions of proteoglycans, chains and depolymerization products with high affinity for antithrombin were found in adipose tissue. The lowest proportions of each of these fractions were found in the peritoneal cavity. The data suggest that there was relatively little biosynthesis of sites with high affinity for antithrombin in peritoneal-cavity mast cells and that heparin catabolism was most active in lungs. Each source of heparin was unique with respect to both biosynthesis and subsequent breakdown of its proteoglycans.     

Transformations of a large aggregate of hydroxycinnamate hydroxylase to lower molecular weight forms by sulfhydryl agents in green leaves of sorghum

The large, partly microsomal aggregate containing 4-hydroxycinnamate hydroxylase activity isolated from green leaves of Sorghum bicolor at pH 6 was obtained instead as intermediate molecular weight forms when green leaves were ground in the presence of 10 mm mercaptoethanol or dithiothreitol. Elution profiles from agarose (Bio-Gel A-15m and A-1.5m) columns indicated that the 4-hydroxycinnamate hydroxylase activity was due either to multiple forms or to a mixture of forms in various stages of dissociation, the largest being eluted just after the void volume from an agarose 1.5m column. The larger form was similar to the major one found previously in etiolated leaves and was precipitated in the same ammonium sulfate fraction. The activity was unstable, but could be reactivated by incubation of the undiluted enzyme extract alone at 30 C prior to the assay. The data indicate that disulfide bonds are involved in the in vivo formation of the large aggregate in green leaves as well as being necessary for the maintenance of optimal activity of smaller polymeric forms in vitro.     

A 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414. Purification, characterization and preparation of an immunoadsorbent for the enzyme.

A 1,4-beta-glucan glucanohydrolase (EC 3.2.1.4) was isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by molecular-sieve chromatography on Bio-Gel P-30, ion-exchange chromatography on DEAE-Sephadex A-50 and isoelectric focusing in a density gradient. Polyacrylamide-gel electrophoresis at two different pH values, analytical isoelectric focusing in a polyacrylamide-gel slab and molecular-sieve chromatography of the reduced and alkylated enzyme in a denaturing medium indicated a homogeneous protein. The enzyme has a mol.wt. of 51,000 and is not a glycoprotein. The pI was found to be 4.66 at 23 degrees C. Antiserum against the purified enzyme was prepared and the amount of enzyme in the original filtrate was determined by rocket immunoelectrophoresis to be about 50mg/liter. An immunoadsorbent made from CNBr-activated sepharose 4B and antiserum affords a rapid and highly specific purification of the enzyme.     

Unlabeled antibody methods in electron microscopy: a comparison of single and multistep procedures using colloidal gold

A comparative study of five unlabeled antibody methods was conducted on the electron microscopic level using bridging techniques and colloidal gold. The study was based on the principles of the single-step colloidal gold (GLAD) method (Larsson L: Nature 282:743, 1979) and the multistep single- and double-bridge techniques used in postembedding immunoperoxidase procedures (PAP) (Sternberger LA: Immunocytochemistry, 2nd ed. Wiley, New York, 1979). Using medullary thyroid carcinoma and the same lot of primary antiserum (goat anti-calcitonin) for each procedure, it was shown that adequate localization of calcitonin with the single-step GLAD method was attainable only at dilutions of 1:100 or lower. The single-bridge technique using goat anti-calcitonin, sheep anti-goat immunoglobulin (Ig)G, and goat anti-calcitonin and antigen-coated gold, respectively, worked well at dilutions of up to 1:5000 but not at dilutions of 1:10,000, while single- and double-bridging techniques utilizing goat anti-calcitonin, sheep (Sh) anti-goat IgG, and sheep anti-goat IgG-coated gold produced good localization at a 1:10,000 dilution of primary antiserum. A two-step method using goat anti-calcitonin and sheep anti-goat IgG-coated gold, respectively, appeared to be the most sensitive technique, with adequate antigen localization occurring at a dilution of 1:25,000. While in our hands the two-step method appeared superior in sensitivity to the single-bridge IgG-coated gold technique, each method has its own advantages depending on the individual needs of the researcher.     

Trypanosoma brucei: preparation and some properties of a multienzyme complex catalysing part of the glycolytic pathway

After grinding Trypanosoma brucei with alumina or silicon carbide, it is possible to prepare a multienzyme complex which catalyses the breakdown of glucose to l-glycerol-3-phosphate and 3-phosphoglycerate. The complex sediments with the postnuclear large granule fraction which pellets at 14,500g; it is also eluted in the void volume during Bio-Gel A-5m column chromatography of a cell homogenate. During isopycnic sucrose gradient centrifugation, the multienzyme complex bands at a density of 1.22 g/ml. Because this is the density of T. brucei microbodies, and because Triton X-100 treatment of the material greatly enhances the activities of its component enzymes, we conclude that the multienzyme complex is probably located in the microbodies of the bloodstream long slender forms of T. brucei.     

Proteoglycan synthesis by rat lung cells cultured in vitro

Cells having a fibroblast-like morphology were cultured from explants of adult rat lung tissue. (35S)Sulfate was incorporated into sulfated proteoglycans in the medium at a linear rate for up to 96 h, while the rate of incorporation into the cell layer increased gradually until reaching a plateau at 40 h. The culture medium contained proteoglycans which migrated as a single peak with Kav = 0.10 on Bio-Gel A-15. Their glycosaminoglycan components (Kav = 0.70 on Bio-Gel A-15) contained predominantly chondroitin sulfate (33 to 44% of the total galactosaminoglycans) or dermatan sulfate chains. Based on the results of chondroitinase AC-II and periodate degradation, disaccharide repeating units of the dermatan sulfate were composed of 36% iduronic acid, 50% 2-sulfoiduronate, and 14% glucuronic acid. A similar composition was found for the dermatan sulfate in the cell fraction. Almost one-half of the sulfate label in the cell fraction was in a heparan sulfate proteoglycan which migrated on Bio-Gel A-15 with Kav = 0.30. The heparan sulfate chains (Kav = 0.81 on Bio-Gel A-15) had few, if any, sulfated N-acetylglucosamine residues and did not contain 2-sulfoiduronic acid in neighboring disaccharide repeat sequences. These results indicate that fibroblast-like lung cells synthesize several types of multichain sulfated proteoglycans which have properties in common with those found in lung tissues.     

Autocrine factor participation in defence of cytotoxic CTLL-2 cells from oxidative stress

The role of autocrine factors (AF) secreted by cytotoxic IL-2-dependent CTLL-2 cells along with pyruvate in cell defense from oxidative stress was investigated. The addition of conditioned medium (CM) containing pyruvate and AF into CTLL-2 cell cultures increased significantly cell survival under oxidative stress condition. The kinetics of hydrogen peroxide removal from cell cultures under oxidative stress in the case of CM addition has been obtained. The removal of H2O2 mostly by means of its reaction with pyruvate that is contained in CM has been shown at the beginning of oxidative stress (up to 15 min). Pyruvate content in CM was determined as 138 +/- 7 microM. Cell filtration on column with Bio-Gel P-10 was used for removal pyruvate from CM. Three fractions of CM (A, B and C) were obtained as a result of gel filtration. Pyruvate was not detected in any fraction. The fraction A was eluted from column as the first one and contained the largest molecules. Cell survival test showed the fraction B to have the highest ability to protect CTLL-2 cells under oxidative stress. The fraction A supported cell survival to a less degree and fraction C was shown to have no protective ability. The addition of the fraction B to the cell cultures resulted in preservation of significantly higher intracellular ATP level in the cells under oxidative stress than in the control ones. Moreover, AF of the fraction B was shown to react directly with hydrogen peroxide and inactivate it in the absence of cells. AF of the fraction A did not have such properties.     

Partial purification of an IGF-II receptor/binding protein from the erythroleukemia cell line K562

We previously reported that insulin-like growth factor II (IGF-11) stimulated clonal growth of an erythroleukemia cell line, K562, in semi-solid agar, an effect not mimicked by insulin-like growth factor I (IGF-1), as IGF-I receptors are generally not expressed in this cell line. Affinity crosslinking of intact K562 cells with ¹²⁵I-IGF-II revealed that the labeled hormone predominantly bound to a protein with a molecular weight of approximately 75 K. We report here the partial purification of the 75 K IGF-II binding protein from K562 cells. Triton X-100-solubilized K562 cells were subjected to Sephacryl-400, followed by Sephacryl-200 chromatography. Fractions of interest were collected and applied to a Sepharose-IGF-II column or an immunoaffinity column. The immuno-affinity column was prepared using an antiserum against placental membrane-derived material eluted from the Sephacryl-400 column in the elution volume, corresponding to the IGF-II binding protein from K562 cells. An affi-gel 10 affinity column, prepared with a protein A purified IgG fraction of this antiserum (antibody-29), retarded proteins showing binding specificity for IGF-II, with apparent molecular weights of 76 K, 87 K, and 70 K under reducing conditions. These protein bands were similar to the proteins retarded in the IGF-II affinity column, when evaluated by affinity crosslinking and SDS-PAGE. Fractionation of the purified material from the antibody-29 affinity column on Superose 12 revealed 6 protein peaks. Affinity crosslinking of the peak fractions from FPLC resulted in single bands with a molecular weight of 75 K under reducing conditions with variable specificity for IGF-II.     

The purification and properties of monoacylglycerol kinase from bovine brain

Monoacylglycerol kinase (MGK) has been purified from bovine brain by six steps: isolation of cytosol, DEAE-cellulose chromatography, ammonium sulfate fractionation (0-40%), Bio-Gel A-1.5m, hydroxylapatite, and ATP-agarose column chromatography. The overall purification was 938 times with a 4.8% yield. The column separations (particularly Bio-Gel A-1.5m) and SDS- and nondenaturing-polyacrylamide gel electrophoresis of enzyme purified from ATP-agarose indicated that MGK exists as a complex (approximately 350 kilodaltons) that is stabilized by 0.5 M NaCl and, on complete dissociation, yields a major protein of 72 kilodaltons. Dithiothreitol, EDTA, and ATP helped to stabilize MGK during purification. The protein peak eluted from hydroxylapatite by 25 mM phosphate activated and stabilized MGK activity. Phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin inhibited MGK. These phospholipids and others activated MGK synergistically with the above protein peak. MGK copurified with diacylglycerol kinase (DGK) throughout giving MGK to DGK ratios of 0.05-0.36. Optimal activity required 0.5 mM 2-monoolein and 10 mM MgCl2. Strong inhibition by p-chloromercuriphenyl sulfonic acid, N-ethyl-maleimide, and 5,5'-dithio-bis(2-nitrobenzoic acid), and prevention of this inhibition by dithiothreitol indicated the involvement of intact SH groups in the action of MGK. Purified MGK showed preference for substrates with unsaturated fatty acids except for 1- or 2-monostearin. Overall the preference favored the selective generation of 1-stearoyl- and 2-arachidonoyl-lysophosphatidic acid.     

Characterization of a glucose polymer from PC12 cells and neuronal cells of rat embryo

A large-sized glucose polymer was isolated by pronase digestion from line PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was included on a column of concanavalin A-Sepharose and could be eluted with 10 mM methyl-alpha-mannoside. Its slight retention in a column of Bio-Gel A-5m suggested that its molecular weight was in the several millions. Glucose was the component monosaccharide and there were two minor lipophilic components present. The polymer was digested with alpha-amylase into a series of oligosaccharides and was cleaved by glucoamylase into glucose residues. The disaccharide obtained by digestion with alpha-amylase was identified as maltose in several HPLC systems and by NMR spectroscopy. NMR measurement revealed the trisaccharide to be maltotriose. Susceptibility of the polymer molecule to alpha-amylase, and the digestion products obtained, indicated a resemblance to glycogen. An analysis for saccharide compositions before and after reduction of the polymer suggested the presence of an aglycon part. Contrary to expectations based on the presence of this moiety, the polymer displayed good solubility in neutral organic solvents. Two-thirds of the glucose polymer was also soluble in 10% TCA. A similar glucose polymer was isolated from neuronal cells of rat embryos metabolically labeled with [1-3H]galactose. Mouse neuroblastoma cells did not synthesize the polymer.     

Degradation of pectic polysaccharides extracted from suspension cultures of carrot by purified exo-polygalacturonase

Highly purified exo-polygalacturonase was obtained from suspension cultures of carrot ( Daucus carota L. cv. Kintoki) by dialysis at pH 5.2, chromatography on DEAE-Sephadex A-50 and on Sephadex G-150, and preparative polyacrylamide disc gel electrophoresis. The enzyme did not attack the isolated carrot cell walls directly, but it had some effect on pectic polysaccharides extracted from the walls. The extracted polysaccharides were fractionated by DEAE-Sephadex A-50 column chromatography yielding four carbohydrate fractions. The major fraction (P-3) was then reacted with the exo-polygalacturonase. The enzyme treatment resulted in hydrolysis of approximately 18% of the glycosyl linkages of fraction P-3 with the release of galacturonic acids. The molecular size estimated by Bio-Gel A-5m gel filtration was not markedly affected by the enzyme action, but the percentage of galacturonosyl residues was clearly reduced. The specific activity of exo-polygalacturonase changed during the growth cycle, in relation to the cell growth.