Characterization of a gonadal factor involved in the control of FSH secretion.

This communication presents evidence for the existence in the ovine testis of proteinaceous factors which suppress LH as well as FSH. Isolation of these factors has been achieved by using three different procedures: cytosol preparation, metaphosphoric acid extraction and ultrafiltration. Chromatography of cytosol or metaphosphoric acid extract on Sephadex G-75 resulted in separation into three protein fractions designated as G-75-I, II and III in order of their elution. When administered to castrated male rats, Fraction G-75-I suppressed circulatory levels of LH (53% inhibition, P less than 0.05) without altering FSH. The most retarded fraction, G-75-III, suppressed FSH (29% inhibition, P less than 0.001) without any concomitant change in LH. When fraction G-75-III was further fractionated on Sephadex G-25, three components were found and two, G-25-II and G-25-III, were biologically active. These fractions were homogeneous on polyacrylamide disc-gel electrophoresis. The FSH-suppressing factor (inhibin) was heat labile and susceptible to trypsin digestion, indicating that it is proteinaceous. Treatment with urea did not reveal any subunits. The molecular weight of this factor, as determined by gel filtration and SDS-urea gel electrophoresis was estimated to be around 1400-1500. The absence of sialic acid and the molecular weight data suggested that the isolated material was a simple protein and probably a small peptide. Gel filtration on Sephadex G-75 of the metaphosphoric acid extracts of liver, kidney, testis and ovary revealed an identical elution pattern for ovarian and testicular inhibin.     

An antigen common to a wide range of bacteria. I. The isolation of a 'common antigen' from Pseudomonas aeruginosa

In crude water-soluble extracts of Pseudomonas aeruginosa 64 antigens can be demonstrated by crossed immunoelectrophoresis in agarose with polyvalent Pseudomonas-immunoglobulin. One of these antigens cross-reacts with antigens prepared from bacteria of a wide range of taxonomic groups. Monospecific immunoglobulins to this antigen (Common Antigen) were produced by immunization with the appropriate immunocomplex extracted from agarose. Common Antigen was purified by the combination of two fractionation methods: Precipitation of the crude extract with 18% (w/v) sodium sulfate, followed by gel filtration of the supernatant on a Sephadex G-200 column. By this method, 35% of Common Antigen from the crude extract was recovered, more than half of the fractions electrophoretically pure. Electrophoresis of reduced Common Antigen on a dodecyl sodium sulfate polyacrylamide gel revealed two protein bands with apparent molecular weights of 59-62 000 and 62-65 000, respectively. The untreated antigen, however, passed a column of Sephadex G-200 with the void volumen, indicating a substance of high molecular weight (> 4-600 000).     

The separation of bovine brain beta-N-acetyl-D-hexosaminidases. Abnormal gel-filtration behaviour of beta-N-acetyl-D-glucosaminidase C.

Bovine brain tissue was extracted and the 50 000g supernatant was separated by electrophoresis, DEAE-Sephadex chromatography and gel filtration on Sephadex G-200 and Bio-Gel P-200. The electrophoretic separation showed that the beta-N-acetyl-D-hexosaminidases (hexosaminidases) of bovine brain tissue were composed of four different fractions. Two fractions (A and B) exerted both glucosaminidase and galactosaminidase activity, a third fraction (C) showed only glucosaminidase activity, whereas a fourth form (D) with specificity towards the galactosaminide moiety was found to be present. DEAE-Sephadex chromatography at pH 7.0 showed that the B form was eluted with the void volume, whereas the A and D forms could be eluted in one peak by raising that salt concentration. The C form could not be detected in the eluate. Gel filtration on Sephadex G-200 showed that the B, A and D forms had almost equal molecular weights. In this case also the C form could not be detected in the column eluates. Gel filtration on Bio-Gel P-200 revealed that the C form was eluted with the void volume.     

Acyl-CoA-ACP-transacylases of Mycobacterium smegmatis. An analytical study

Homogenates of Mycobacterium smegmatis have been prepared by sonication of bacteria in 0,1 M phosphate buffer, pH 7, containing 0,001 M EDTA and 0,01 M 2-mercaptoethanol. The 105 000 g supernatant of these homogenates has been fractionated by ammonium sulfate precipitation. The fraction precipitating between 25 and 65 per cent saturation in ammonium sulfate shows palmityl-CoA-ACP-transacylase, malonyl-CoA-ACP-transacylase and acetyl-CoA-ACP-transacylase activities. These activities are not associated, essentially, to a complexe of high molecular weight as they are retained on the column for 80 per cent or more upon gel filtration.By chromatographic procedures involving Sephadex G-150 gel filtration, and chromatography on DEAE-Sephadex A-50 the malonyl-CoA-ACP-transacylase activity is separated from the other two activities. The palmityl-CoA-ACP-transacylase activity and the acetyl-CoA-ACP-transacylase activity show two close but distinct major peaks either upon gel filtration on Sephadex G-150 or upon DEAE-Sephadex A-50 chromatography indicating thus that these two activities are attribuable to two distinct molecular entities. Besides the major peaks for these two activities, a supplementary minor peak for each activity is observed.An estimate of the approximate range of the apparent molecular weights for palmityl-CoA-ACP-transacylase, malonyl-CoA-ACP-transacylase and acetyl-CoA-ACP-transacylase has been made in using the curve of the K_av values of known proteins plotted against the logarithms of their molecular weights. The molecular weights thus estimated for these three enzymes are 100 000, 32 000 and 85 000 respectively.The void volume fractions of the chromatography of the homogenates on Sephadex G-150 present 20 per cent or less of the enzyme activity of the homogenates. Chromatography of the mixture of the void volume fractions on DEAE-Sephadex A-50 shows that the malonyl-CoA-ACP-transacylase activity associated to these fractions behaves differently from that of the fractions retained upon gel filtration; this may be taken as an indication for the presence of a complexe of the fatty acid synthetase present as a minor component of the homogenates.     

Purification of papain-solubilized histocompatibility antigens from a cultured human lymphoblastoid line, RPMI 4265.

HL-A antigens having specificities HL-A2, HL-A7, HL-A12 have been solubilized by papain treatment of membrane preparations from the cultured human lymphoblastoid cell line RPMI 4265 and purified about 80-fold by chromatography on carboxymethylcellulose, Sephadex G-150, and diethylaminoethylcellulose columns. Separation of HL-A2 from a mixture of HL-A7 and HL-A12 was achieved on the final DEAE-cellulose column. The yield was about 1 mg of protein of each antigen preparation per 100 g of packed, frozen cells. On sodium dodecyl sulfate gel electrophoresis both preparations showed two polypeptide bands. The smaller subunit of 12,000 daltons is common to all HL-A preparations and has been shown to be identical with beta2-microglobulin. The larger subunit is a glycopeptide and in the HL-A7, 12 preparation was resolved into a duplex of 34,000 and 37,000 daltons. The HL-A2 preparation showed a single band at 34,000 daltons. On isoelectric focusing under nondenaturing conditions, the preparation showed multiple bands, all of which contained both subunits and retained antigenic activity. On isoelectric focusing in the presence of 6 M urea a single band for beta2-microglobulin and multiple bands for the larger subunit were seen. This charge heterogeneity of the larger subunit has been shown to be due to variable amounts of sialic acid. When HL-A antigen preparations were subjected to Sephadex G-100 chromatography in the presence of 3 M KCl, no separation of the two subunits was observed.     

IUB commission of editors of biochemical journals the citation of bibliographic references in biochemical journals recommendations (1971)

A pentose-rich acidic glycoprotein was isolated from protease digested bovine vitreous humor by fractionation on an AG1-X2 column using NaCl solution gradient.The material eluted at 0.35 M NaCl (glycoprotein) was electrophoretically heterogeneous at pH 8.6 after partial purification on Sephadex G-25. Gel filtration on G-100 resolved the glycoprotein into two fractions. These fractions differ in molecular weight; mol. wt approx. 95 000 material consisted of two components on electrophoresis and mol. wt approx. 28 000 material showed only a single component on electrophoresis. The lower molecular weight component was re-chromatographed on Sephadex G-100 yielding a single orcinol positive component which gave a homogeneous band on gel electrophoresis.Quantitative analysis of this material gave 30% protein, 7.0% pentose, 18.7% glucosamine, 9.2% galactosamine, 10.9% hexuronic acid and 16.1% hexose.Treatment with 0.5 M NaOH at 20°C for 24 h resulted in a 50% decrease in the threonine content suggesting the possible involvement of this amino acid in the protein-carbohydrate linkage group.Paper chromatography of the fraction hydrolysate demonstrated the presence of glucurone, xylose, arabinose, glucose and galactose.     

An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. II. Purification and characterization of multi-forms of RNase T2

RNase T2 bound to an affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, specifically at pH 4.5. The colorless enzyme was eluted only by the simultaneous addition of 2'(3')-AMP (1 mM) and NaCl (greater than 1 M) at pH 4.5. By applying this affinity chromatography to the purification of RNase T2, pure enzyme with a specific activity of 60 was obtained in only four steps and the yield was about 10 times higher than that of the previous purification method. This enzyme preparation was found to be heterogeneous in molecular weight and was separated into two fractions on Sephadex G-75 gel filtration. As the smaller enzyme with a molecular weight of 36,000 was identical with RNase T2 in every property examined, we tentatively designated the larger one with an apparent molecular weight of 80,000 as high molecular weight RNase T2 (RNase T2-L). RNase T2-L was still heterogeneous and was separated into five fractions, RNases T2-L 1-5, by repeated Sephadex G-150 gel filtration. The amino acid and carbohydrate analyses revealed that each of these fractions has a protein moiety in common with RNase T2 and the heterogeneities were due to the carbohydrate content, mainly galactose content.     

Induction of reaginic (IgE) gonococcal antibodies in the rat by a common antigen of Neisseria gonorrhoeae.

An antigen (ZAB) common to Neisseria gonorrhoeae was prepared by stepwise elution of a crude gonococcal antigen (ZA) from columns of diethylaminoethyl cellulose employing 0.02 M phosphate buffers, pH 7.6, containing increasing concentrations of sodium chloride. Rats immunized with ZAB produced reaginic (IgE) antibody which cross-reacted with ZA prepared from eight gonococcal strains by the passive cutaneous anaphylaxis (PCA) test. Heating of the sera at 56 degrees C for 4 h destroyed the PCA activity. The PCA activity of the anti-ZAB rat serum was removed after absorption with ZAB antigen or with rabbit anti-rat IgE but not after absorption with gonococcal lipopolysaccharide or with heat-killed or formalinized gonococci. Treatment of ZAB with trypsin or heating at 100 degrees C for 30 min destroyed or reduced the antigenic activity respectively. Further purification of ZAB by filtration through Sephadex G-100 gave a preparation (ZAB2) which contained the common antigen as shown by the cross-reactivity of anti-ZAB2 rat serum with seven stains of N. gonorrhoeae. Fraction ZAB2 contained material which had a molecular weight less than 13,700 and was associated with the presence of material absorbing at 260 nm. The results of this study indicate that a low molecular weight antigen, which appears to be protein in nature and associated with nuclei acid, is common to the gonococcus and is the main antigenic component inducing reaginic (IgE) antibody in the rat.     

Purification of soluble hepatocellular carcinoma extracts.

When soluble hepatocellular carcinoma (HCC) extract prepared by 3 M KCl solution was passed through a column of Sephadex G-200, four fractions (designated as I, II, III, IV) could be obtained from the elution profile. By leukocyte migration inhibition assay, the active component of soluble HCC extract which represented tumor-associated antigen was shown to be contained in fractions I and II.     

Purification and characterization of fish surface mucin

Fish surface mucin from Pampus argenteus was extracted with different organic solvents and the residue passed through Sephadex G-200. The major peak was purified by DEAE-Cellulose chromatography and five fractions were obtained. Carbohydrate and protein contents showed that major peak is a glycoprotein. Rechromatography of this component on the Sephadex G-200 column gave a single peak, with an estimated minimal molecular weight of 6.9 X 10(5). Analysis of individual sugar components revealed the presence of galactose, glucose, mannose, arabinose, N-acetyl glucosamine, N-acetyl galactosamine and sialic acid. The most represented amino acids are threonine, serine, proline, glutamic acid and glycine. The N-terminal amino acid end was blocked. Nearly 47% of sulphate was acid labile. Sialic acid and fucose were released rapidly by mild acid hydrolysis. The presence of blood group-A activity suggests that some kind of terminal alpha-Gal-NAC may be present.     

Schistosoma mansoni: antigenic heterogeneity of excretions and secretions

The excretory-secretory antigens of adult Schistosoma mansoni were obtained by in vitro cultivation of worms in a chemically-defined medium. The protein output in this system was low, 0.2–0.4 μg of proteinworm/48 hr. The composition of the crude culture antigen (CA) was approximately 80% protein, 15% carbohydrate and 5% nucleic acid. Disc gel electrophoresis of CA revealed the presence of at least 15 protein components, many with carbohydrate moieties. Three major fractions were obtained by gel filtration on Sephadex G-200. Fraction I contained the bulk of the glycoprotein material. Immunoelectrophoresis of CA with hyperimmune rabbit serum indicated the presence of at least 6 antigens, most of which eluted in Fraction II. Serum from infected mice and monkeys, but not from rabbits and rats, reacted with CA and its fractions, especially Fraction II, on immunodiffusion analysis. Comparison of CA with other adult worm extracts by immunodiffusion techniques showed that most of the excretory-secretory antigens could be obtained by either freezing and thawing or by extraction with 3 M KCl. The P.K.-type activity of CA was considerably greater than that of a lipid-free adult worm antigen. Both Fractions I and II had the P.K.-type activity. An antigen capable of eliciting macrophage migration inhibition factor from infected rat lymphocytes was detected in CA, although the lymphocyte toxicity of CA was high at concentrations above 10 μg/ml.     

Grouping antigens of four Lactobacillus species and their characteristics.

Antigenic analyses of Lactobacillus bulgaricus, Lactobacillus lactis, Lactobacillus brevis and Lactobacillus buchneri were carried out by double immunodiffusion in agar. Antigens were extracted from whole cells and cell wall preparations with cold trichloroacetic acid. Most strains of the four species possessed antigen 9 in their cell walls. Another antigen, antigen 10, was found in the cell walls of all the strains of L. brevis and L. buchneri, and in some strains of L. lactis, but not in L. bulgaricus. Fractionation of the antigens was attempted using the cell wall extracts of L. lactis L-10 with only antigen 9 and of L. brevis X-1 with both antigens 9 and 10. The partially purified fractions of antigen 9 and of the complex of antigens 9 and 10 were obtained by zone electrophoresis. However, antigen 10 from the complex could not be separated by the same method or gel filtration on Sephadex G-100 since the two antigens 9 and 10 of the complex always behaved together. The fraction of antigen 9 consisted almost entirely of glycerol and glucose as sugar components, the molar ratio being 2: 1. The complex of antigens 9 and 10 also consisted of the same sugars, and the molar ratio of glycerol: glucose was 4: 1. Inhibition tests indicated that the immunodominant component of antigen 9 was a-methylglucoside (glucose), and most probably the determinant is a glycosylated glycerol teichoic acid. It was considered that the determinant of antigen 10 is a glycerol teichoic acid although glucosamine and galactosamine inhibited effectively the reaction between antigen 10 and its antibody.     

The antigenic structure ofFrancisella tularensis

The ether antigen ofFrancisella tularensis was fractionated using DEAE-cellulose and CM-cellulose chromatography, and Sephadex G-200 gel filtration. The CM-cellulose chromatography did not appear to be a suitable method for separation of individual components of the complex ether antigen. Most of the polysaccharide material and substances with a high phosphorus content were eluted already in the first peak of the elution curve. This method could be used only to separate a component, yielding one immunoelectrophoretic precipitin line, localized towards the cathode. On the other hand, the DEAE-cellulose chromatography, and particularly the Sephadex G-200 gel filtration (especially when recycling was introduced), yielded a clear separation of relatively clean components of the ether antigen. The present work provides a comparison between the immunochemical (immunoelectrophoretic) properties of fractions obtained by these methods and components isolated by salting out with ammonium sulphate, ethanol or trichloracetic acid precipitation.     

Mechanisms of insulin degradation by isolated rat adipocytes

Summary We have examined some of the chemical and biological characteristics of the insulin-derived cell-associated radioactivity following incubation of isolated adipocytes with ¹²⁵I-insulin (10^–10 M) for one hour at 37 °C S ephadex G-50 chromatography of the cell-associated radioactivity demonstrated three peaks: peak I eluted with the void volume and consisted of large molecular weight material; peak II comigrated with 1251-insulin; and peak III consisted of small molecular weight degradation products (probably iodotyrosine). When the insulin peak (peak II) was divided into fourths, it was found that the binding and biologic activity of this material was not homogenous; thus, binding and biologic activity (relative to native insulin) fell markedly from the earliest to the latest eluting fractions of this peak. Furthermore, when the entire peak 11 material was applied to DEAE-Sephacel and eluted with a 0.01–0.2 M NaCl gradient, three distinct peaks were observed. These peaks were all 90% TCA precipitable, whereas the ability of the latter two eluting peaks to precipitate with anti-insulin antiserum was markedly reduced. When similar experiments were performed with chloroquine-treated cells, a large increase in cell-associated radioactivity was observed, and Sephadex G-50 chromatography demonstrated that this increase was entirely confined to peaks I and II. When the insulin peak (peak II) was divided into fourths, it was found that chloroquine markedly inhibited the decreased binding and biologic activity, from the earliest to the latest eluting fraction of this peak. Furthermore, when the peak II material (Sephadex G-50) from chloroquine-treated cells was chromatographed on DEAE-Sephacel, this material eluted in a single peak which was 95% TCA precipitable and 106% precipitable by anti-insulin antiserum. In conclusion, these studies demonstrate that: 1) intermediate insulin-derived products with reduced binding and biologic activity are generated in the process of cellular insulin degradation, and 2) the formation of these intermediate products is mediated by a chloroquine-sensitive pathway.     

The induction of cellular immunity with soluble murine histocompatibility antigens

Soluble transplantation antigens have been prepared from various lymphoid organs of the mouse strains A and C57BL. These preparations have been partially characterized by gel filtration on Sephadex G-200 and G-100. The distribution of various antigenic activities, such as precipitation with rabbit antisera, inhibition of the cytotoxic reactions of heterologous antisera and of alloantibodies, differed considerably among the chromatographic fractions. The soluble antigen preparations retained their antigenic and immunogenic properties, as demonstrated by their ability to block the cytotoxic reactions of alloantisera and to modify tumor growth in immunized recipients. Immunization of normal recipients with the immunogenic transplantation antigen preparations led to the production of sensitized lymphocytes, capable of destroying allogeneic target cells in vitro. Sensitized lymphocytes appeared in the regional lymph nodes after a single injection of 200–300 μg of the antigen preparation, reaching a peak level between 9 and 12 days. On reimmunization, the cytolytic activity of lymph node cells increased considerably and sensitized lymphocytes also appeared in the spleens of immunized animals.     

Guerreiro Machado reaction. Chromatographic analysis of Trypanosoma cruzi antigens

The aqueous extract of Trypanosoma cruzi, previously treated by benzene, was filtered through a column of Sephadex G-200. The eluted fractions were tested by complement-fixation with the chagasic reference serum. It was found that the CF antigens were eluted in the first fractions and the contaminants in the last ones. The aqueous antigen, when dried and extracted with methanol, showed a very high titer in the complement-fixation quantitative test. This antigen is free of contaminants and can be recommended for use in the serological test for the diagnosis of Chagas' disease.     

Partial characterization of a fraction from human follicular fluid that initiates the human sperm acrosome reaction in vitro

A human follicular fluid (HFF) fraction prepared by Sephadex G-75 column chromatography has been previously shown by this laboratory to initiate the human sperm acrosome reaction (AR) in vitro. In the present report, the apparent molecular weight (MW) of this AR activity determined by a longer G-75 column than was used in the previous work was 50,000 ± 5,106. The G-75 Sephadex void volume fractions of some but not all HFF samples were also found to contain some AR-initiating activity. The occasional void volume activity was less potent than that of the 50,000 MW fraction and was not studied further. Further characterization of the 50,000 MW fraction was carried out. A time-course study demonstrated that maximum AR were obtained within 5 min following the addition of the 50,000 MW fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining revealed that the 50,000 MW fraction was still a relatively crude preparation. Treatment of the 50,000 MW fraction with chloroform:methanol did not extract the AR-initiating activity into the lipid phase. The AR-initiating activity of the untreated 50,000 MW fraction was precipitated when it was boiled, but the activity was partially resistant to boiling after overnight incubation. Treatment of the 50,000 MW fraction with pronase E or with several glycosaminoglycan hydrolases did not destroy the activity. Pronase treatment resulted in a higher amount of boiling-resistant AR-initiating activity. The AR-initiating activity of the untreated 50,000 MW fraction was partially dialyzable, but the activity of an undialyzed fraction did not pass through an ultrafiltration membrane with a 10,000 MW cut-off. However, treatment of the 50,000 MW fraction with protease, peptide:N-glycosidase F, and to a lesser extent chondroitinase ABC yielded an active lower MW activity which could pass through such an ultrafiltration membrane. The lower MW activity released by peptide:N-glycosidase F eluted in the included volume (5,000–1,000) of a Sephadex G-25 column. Neutral hexose but not protein or peptide was detected in the G-25 peak of AR-initiating activity. These results suggest that the AR-initiating activity present in the 50,000 MW fraction of HFF: (1) is present either as two different AR factors (a high-MW factor and a low-MW, noncovalently bound factor) or as a single factor responsible for both the nondialyzable and dialyzable AR-initiating activities (the latter being enzymatically released from the former), and (2) may be at least partially associated with N-linked oligosaccharides of a glycoprotein or proteoglycan.     

The occurence of a neutral protease and its inhibitor in rat peritoneal macrophages.

The lysate of the glycogen-induced macrophages in rat peritoneal exudate was fractionated by centrifugation and extraction into a water extract, 1 M KCl extract and residue fractions. Approximately 50% of the neutral protease activity toward casein in the lysate was recovered in the KCl extract fraction, which was practically devoid of acid protease, cathepsin D. The pH optimum of the neutral protease toward casein and urea-denatured hemoglobin was pH 8.5. The activity was inhibited strongly by DFP or chymostatin and only partially by HgCl2 or PCMB. Addition of a salt to the reaction medium caused enhancement of the activity with an optimum concentration of 0.25 M: KCl, KBr, KI, NaCl, NaBr, NaI, and MgCl2 were all almost equally effective. When the enzyme preparation was filtered through a column of Sephadex G-75 gel in the presence of 1 M KCl, a larger molecular weight fraction at the void volume was obtained in addition to a smaller molecular weight fraction showing a caseinolytic activity insensitive to KCl concentration. The former was found to have a specific inhibitory effect on the latter activity.     

Beta-endorphin-like and adrenocorticotropin-like materials in turtle heart and intestine

1. Turtle heart and intestine acetone powders were extracted with an acetone-water-HCl mixture. An acid acetone powder resulted by adding a copious volume of acetone to the extract. The powder was subjected to gel filtration on Sephadex G-25 and ion exchange chromatography on CM-cellulose. 2. In turtle heart, corticotropin-like bioactivity was distributed among chromatographic fractions (derived from material unretarded on Sephadex G-25) unadsorbed and adsorbed on CM-cellulose. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were adsorbed on CM-cellulose. 3. In turtle intestine, corticotropin-like bioactivity was absent. Opiate receptor binding activity was present in fractions unretarded as well as in fractions retarded on Sephadex G-25, indicating a molecular weight of greater and smaller than 5000 respectively. 4. The highest opiate receptor binding activity and beta-endorphin-like immunoreactivity were found in a fraction adsorbed on CM-cellulose.     

Androphilic proteins in cytosols of human benign prostatic hypertrophy.

To examine the properties of androphilic proteins in human benign prostatic hypertrophy, the binding capacity and affinity of the proteins were determined after acetone-treatment, ammonium sulfate precipitation and chromatographies of DEAE and Sephadex G-200. Androphilic proteins in the extract of acetone-dried cytosol from the hypertrophic human prostate was precipitated at 30-50% saturation of ammonium sulfate. The binding of this fraction to dihydrotestosterone and testosterone was high affinity, but the binidng to estradiol-17 beta was the one of non-specific. Androphilic proteins in the 30-50% fraction were eluted from DEAE-cellulose column by buffer containing 0.05 M KCL. On Sephadex G-200 chromatography of 30-50% fraction, the androphilic proteins were observed in three peaks; one was eluted in the void volume and other two were eluted at the sites of IgG and albumin. The amount and ratio of proteins eluted in the void volume and the site of IgG from Sephadex G-200 column were variable in individual tissue samples. The chromatographic behavior of the 30-50% fraction in Sephadex G-200 was not changed significantly by introducing 0.4 M KCl in the system. Polyacrylamide gel electrophoresis was applied for further separation of the proteins.