Affi-Gel Blue Chromatography Research Articles

Partial purification and characterization of CA19-9 antigen from the ascitic fluid of a patient with pancreatic cancer

CA19-9 immunoreactive protein was partially purified from the ascitic fluid of a patient with pancreatic cancer by perchloric acid fractionation, gel chromatography and Affi-gel Blue column chromatography, resulting in a purified sample of 5.0 x 10(6) CA19-9 units per milligram of protein (3700-fold purification). Western blotting analysis of this purified sample revealed a single band of molecular weight 210 kDa. Although the original ascitic fluid showed a high CA125 immunoreactivity, this purified sample had no CA125 immunoreactivity. The elution pattern for CA19-9 activity on Affi-gel Blue column is quite distinct from that for CA125. These results suggest that CA19-9 antigen in carcinoma patients may be identical or very similar to that recently purified from the culture media of the colorectal cell line SW1116 and is distinct from CA125 antigen.

Dephosphorylation of Neuromodulin by Calcineurin

Neuromodulin (p57, GAP-43, F1, B-50) is a major neural-specific, calmodulin binding protein found in brain, spinal cord, and retina that is associated with membranes. Phosphorylation of neuromodulin by protein kinase C causes a significant reduction in its affinity for calmodulin (Alexander, K. A., Cimler, B. M., Meirer, K. E., and Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113). It has been proposed that neuromodulin may function to bind and concentrate calmodulin at specific sites within neurons and that activation of protein kinase C causes the release of free calmodulin at high concentrations near its target proteins. It was the goal of this study to determine whether bovine brain contains a phosphoprotein phosphatase that will utilize phosphoneuromodulin as a substrate. Phosphatase activity for phosphoneuromodulin was partially purified from a bovine brain extract using DEAE-Sephacel and Sephacryl S-200 gel filtration chromatography. The neuromodulin phosphatase activity was resolved into two peaks by Affi-Gel Blue chromatography. One of these phosphatases, which represented approximately 60% of the total neuromodulin phosphatase activity, was tentatively identified as calcineurin by its requirement for Ca2+ and calmodulin (CaM) and inhibition of its activity by chlorpromazine. Therefore, bovine brain calcineurin was purified to homogeneity and examined for its phosphatase activity against bovine phosphoneuromodulin. Calcineurin rapidly dephosphorylated phosphoneuromodulin in the presence of micromolar Ca2+ and 3 microM CaM. The apparent Km and Vmax for the dephosphorylation of neuromodulin, measured in the presence of micromolar Ca2+ and 2 microM CaM, were 2.5 microM and 70 nmol Pi/mg/min, respectively, compared to a Km and Vmax of 4 microM and 55 nmol Pi/mg/min, respectively, for myosin light chain under the same conditions. Dephosphorylation of neuromodulin by calcineurin was stimulated 50-fold by calmodulin in the presence of micromolar free Ca2+. Half-maximal stimulation was observed at a calmodulin concentration of 0.5 microM. We propose that phosphoneuromodulin may be a physiologically important substrate for calcineurin and that calcineurin and protein kinase C may regulate the levels of free calmodulin available in neurons.

Affinity Separation of Human Plasma Gelsolin on Affi-Gel Blue1

Human plasma gelsolin was specifically eluted with 1 mM adenosine 5'-triphosphate from an Affi-Gel Blue column. Since the ionic strength of sodium chloride required to elute the protein from the dye column was much higher than that of 1 mM adenosine 5'-triphosphate, the binding of plasma gelsolin with the dye-ligand appeared to be biospecific. Taking advantage of this affinity interaction, we have developed a revised purification method of human plasma gelsolin. The purification included ammonium sulfate precipitation, diethylaminoethyl-Sepharose chromatography, Affi-Gel Blue chromatography, and Phenyl-Sepharose chromatography. The method allowed a reproducible purification of the protein to apparent homogeneity, producing a 331-fold purification with a yield of 6%.

Bovine mitochondrial rhodanese is a phosphoprotein

The mitochondrial sulfurtransferase, rhodanese, has been analyzed for phosphate content. Significant amounts of protein-bound phosphate (30-40%) were measured in the six rhodanese preparations examined. Chromatographic experiments followed by phosphate analyses done on two of the preparations indicated that rhodanese A and rhodanese B, two enzyme forms that were previously resolved on DEAE-Sephadex by Blumenthal and Heinrikson (Blumenthal, K., and Heinrikson, R. L. (1971) J. Biol. Chem. 240, 2430-2437), correspond to dephospho- and phosphorhodanese, respectively. The phosphorylation of rhodanese by [gamma-32P]ATP is catalyzed by cAMP-dependent protein kinase. The stoichiometry of 32P incorporation based on the amount of dephosphorhodanese in the enzyme preparation approaches 1.0. The phosphorylation site is accessible in rhodanese that is free of substrate sulfur but not in the covalent enzyme-sulfur intermediate which is formed as an obligatory step during the course of catalysis. Because the cellular localization of cAMP-dependent protein kinase makes it unlikely as the physiologic modulator of rhodanese activity, liver extracts have been tested for a rhodanese kinase that does not require cAMP. Rhodanese kinase activity which is independent of cAMP is observed in extract fractions resolved by Affi-Gel Blue chromatography and freed from endogenous rhodanese by chromatography on Sephadex G-100. These results together with previous findings from this and other laboratories have led to a working model of a bicyclic cascade system that can modulate the rate of mitochondrial respiration. The essence of the model is a transduction and amplification of cellular signals into the altered covalent phosphorylation of rhodanese. Rhodanese, in turn, serves as a converter enzyme which directly alters the rate of the respiratory chain and, thus, ATP production by the reversible sulfuration of key iron-sulfur centers. The model, when expanded to include signal pathways initiated by hormones or neurotransmitters, represents a mechanism by which mitochondria can recognize and meet changing energy demands.

Partial purification of human urinary megakaryocyte colony-stimulating factor.

Urinary extracts from patients with aplastic anemia are known to promote murine megakaryocytopoiesis. In this report, we show a simple method for the partial purification of megakaryocyte colony-stimulating factor from human urine. A four-step purification procedure, which included ethanol precipitation, CM Affi-Gel Blue chromatography, wheat germ agglutinin-Sepharose chromatography and high-resolution hydroxyapatite chromatography, resulted in an about 430- to 630-fold increase of specific activity. The final fractions were still contaminated with erythropoietin, but the contaminated content of erythropoietin was not enough to stimulate mouse megakaryocytopoiesis in our culture system. We also demonstrate that human urinary extracts stimulated human megakaryocyte colony formation.

Characterization of the soluble cholinesterase from Squilla mantis

1. 1. The partial purification of the soluble cholinesterase from Squilla mantis is described. 2. 2. Affi-Gel Blue chromatography has represented the most efficient step in the purification of the enzyme. 3. 3. A single form of cholinesterase is present in the soluble fraction (70,000 g supernatant) of Squilla mantis homogenate. The isoelectric point is 5.3. 4. 4. Although propionylthiocholine was hydrolyzed at a higher rate than other substrates, V max/ K m values indicated substrates containing acetic acid (acetylthiocholine and acetyl-β-methylthiocholine) showed a better affinity for the enzyme. 5. 5. Tetramethyl-, tetraethyl-, tetrapropyl- and tetrabutyl-ammonium ions are all linear competitive inhibitors of the hydrolysis of acetyl- and propionylthiocholine. 6. 6. Some kinetic, structural and phylogenetic features of the enzyme are discussed.

Effects of surfactants on human serum biotinidase

Isoelectric focusing (IEF) of purified biotinidase and lipoamidase (sialylated glycoprotein enzymes) was found to be reproducible and reliable when non-ionic detergent, Nonidet P-40 (NP-40), was added at 1% (v/v). Applications of the IEF containing 1% NP-40 on biotinidase and lipoamidase purifications were also successful; i.e. human serum biotinidase was purified by IEF after Affi-Gel Blue chromatography in the final step, and microsomal biotinidase and lipoamidase from guinea pig liver and kidney were purified by the preparative liquid IEF of Rotofor cell with 1% NP-40 followed by the final gel-permeation chromatography with Sephadex G-200. Thus, this high-recovery IEF with non-ionic detergent of NP-40 at 1% is shown to be a practical and reproducible method applicable to analysis and purification for several biotinidases and lipoamidases, and also expected to be generally applicable to the other relatively hydrophobic proteins.

Characteristics and behavior during partial purification of estrogen sulfotransferase of guinea pig liver and chorion

Some characteristics of estrogen sulfotransferases from guinea pig liver and chorion were compared. Liver cytosolic activity was stimulated 10-fold by 25 mM monothiolglycerol and 2-fold by 15 mM MgCl 2 or CaCl 2, similar to that found previously for chorion. Liver and chorion activities were each eluted as a single peak from fast protein liquid chromatography (FPLC) gel filtration columns at apparent molecular weights of 52 300 and 50 000, respectively. Each was eluted during FPLC anion exchange under single, wide peaks with low recoveries. Liver sulfotransferase activity was eluted from Affi-gel Blue columns in the form of several peaks whereas the chorion activity behaved as a single species. The enzymes from both tissues, when partially purified by gel filtration followed by anion exchange, acted upon estrone and estradiol at the 3-position but activity toward dehydroepiandrosterone and testosterone was minimal or undetectable. Affi-gel Blue chromatography followed by FPLC gel filtration resulted in increases in specific activity of 26-and 90-fold for liver and chorion, respectively. Both enzymes were eluted fromagarose-hexane-adenosine 3′,5′-diphosphate (PAP-agarose) columns as single peaks. Average increases in specific activity for this column step were 40-fold and 96-fold for the entire eluted peaks of liver and chorion enzyme, respectively. Individual fractions from the PAP-agarose column indicated a specific activity increase of as much as 60-fold for liver and 208-fold for chorion. These latter were markedly unstable and it was not possible to obtain further purification by additional steps. Velocity versus substrate concentration curves for the partially purified enzymes showed complex kinetics, particularly with estradiol as substrate.

Partial purification and characterization of a serine endopeptidase from rat liver plasma membranes

A serine endopeptidase was partially purified from rat liver plasma membranes by using a four-step procedure: (i) solubilization with N-lauroylsarcosine; (ii) Ultrogel AcA-34 chromatography; (iii) CM Affi-Gel blue chromatography; (iv) agarose-soybean trypsin inhibitor chromatography. This enzyme was found to hydrolyze casein and various chromogenic peptide substrates; highest activity occurred with H- d-Val-Leu-Arg -p- nitroanilide , reported to be a specific substrate for human glandular kallikreins. The enzyme was heat-sensitive, showed a pH otpimum between 8.0 and 9.0 and was inhibited by D-Phe- L-Phe- L-Arg-CH 2Cl, aprotinin, diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, phenylmethylsulphonyl fluoride, leupeptin, antipain and dithiothreitol. This liver plasma membrane proteinase has an apparent molecular weight of about 30 000 as determined by Ultrogel AcA-34 chromatography and by autoradiography of [ 3H]DFP-labelled protein electrophoresis.

Dog inactive renin: biochemical characterization and secretion into renal plasma and lymph.

It has been suggested that the dog is a useful model for studies of inactive renin (IR). However, the nature and origin of the trypsin-activated angiotensin-forming activity in dog plasma have not been fully defined. We characterized dog IR using renin-specific antibody, immunoaffinity, and Affigel blue chromatography. Activated IR resembles renal and plasma active renin in biochemical and immunological properties. IR was detected in renal lymph, arterial and venous plasma, as well as renal extracts. At basal state, there was a significant renal venous-arterial gradient of IR indicating secretion from the kidney. Furthermore, a sixfold higher concentration of IR was demonstrated in renal lymph than in plasma. Our data provides evidence for two possible routes of IR secretion from the kidney and supports the contention that the dog is a good model for studies of IR secretion and regulation.

56] Purification of mouse monoclonal antibodies from ascitic fluid by DEAE affi-gel blue chromatography

56] Purification of mouse monoclonal antibodies from ascitic fluid by DEAE affi-gel blue chromatography

Purification of bovine .ALPHA.-fetoprotein on DEAE Affi-Gel Blue and Chromatofocusing.

Bovine a-fetoprotein (AFP) was purified from bovine fetal serum in an overall yield of 10% of initial AFP content in the serum by DEAE Affi-Gel Blue chromatography and Chromatofocusing. The purified AFP was found to be a homogenous protein in two procedures used polyacrylamide gel electrophoresis and immunoelectrophoresis against rabbit anti-bovine AFP. These results suggest that the two chromatography systems are sufficient and reproducible for the purification of AFP in bovine serum.

Highly Specific and Sensitive Hybridoma Antibodies against the α-Subunit of Human Glycoprotein Hormones*

The free alpha-subunit of human CG (hCG-alpha) has been detected in pregnancy as well as in several endocrine and nonendocrine tumors. In order to facilitate the detection of these tumors, we have developed, by hybridoma technology, very sensitive and highly specific antibodies to hCG-alpha, A2D4 and A2-58. Employing these antibodies, assays were developed to detect picogram levels of the free alpha-subunit. These antibodies have negligible cross-reactivity with intact hCG (A2D4, 0.17%; and A2-58, 1.75%). Pituitary hormone standards of human LH, human FSH, and human TSH from NIH showed cross-reactivity with these antibodies. However, the cross-reactivity was found to be due to the contamination of the hormone preparations with alpha-subunit. The antibodies have no effect on the binding of hCG to its receptor. They cross-react to the same extent with the deglycosylated hCG-alpha and alpha-subunits of all other human glycoprotein hormones but do not react with alpha-subunits of glycoprotein hormones from other species. We have purified these antibodies to homogeneity by DEAE Affi-Gel Blue and affinity chromatography. Both these antibodies belong to immunoglobulin M subclass.

Radioimmunoassays for the detection of antibodies to treponemal polypeptide antigens in serum.

Cross-reacting treponemal antigens are potentially important candidates for serodiagnostic assays in syphilitic infections. Based on the idea that the organelles for locomotion in virulent and avirulent treponemes might be composed of similar subunits, we attempted to purify the flagellar antigens of Treponema phagedenis biotype Reiter and Treponema refringens for use in radioimmunoassays. With a combination of physical and chemical methods, the major protein subunit of purified flagellar preparations exhibited a mass of approximately 37 kilodaltons (kd) on sodium dodecyl sulfate-polyacrylamide gels. These 37-kd materials, with weight estimates comparable to those of other flagellin molecules, were further purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroelution. Human and rabbit sera, alone or subjected to DEAE Affi-Gel blue chromatography, were subsequently tested in radioimmunoassays employing each of the purified preparations. Even though sera from patients with secondary syphilis and from experimentally infected animals at 3 to 4 weeks postinfection were reactive in radioimmunoassays employing the 37-kd flagellar antigens, the assays were relatively insensitive for detection of immunoglobulin G responses in the early stages of human infection. Detection of immunoglobulin G antibodies in sera obtained early in the course of natural or experimental infection was possible with electroeluted 33- to 64-kd materials from both avirulent and virulent treponemes.

Monoclonal antibodies against rat saliva and salivary gland antigens.

Hybridomas were produced by the fusion of NS1 myeloma cells with spleen cells of a BALB/c mouse immunized with rat submandibular saliva. Growth of hybridomas was evident in 60/96 wells, and colonies secreting antibodies against saliva components were identified in 20 wells by using a solid phase enzyme-linked immunoassay. Cloning of cells from 12 wells yielded originally 43 hybridoma cell lines secreting anti-saliva antibodies. After recloning, one hybridoma (4Cl3) was selected for further studies. The hybridoma (4Cl3) cells were grown as ascites tumors, and the antibodies were purified from the ascitic fluid by diethylaminoethyl Affi-gel Blue chromatography. The purified antibody (MA4), immunoglobulin G1, immunoprecipitated a 39K dalton protein from submandibular saliva, and also reacted with a protein of the same electrophoretic mobility on immunoblots. From extracts of submandibular gland slices, incubated with [3H]leucine, the antibody again immunoprecipitated a 39K protein, indicating that this protein is synthesized in the gland. MA4 was used for immunocytochemical stainings of submandibular glands of rats of different ages. In general, immunostaining was seen only in acinar cells. Thus, there was no staining in the glands of 1-day-old rats that lack differentiated acinar cells. In the glands of 1- to 4-week-old rats the number of immunoreactive cells and the extent of immunostaining paralleled the differentiation of the acinar cells. In the glands of adult rats a uniform staining of the secretory granules of the acinar cells was observed. The immunoreactive 39K protein seemed to be restricted to the acinar cells in the submandibular gland; there was no immunostaining in the parotid, sublingual, or lingual salivary glands, or in the pancreas, colon, and duodenum. Stimulation of saliva secretion by isoproterenol resulted in a virtual depletion of the antigen from the acinar cells. These results indicate the feasibility of producing mouse hybridomas that secrete antibodies against rat saliva components. The monoclonal antibody at hand will be useful in analyzing the differentiation of the acinar cells, and the factors that influence this differentiation process.

Isolation, characterization and clinical evaluation of the gamma-glutamyltransferase associated with hepatocellular carcinoma.

Sera from 24 patients with hepatocellular carcinoma (HCC), 30 patients with hepatobiliary diseases other than HCC and 5 normal subjects were analyzed for gamma-glutamyltransferase (GGT) isozymes. In ultracentrifugation, GGT I' was recovered in the non-lipoprotein fraction (the residue), together with GGTs I'', II', I and X. GGTs III to IX were recovered in lipoprotein fractions. GGTs in the lipoprotein fractions were removed beforehand by Affi-Gel Blue chromatography, leaving GGTs I', I'', II', I and X in the non-bound fraction, which was subjected to Con A-Sepharose chromatography. From the double affinity chromatography (DAC), GGTs I' and II' were recovered in the unbound fraction, and GGTs I, I'', II' and X in the bound fraction. GGT activities in the unbound fractions of sera from HCC patients were generally higher than those from patients with other benign hepatobiliary diseases. When the GGT activity of the unbound fractions in DAC was expressed as a percent of the sum of the unbound and bound activities (U/(U + B)) and 22% was set as the lower limit of positive values, 54% of the HCC cases had positive values, while none of the patients with hepatobiliary diseases other than HCC had positive values. The U/(U + B) ratio of GGT in DAC appears to be a clinically useful test for screening HCC.

Concomitant purification of three porcine heart mitochondrial enzymes: Citrate synthase, aspartate aminotransferase, and malate dehydrogenase

The mitochondrial enzymes citrate synthase, malate dehydrogenase, and aspartate amino-transferase were purified to homogeneity from porcine hearts by use of Bio-Rex 70, carboxy-methylcellulose CM32, and Affi-Gel blue chromatography. This procedure provides relatively rapid, large-scale preparation of the three enzymes based on their differential binding to commercially available cation-exchange resins followed by a final affinity chromatography step.

Role of steroids in secretion—modulating effect of triamcinolone and estradiol on protein synthesis and secretion from the rat exocrine pancreas

Following adrenalectomy of male rats, or adrenalectomy and ovariectomy of females, there was marked depletion of zymogen granules in acinar cells of the pancreas. Within 9 h after treatment with either triamcinolone or 17β-estradiol, complete restoration of these secretory vesicles was observed. This repletion was not inhibited by actinomycin-D. Supernatant fractions (100,000 g, 60min) of rat pancreas, from both normal and surgically altered animals, contained proteins that bound [ 3H]-triamcinolone and [ 3H]-estradiol, suggesting that the action of these hormones is exerted directly on the pancreas. Binding of both steroid hormones required the presence of an additional coligand referred to as accessory factor. In addition, the binding proteins for [ 3H]-triamcinolone and [ 3H]-estradiol eluted in similar positions after Sephadex G-200 and CM Affi-gel Blue chromatography. It is uncertain, however, whether a single protein binds both steroid hormones since they had different binding isotherms. Scatchard analysis of binding of [ 3H]-estradiol yielded a single straight line of negative slope from which it was calculated that there were about 4.4 pmol of binding sites per mg protein, having an average apparent K d of about 5 × 10 −8M. Similar analysis of the data for [ 3H]-triamcinolone yielded a straight line of zero slope indicating nonsaturable binding of hormone at concentrations as high as 10μM. Since both [ 14C]- L-leucine incorporation into protein and amylase secretion were affected markedly by the steroid-hormonal status of the animal, it is presumed that steroid-bound complexes in acinar cells of the pancreas modulate synthesis and secretion of protein.

Purification of N-acetyl-L-glutamate synthetase from rat liver mitochondria and substrate and activator specificity of the enzyme.

N-Acetylglutamate synthetase was purified 33,000-fold to apparent homogeneity from the matrix fraction of rat liver mitochondria. The purification procedure involved treatment of mitochondria with digitonin, ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, Affi-Gel blue chromatography, acetylglutaminyl Bio-Gel chromatography, sucrose density gradient centrifugation, and isoelectric focusing. As the enzyme lost activity in contact with a glass surface, glassware coated with silicone was used throughout the purification. Triton X-100 stabilized the enzyme and was included in most buffers at a concentration of 0.1% (w/v). The purified acetylglutamate synthetase has a specific activity of 92.4 mumol min-1 (mg of protein)-1, in the presence of 1 mM L-arginine. The enzyme had a sedimentation coefficient (S20,w) of 8.1 S and its molecular weight was estimated to be about 160,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the enzyme migrated as a single protein band with Mr = 57,000, indicating a composition of three subunits of similar size. The purified acetylglutamate synthetase showed a high substrate specificity for L-glutamate and acetyl-CoA, confirming the previous results with a less purified preparation (Shigesada, K., and Tatibana, M. (1978) Eur. J. Biochem. 84, 285-291). Among other 14 acyl-CoAs tested, only propionyl-CoA could substitute for acetyl-CoA, as an acyl donor, with a reaction rate 4.3% of that with acetyl-CoA at 0.5 mM. Among amino acids other than L-glutamate and derivatives or analogues of glutamate tested, the following served as an acyl acceptor: glycine at a rate 2.7% of that with L-glutamate at 1.0 mM, L-glutamine (5.0%), L-glutamate gamma-hydroxamate (15.5%), DL-alpha-aminoadipate (5.2%), and DL-alpha-aminopimelate (4.0%). The purified enzyme is markedly stimulated by L-arginine. The specificity of L-arginine as a low molecular weight activator was strict; only L-argininic acid could activate the enzyme to a lesser extent. Cationic polypeptides, such as protamine, polyarginine and polylysine, also activated the synthetase. The effect was additive to that of arginine.

Specific binding of [ 3H]-estradiol to the cytosol of rat pancreas: Alteration of the apparent number of binding sites by an endogenous factor and oligopeptide derivatives

The supernatant fraction of rat pancreas contains an estrogen-binding protein that differs in several ways from the one found in uterus: (1) binding of 17β-[ 3H]-estradiol in pancreas was non-saturable at hormone concentrations ranging from 2–50 nM; (2) diethylstilbestrol (as well as 17α-estradiol) was only about half as effective as unlabeled 17β-estradiol in competing for these specific binding sites; and (3) the binding reaction in pancreas requires the presence of a coligand, referred to as accessory factor. Accessory factor is an endogenous substance isolated from rat pancreas that is required for specific binding of [ 3H]-estradiol to a soluble protein present in this tissue. The activity of this factor can be mimicked by the synthetic oligopeptide N-benzoyl- l-phenylalanyl- l-valyl- l-argininyl- p-nitroanilide, or the simpler derivative N-benzoyl- l-argininyl- p-nitroanilide. Removal of endogenous accessory factor by CM Affi-gel Blue chromatography renders the estrogen-binding protein incompetent. Using such preparations, the capacity to reactivate binding of [ 3H]-estradiol by partially purified accessory factor and synthetic oligopeptide was compared. When specific binding of [ 3H]-estradiol was determined at concentrations ranging from 2–50 nM in the presence of limiting amounts of accessory factor, a saturable isotherm was obtained. In the presence of increasing amounts of accessory factor, specific binding was nonsaturable as was observed with the initial cytosol. Scatchard analysis indicated: (a) that the number of binding sites for [ 3H]-estradiol increased as a function of accessory factor concentration; and (b) the affinity of these sites for [ 3H]-estradiol declined concomitantly. As a working hypothesis it is suggested that binding of [ 3H]-estradiol in the presence of limiting amounts of accessory factor results in a ternary complex (binding protein: [ 3H]-estradiol: and accessory factor). Upon further addition of factor a supramolecular structure seems to form that is nonsaturable with respect to binding of [ 3H]-estradiol at concentrations as high as 50 nM. One possible function of this binding system is that the coligand (accessory factor) may enable the cell to concentrate steroid, and depending on the extent of steroid binding, regulate the intensity of the reaction in which the binding complex participates.