DEAE-cellulose Ion Exchange Column Research Articles

WITHDRAWN: Partial purification of pepsin enzyme produced by Staphylococcus sciuri and Pythium sp. using whey

Abstract The study included purification of pepsin enzyme EC: 3.4.23.1 that produced by using a mixed inoculum consisting of Staphylococcus sciuri and Pythium sp. and using the whey as a local carbon source obtained from a previous study. Pepsin was purified first by using salts of ammonium sulphate with saturation rates ranging between (30–80)%, and then the process of dialysis with distilled water, the process was performed with distilled water and the process of purification of the enzyme was completed using the DEAE-Cellulose ion exchange column and then the gel filtration using the Sephadex G-300 column. The enzymatic activity was 1.998 U/ml at 3.714 times, with 39.1%. The optimal pH for the partially purified pepsin was 3.0 and the optimal temperature for t pepsin activity was 35 Co and 5% of casein.

Purification and characterization of thiol dependent, oxidation-stable serine alkaline protease from thermophilic Bacillus sp.

Alkaline serine protease was purified to homogeneity from culture supernatant of a thermophilic, alkaliphilic Bacillus sp. by 80% ammonium sulphate precipitation followed by CM-cellulose and DEAE-cellulose ion exchange column chromatography. The enzyme was purified up to 16.5-fold with 6900 U/mg activity. The protease exhibited maximum activity towards casein at pH 8.0 and at 80 °C. The enzyme was stable at pH 8.0 and 80 °C temperature up to 2 h. The Ca2+ and Mn2+ enhanced the proteolytic activity up to 44% and 36% as compared to control, respectively. However, Zn2+, K+, Ba2+, Co2+, Hg2+ and Cu2+ significantly reduced the enzyme activity. PMSF (phenyl methyl sulphonyl fluoride) completely inhibited the protease activity, whereas the activity of protease was stimulated up to two folds in the presence of 5 mM 2-mercaptoethanol. The enzyme was also stable in surfactant (Tween-80) and other commercial detergents (SDS, Triton X-100).

Purification and characterization of alkaline xylanase from Thermoascus aurantiacus var. levisporus KKU-PN-I2-1 cultivated by solid-state fermentation

Alkaline xylanase from Thermoascus aurantiacus var. levisporus KKU-PN-I2-1 was purified and characterized. The enzyme was enriched to apparent homogeneity by ammonium sulfate precipitation, Sephadex G-100 gel filtration and DEAE-cellulose ion-exchange column chromatography; it was purified 14.5 fold, with 2.3% recovery of the total activity and a specific activity of 2.56 × 103 mU/mg protein. Xylanase appeared to be monomeric, with a molecular weight of 27 kDa and isoelectric point (pI) of 7.2. Purified xylanase exhibited an optimum pH and temperature of 9.0 and 60 °C, respectively. The enzyme retained more than 70% of its original activity in the pH range of 7.0–9.0. In thermostability tests, xylanase retained more than 70% of its original activity after 90 min incubation at temperatures up to 50 °C. The enzyme had a Km of 40.9 mol/L and Vmax of 6.2 μmol/min/mg. Xylanase was inhibited by Hg2+ and stimulated by Cu2+, EDTA, Mg2+ and Co2+ at 1 mM. The purified xylanase only showed activity on xylan and hydrolyzed beechwood xylan to yield mainly xylotetraose and xylobiose, suggesting that it is an endo-xylanase.

Purification and characterization of novel organic solvent tolerant 98 kDa alkaline protease from isolated Stenotrophomonas maltophilia strain SK

Ability of microorganisms to grow at alkaline pH makes them an attractive target for several industrial applications. Thus, search for new extremozyme producing microorganisms must be a continuous exercise. Hence, we isolated a potent alkaline protease producing bacteria from slaughter house soil. The morphological, biochemical and 16S rDNA gene sequencing studies revealed that the isolated bacteria is Stenotrophomonas maltophilia strain SK. Alkaline protease from S. maltophilia strain SK was purified by using ammonium sulphate precipitation and DEAE-cellulose ion exchange column chromatography. The purified enzyme was optimally active at pH 9.0 and temperature 40°C with broad substrate specificity. It was observed that the metal ions such as Ca++, Mg++ and Fe+++ completely repressed the enzyme activity. The enzyme was stable in presence of various water miscible solvents like ethanol, methanol, isopropanol at 25% (v/v) concentration and less stable at 37.5% (v/v) concentration. These robust properties of enzyme might be applicable for various applications in detergent and pharmaceutical industries.

Characterization of alkaline phosphatases activities in uterine secretion of buffaloes

Alkaline phosphatases is reported as a predominant protein in buffalo uterine luminal secretion during the late- luteal phase of the cycle. Since the biochemical properties of this important protein are not known, an experiment was designed to purify and characterize the alkaline phosphatase from buffalo uterine secretion. The enzyme could be purified from uterine luminal fluid by 2 chromatography steps using DEAE cellulose ion-exchange and Sephacryl S-200 HR gel-filtration columns. The molecular weight of the purified protein was 179 kDa by non-reducing SDS- PAGE. Reduction by ß-metcaptoethanol yielded 136kDa and 43kDa bands indicated presence of 2 separable subunits of this protein. Complete inhibition of activity by ß-mercaptoethanol and partial inhibition by iodoacetamide indicated that both the subunits are essential for bioactivity. Three isoforms with pI values 5.9, 6.0 and 6.2 were detected by iso-electric focusing. Purified enzyme showed optimum activities at 37°C in pH ranges 10.5 to 11.0. It is a temperature sensitive enzyme as evidenced by a loss of 91% enzyme activity within 28 h of room temperature storage. The loss of activity was slow (12% per day) at 4p C storage indicating importance of cold chain maintenance while processing. The SDS proved to be a better detergent compared to Triton X100 as less inhibition (46% vs 76%) of enzyme activities was observed with the former. Inhibition of activities by metal chelators confirmed that it was metallo- phosphatase. Further study would help exploring its role in buffalo uterine microenvironment.

English

Phaeogyroporus portentosus is widely consumed as food in Thailand, but its potential bioactivities remain largely unknown. Here, the bioactivities of the hot water-extractable polysaccharide-protein complex (PPC) were evaluated after precipitating with ethanol. This crude PPC extract was then fractionated by DEAE-cellulose ion exchange column chromatography, and subsequently further purified by Superdex G-200 gel filtration column chromatography, giving an abundant polysaccharide fraction (PPC-P11) and a much less common fraction (PPC-P12) that was obtained in an insufficient amount for further analysis. The PPC-P11 fraction was characterized for its molecular weight and its functional groups were determined by 13C and 1H NMR spectroscopy, FT-IR spectroscopy and gel permeation chromatography (GPC) respectively. It contained a high amount of glucose with an average molecular weight of 294.7 kDa (as compared to < 21.5 kDa by reducing SDS-PAGE analysis). The NMR signal observed at 103.5 ppm was assigned to the C1 of beta-glucose, and those between 20 and 40 ppm suggest the presence of a glucan-protein compound. PPC-P11 was found to display a dose-dependent antioxidant capacity in each of the four complementary test systems assayed (hydrogen peroxide, DPPH, ABTS and nitric oxide free radical scavenging) with quite low IC50 values (< 100 µg/mL). Moreover, a relatively strong in vitro antiproliferative effect was found against five human cell lines, with IC50 values ranging from 1.178 ± 0.129 µg/mL (breast cancer; BT474) to 5.183 ± 0.229 µg/mL (hepatoma cancer; HEP-G2). PPC-P11 from P. portentosus is, therefore, a potential antioxidant source for both health medicines and the food industry.   Key words: Antioxidation, antiproliferation, polysaccharide-protein complex,Phaeogyroporus portentosus

Protection against Dental Carries by Passive Immunization with Hen Egg Yolk Antibody Using Cell Associated Glucosyltransferase of Streptococcus mutans

Background: Dental caries is one of the most frequently encountered diseases. Of the oral bacteria, Streptococcus mutans is considered to be causative agent of dental caries in humans. Aim: This study aims at formulating a dental composition using Chicken Egg yolk Antibodies (IgY). Methodology: IgY was raised in 22 weeks old white leghorn chicken against CA-GTF antigen of Streptococcus mutans. The level of the antibody in serum was monitored and booster doses were given whenever necessary. The antibodies were purified from the egg yolk using PEG and Ammonium sulphate precipitation and DEAE cellulose ion exchange column chromatography. Results and discussion: High titre of more than 1:10000 antibodies were detected by Indirect ELISA at 150th day of observation. The IgY concentration in egg yolk was increased during the immunization period and reached maximum of 3.9 mg/ml. The minimum agglutination concentration of IgY against whole cell antigen of S. mutans was found to be 1:625. Similarly, when pre-immune IgY (control) were used no agglutination were observed. There was a decrease in Streptococcus mutans growth with increasing concentration of antibodies (IgY). The growth was not distinct when 25 μg/ml of IgY was added to the culture. Similarly, GTF activity was quantified by phenol-sulfuric acid method. Conclusion: Total glucan synthesis by cell-associated GTF preparation from Streptococcus mutans was significantly inhibited by the addition of antibodies to CA-GTF. This raises the possibility of conferring passive protection against Streptococcus mutans-induced dental caries by using IgY from the eggs of the hens hyperimmune to Streptococcus mutans CA-GTF antigen. IgY specific to Streptococcus mutans can prevent the tooth decay by immobilizing bacteria and disabling bacteria’s ability to convert sugar into acid, thereby preventing dental caries.

Direct-binding quartz crystal microbalance immunosensor to detect carp metallothionein

Metallothionein (MT) is a valuable biomarker against xenobiotic heavy metal contamination in organs and blood of fish. In this study, MT was induced in carp blood by cadmium injection and was purified to homogeneity by DEAE-cellulose ion exchange column chromatography to be used dually as a standard and immunogen. A sensitive detection on carp MT was then followed using a batch-type direct-binding quartz crystal microbalance immunosensor system installed with a well cell. Among antibody immobilization methods tested, two methods that were carried out via a heterobifunctional thiolation cross-linker, and via the cross-linker and protein G showed their respective advantages in simplicity and sensitivity. When analyzed using the immunosensor for carp MT in the concentration range of 50–4000 ng/mL, the limit of detection around 250 ng/mL was obtained. Based on its reasonable sensitivity and real-time presentation for the sensor signal, the immunosensor of this study was presumed as a screening tool to monitor possible heavy metal contamination in a fish farm and imported fish.

Partial characterization of proteins from baru (Dipteryx alataVog) seeds

Baru (Dipteryx alata Vog.) is a fruit distributed throughout the Brazilian savanna and contains a seed with a high protein content, whose properties have been rarely explored. The purpose of this study was to characterize this protein, especially by isolation and quantifying its fractions and measuring some of its molecular properties. Baru seeds contain 244 g kg(-1) protein on a dry weight basis. Solubility profiles showed a preponderance of globulins. This fraction dominated the seed composition, with 61.7 wt% of the total soluble proteins. Albumins and glutelins accounted for 14 and 3.3 wt%, respectively. SDS-PAGE resolution of albumin and globulin showed main bands with molecular weights of 84 kDa and 64, 66 and 73 kDa, respectively. The total protein of the flour and the globulin showed values of in vitro digestibility of 85.59% and 90.54%, relative to casein. Total globulin produced only one chromatographic peak, both on Sepharose CL-6B gel filtration and on DEAE-cellulose ion-exchange columns, eluted at a concentration of 0.12 mol L(-1) NaCl. The baru seed had high protein content with large quantities of storage proteins. The chromatographic and solubility profiles indicate the predominance of a fraction with characteristics of a legumin-type protein.

Cell proliferative effect of polyxyloses extracted from the rhizomes of wild turmeric, Curcuma aromatica

Hot water-soluble crude polysaccharides were extracted from the rhizomes of wild turmeric, Curcuma aromatica Salisb. (Zingiberaceae), using dry grinding, boiling water extraction, and then ethanol precipitation. The crude polysaccharide extract was then fractionated by DEAE-cellulose ion exchange column chromatography, and subsequently further purified by Superdex G-200 gel filtration column chromatography, giving two relatively abundant polysaccharide fractions, called P11 and P21, and a much less common fraction P22 obtained in insufficient amounts for further analysis. The two main polysaccharide fractions were evaluated for monosaccharide composition by acid hydrolysis and high performance liquid chromatography (HPLC), whilst the molecular weight and functional groups were determined by gel permeable chromatography (GPC) and FT-IR, respectively. Fractions P11 and P21 were found to be polyxyloses with molecular weight-averages of 469,171 and 157,665 Da, respectively. P11 (100 μg/mL) could significantly induce human gingival fibroblast cells proliferation by 30%, while P21 (100 μg/mL) could significantly inhibit gingival fibroblast cells proliferation by 92%. The in vitro human primary gingival fibroblast cell proliferation in cell culture at a concentration of 100 μg/mL.

Comparison of Antivenom potential of chicken Egg yolk Antibodies Generated against Bentonite and Adjuvant coated Echis carinatus venom

The Chicken egg yolk antibodies generated against Bentonite and Adjuvant (FCA) coated Echis carinatus venom was evaluated for their anti-venom potential. Antibodies are extracted from immunized chicken egg yolk by the method described by Polson et al.(1980) and further purified by DEAE cellulose ion exchange column chromatography, which gave pure (180 - 200KDa) specific antibodies against venom. Inhibition of lethal, edema, procoagulant, and phospholipase A2 activities of Echis carinatus venom was determined. We found that 1.27mg of IgY (generated against FCA coated venom) and 1.33mg of IgY (generated against Bentonite coated venom) was able to completely neutralize the lethal activity (2LD 50) of Echis carinatus venom. Both chicken egg yolk antibodies effectively inhibited procoagulant activity and partially inhibited the Edema forming activity. IgY generated using Adjuvant (FCA) coated venoms showed very high anti-phospholipase A2 activity. In general, the results demonstrated the effectiveness of chicken egg yolk antibodies raised against adjuvant and bentonite coated venoms in neutralizing various pharamacological effects of Echis carinatusvenom.Keywords: Venoms, FCA, Bentonite, Chicken antibodies (IgY), PLA 2

Neutralization of the pharmacological effects of Cobra and Krait venoms by chicken egg yolk antibodies

Five-month-old white leghorn chickens were immunized with 50 μg of Common Cobra ( Naja naja) and 30 μg of Krait venoms ( Bungarus caeruleus) to generate antivenom antibodies against the venom antigen. Chickens received booster doses of increasing concentrations of venom at 14 days time intervals to raise the antivenom level in egg yolk. The antivenom from immunized chicken egg yolk was extracted by polyethylene glycol (PEG) and ammonium sulphate precipitation method which was further purified by DEAE cellulose ion exchange column chromatography. A high molecular weight protein of 180 kDa was detected by electrophoretic analysis which shows the purity of antivenom generated in chicken. Antibodies generated were specific and sensitive to the venom antigen. Various pharmacological activities of Cobra and Krait venoms were carried out by both in-vivo and in-vitro methods. The neutralization of lethality, hemorrhagic, edema, PLA 2 and procoagulant activity was evaluated in assays involving pre-incubation of venom and antivenom prior to testing. The antivenom was effective in neutralizing the toxic and enzymatic activities of venom. The LD 50 of venom for 18 g of mice was found to be 10 μg for Cobra and 3 μg for Krait venoms. The median effective dose (ED 50) of anti-Cobra venom was 4.48 mg/5LD 50 and 1.0 ml neutralized 0.127 mg of Cobra venom and the median effective dose (ED 50) of anti-Krait venom was 3.18 mg/5LD 50 and 1.0 ml neutralized 0.051 mg of Krait venom. The results indicate that antivenom generated in chicken could be used for therapeutic purposes in case of snakebite envenomation.

Studies on pharmacological effects of Russell's viper and Saw-scaled viper venom and its neutralization by chicken egg yolk antibodies

Antivenom antibodies were raised in 24-week-old white leghorn chickens against hemotoxic venoms of Russell's viper and Saw-scaled viper snakes. Booster injections of increasing concentrations of venom were given at 14days of time interval to raise the antivenom level in egg yolk. Antibodies were extracted from immunized chicken egg yolk by Polson et al. (Polson A., Von Wechmar M.B., Van Regenmortel M.H.V. Isolation of viral IgY antibodies from yolks of immunized hens. Immunological Communications 1980; 9:475–493.) and further purified by DEAE cellulose ion exchange column chromatography, which gave pure (180–200kDa) specific antibodies against venom. High titre of more than 1:10,000 antibodies were detected by ELISA at the 135th day of observation. The lethal toxicity and various pharmacological activities like hemorrhagic activity, phospholipase activity, edema and procoagulant activities of venom were carried out by both in vivo and in vitro methods. The effectiveness of antivenom in neutralizing these effects was carried out involving pre-incubation type experiments. The median effective dose (ED 50) for Russell's viper venom was 0.96mg/2LD 50/18g mice and for Saw-scaled viper venom it was 1.28mg/2LD 50/18g mice. One millilitre of specific antivenom was effective in neutralizing 0.110mg of Russell's viper and 0.137mg of Saw-scaled viper venoms respectively (PD 50). Antivenom was effective in neutralization assays in a dose dependent manner. The results indicate that antibodies raised in chicken could effectively neutralize the pharmacological effects induced by venoms and chickens therefore present an alternative and cheaper source of specific antibody generation.

Purification and characterization of milk clotting enzyme from goat ( Capra hircus)

Chymosin, the major component of rennet (milk clotting enzyme), is an acid protease produced in the fourth stomach of milk-fed ruminants including goat and sheep in the form of an inactive precursor prochymosin. It is responsible for hydrolysis of κ-casein chain in casein micelles of milk and therefore, used as milk coagulant in cheese preparation. The present investigation was undertaken to purify and characterize goat ( Capra hircus) chymosin for its suitability as milk coagulant. The enzyme was extracted from abomasal tissue of kid and purified nearly 30-fold using anion exchanger and gel filtration chromatography. Goat chymosin resolved into three major active peaks, indicating possible heterogeneity when passed through DEAE-cellulose ion exchange column. The purified enzyme had a molecular mass of 36 kDa on SDS-PAGE, which was further confirmed by Western blot analysis. The purified enzyme preparation was stable up to 55 °C with maximum activity at 30 °C. The milk clotting activity was decreased steadily as pH is increased and indicated maximum activity at pH 5.5. Proteolytic activity of goat chymosin increased with incubation time at 37 °C. Goat chymosin was found to be more thermostable than cattle chymosin and equally stable to buffalo chymosin.

Three glycoproteins with antimutagenic activity identified in Lactobacillus plantarum KLAB21.

Antimutagenic substances were purified from a culture supernatant of Lactobacillus plantarum KLAB21 cells isolated from kimchi, a Korean traditional fermented vegetable, and their characteristics were investigated. The antimutagenic substances were separated into two fractions by DEAE-cellulose ion-exchange column chromatography, which were designated the R1 and R2 fractions. The R1 fraction was then divided into two fractions again by Sephadex G200 gel filtration chromatography, and the fractions were designated R1-1 and R1-2. All three fractions were further purified using a Sepharose CL-6B gel filtration column. All the purified fractions were successfully stained with fuchsin as well as Coomassie brilliant blue, suggesting that they are glycoproteins. The purified fractions were confirmed to possess antimutagenic activity against N-methyl-N'-nitro-N-nitrosoguanidine on Salmonella enterica serovar Typhimurium TA100 cells. Their molecular masses were determined to be 16 (R1-1), 11 (R1-2), and 14 (R2) kDa on the Sepharose CL-6B column. Total sugar contents were 8.4% (R1-1), 7.3% (R1-2), and 9.4% (R2). The amino acid compositions of the fractions were different from each other; the major amino acids were glutamic acid (21.5%) and phenylalanine (17.1%) in the R1-1 fraction and glycine (41.3%) in the R1-2 fraction, but valine (31%) and phenylalanine (22.6%) were the major amino acids in the R2 fraction.

Inhibition of various isoforms of rat liver glutathione S‐transferases by tannic acid and butein

Glutathione S-transferases (EC.2.5.1.18, GSTs) were purified from rat liver by S-hexylglutathione affinity chromatography and six isoforms, namely C-1, C-2, C-3, C-4, A-2 and A-1, were isolated by CM-cellulose and DEAE-cellulose ion-exchange columns. Tannic acid and butein showed varying degrees of inhibition on the six individual GST isoforms. When 1-chloro-2,4-dinitrobenzene (CDNB) was used as a substrate, butein exerted significantly more potent inhibition on the cationic isoforms C-2, C-3 and C-4 with IC50 values of 6.8, 8.5 and 8.0 muM respectively. All the isoforms showed lower activity towards p-nitrobenzyt chloride when compared to CDNB and inhibition of the p-nitrobenzyl chloride-activity by tannic acid and butein was also weaker. The inhibitory effects of tannic acid and butein on each isoform decreased generally with increasing pH in the range of 6.0 to 8.0. The optimum pHs for inhibitions by tannic acid and butein on the six individual isoforms lie in the pH range of 6.0 to 6.5.

Analysis of cancer-associated colonic mucin by ion-exchange chromatography: evidence for a mucin species of lower molecular charge and weight in cancer

Cancer-associated mucins in the colon are antigenically distinct and glycosylated differently from their normal counterparts. Mucin-rich glycoconjugate preparations were made from nine non-neoplastic colons, seven colon cancers, and two different xenografts from mucin-producing human colon cancer cell lines, and radiolabeled with 3H. The preparation was applied to a DEAE-cellulose ion-exchange column, and eluted with a discontinuous ascending NaCl gradient resulting in seven discrete fractions or ‘species’. Over half of the 3H-labeled glycoconjugates from specimens of non-neoplastic colonic epithelium eluted in fraction V (eluted with 0.25 NaCl). Significantly less of the 3H-labeled glycoconjugates from specimens of colon cancer eluted in fraction V (34%, P<0.0005), and there were significant increases in glycoconjugates eluted in fractions IV ( P<0.008), III( P<0.0005), and II ( P<0.028). Additional samples were prepared without the radiolabeling procedures, chromatographed on a DEAE-cellulose ion-exchange column, and analyzed for monosaccharide content. Each of the fractions contained the monosaccharides expected in mucin-type glycoproteins, but only sialic acid was differentially expressed in the seven fractions or ‘species’, occurring principally in the more charged species. However, differences in sialic acid content were not sufficient to explain the differences in retention on the ion-exchange column, nor were differences in O-acetylation of the mucins. Mucin-type glycoconjugates from colon cancers are relatively less charged than those from the normal colon, and elute at lower ionic strengths. Of interest, cancer-associated mucins appear to be a lower molecular weight than their normal counterparts. Additional studies of oligosaccharide and apomucin structure will be required to explain the molecular basis of these differences in charge.

Acid stable trypsin inhibitor in bile

Acid stable trypsin inhibitor having the same antigenicity as urinary trypsin inhibitor was first identified in the bile of patients with malignant tumors (biliary tract carcinoma or pancreas head carcinoma) and gallstones. Bile trypsin inhibitor from malignant tumor patients was partially purified by DEAE cellulose ion exchange column chromatography. Two molecular forms of the inhibitor were identified. The main form had a molecular weight of about 86000 and the minor one a molecular weight of 31000 as determined by gel filtration. Using isoelectric focussing, the larger molecular form gave a p I value of 2.0 and the smaller form, a p I value of 5.1. The isolated larger form migrated on the slightly cationic side of human serum albumin by analytical polyacrylamide gel electrophoresis. The larger form reacted and fused with anti-urinary trypsin inhibitor serum and strongly inhibited trypsin, partially inhibited chymotrypsin and plasmin, but did not inhibit urokinase. The clinical significance of acid stable trypsin inhibitor is discussed.

Crystallization and preliminary data of Indian buffalo erythrocyte carbonic anhydrase

The most abundant anhydrase isoenzyme from the erythrocyte of Indian buffalo has been purified using affinity gel and DEAE-cellulose ion-exchange columns and single crystals suitable for X-ray diffraction studies have been obtained. The unit cell dimensions are a = 46.8 A ̊ , b = 104.5 A ̊ , c = 60.4 A ̊ , β = 91.2 ° and the space group is P2 1 with two molecules per asymmetric unit.

Physical separation of aortic corticoid receptors with type I and type II specificities

Previous gel filtration binding assay studies indicated that rat vascular smooth muscle cells contained corticoid receptor I and corticoid receptor II sites which could be distinguished on the basis of their relative affinities for aldosterone and dexamethasone. Ion-exchange chromatography experiments were designed to separate the two sites for further studies on their physical characteristics and role in vascular smooth muscle cell physiology. Cultured aortic cells were incubated with 5–10nM 3H steroid alone or in the presence of 10-fold non-radioactive steroid competitor for 30 min at 37°C. Following cell lysis, total cellular protein-bound steroid was isolated using Sephadex G-25 and applied to a DEAE-cellulose ion-exchange column. Three peaks of radioactivity were eluted using a 1–200 mM sodium phosphate gradient: peak T (30–38 mM), peak II (52–64 mM), and peak III (92–102 mM). Peaks I and II contained 60% of the eluted radioactivity and exhibited the same steroid specificity as corticoid receptor II sites (dexfimethasone aldosterone). Peak III contained 40% of the eluted radioactivity and exhibited the same steroid specificity as corticoid receptor I sites (aldosterone dexamethasone). These studies support the binding assay data on steroid specificity and relative proportion of type I and II sites. They also document the existence of type I and II corticoid receptors with different physicochemical characteristics in rat aortic smooth muscle cells.