Avidin Column Research Articles

Analysis of the envelope proteins of heat-shocked Vibrio parahaemolyticus cells by immunoblotting and biotin-labeling methods.

Vibrio parahaemolyticus, a common enteropathogen in tropical and subtropical coastal regions, exhibits significant adaptive acid tolerance response and heat-shock response, and the envelope proteins induced by stresses are suggested to be associated with virulence. This work examined the heat-shock proteins located in the envelope of V. parahaemolyticus by two rapid methods; namely, the immunoblotting and biotin-labeling methods. The bacterial cells were cultured at 25 C and heat shocked at 37 or 42 C for 1 or 2 hr. The cells were first lysed, then proteins were separated by gel electrophoresis and probed with antiserum raised against heat-shocked cells. Next, the heat-shocked cells were examined by labeling with water soluble sulfo-NHS-LC-biotin. Proteins of 33, 61, 66, 71, 78, 92 and 101 kDa were induced, while 55, 86, 102, 120 and 160 kDa proteins were markedly enhanced in the envelope of the heat-shocked V. parahaemolyticus cells. The biotin tagged envelope proteins were purified using a monomeric avidin column, and the N-terminal sequence was determined and compared with other high identity protein sequences. The sequence results suggest that Vph1 (55 kDa), Vph2 (46 kDa) and Vph3 (42 kDa) are de novo synthesized heat-shock proteins located in the envelope of this pathogen, and the functions of these proteins in stress protection and virulence have yet to be determined.

Global Profiling of the Cell Surface Proteome of Cancer Cells Uncovers an Abundance of Proteins with Chaperone Function

There is currently limited data available pertaining to the global characterization of the cell surface proteome. We have implemented a strategy for the comprehensive profiling and identification of surface membrane proteins. This strategy has been applied to cancer cells, including the SH-SY5Y neuroblastoma, the A549 lung adenocarcinoma, the LoVo colon adenocarcinoma, and the Sup-B15 acute lymphoblastic leukemia (B cell) cell lines and ovarian tumor cells. Surface membrane proteins of viable, intact cells were subjected to biotinylation then affinity-captured and purified on monomeric avidin columns. The biotinylated proteins were eluted from the monomeric avidin columns as intact proteins and were subsequently separated by two-dimensional PAGE, transferred to polyvinylidene difluoride membranes, and visualized by hybridization with streptavidin-horseradish peroxidase. Highly reproducible, but distinct, two-dimensional patterns consisting of several hundred biotinylated proteins were obtained for the different cell populations analyzed. Identification of a subset of biotinylated proteins among the different cell populations analyzed using matrix-assisted laser desorption ionization and tandem mass spectrometry uncovered proteins with a restricted expression pattern in some cell line(s), such as CD87 and the activin receptor type IIB. We also identified more widely expressed proteins, such as CD98, and a sushi repeat-containing protein, a member of the selectin family. Remarkably, a set of proteins identified as chaperone proteins were found to be highly abundant on the cell surface, including GRP78, GRP75, HSP70, HSP60, HSP54, HSP27, and protein disulfide isomerase. Comprehensive profiling of the cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns in this compartment.

Trifunctional conjugation reagents. Reagents that contain a biotin and a radiometal chelation moiety for application to extracorporeal affinity adsorption of radiolabeled antibodies.

A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with (111)In and (90)Y, so the chelates CHX-A' '-DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with (111)In and (90)Y was facile at room temperature for conjugates containing the CHX-A' ', but elevated temperature (e.g., 45 degrees C) was required to obtain good yields with the DOTA chelate. Stability of (90)Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the (90)Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A' ' containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.

Identification of a region in alcohol dehydrogenase that binds to α-crystallin during chaperone action

α-Crystallin, the major eye lens protein and a member of the small heat-shock protein family, has been shown to protect the aggregation of several proteins and enzymes under denaturing conditions. The region(s) in the denaturing proteins that interact with α-crystallin during chaperone action has not been identified. Determination of these sites would explain the wide chaperoning action (promiscuity) of α-crystallin. In the present study, using two different methods, we have identified a sequence in yeast alcohol dehydrogenase (ADH) that binds to α-crystallin during chaperone-like action. The first method involved the incubation of α-crystallin with ADH peptides at 48 °C for 1 h followed by separation and analysis of bound peptides. In the second method, α-crystallin was first derivatized with a photoactive trifunctional cross-linker, sulfosuccinimidyl-2[6-(biotinamido)-2-( p-azidobenzamido)-hexanoamido]ethyl-1,3di-thiopropionate (sulfo-SBED), and then complexed with ADH at 48 °C for 1 h in the dark. The complex was photolyzed and digested with protease, and the biotinylated peptide fragments were isolated using an avidin column and then analyzed. The amino acid sequencing and mass spectral analysis revealed the sequence YSGVCHTDLHAWHGDWPLPVK (yeast ADH 40–60) as the α-crystallin binding site in ADH. The interaction was further confirmed by demonstrating complex formation between α-crystallin and a synthetic peptide representing the binding site of ADH.

Bacterial Expression of in Vivo-Biotinylated Aequorin for Direct Application to Bioluminometric Hybridization Assays

We have constructed a plasmid suitable for bacterial expression of in vivo-biotinylated photoprotein aequorin. The biotin tag facilitates the isolation of aequorin from crude cell extract and the direct complexation of aequorin with streptavidin for the development of highly sensitive hybridization assays, thereby avoiding the need for chemical crosslinking. The plasmid contains a biotin-acceptor coding sequence fused to an apoaequorin gene. The birA gene, encoding biotin protein ligase (BPL), is inserted downstream of the apoaequorin sequence. BPL biotinylates, posttranslationally, the acceptor domain at a unique position. Functional aequorin is generated by incubating the lysate with coelenterazine and is purified by using a monomeric avidin column that allows elution under nondenaturing conditions. The biotinylated aequorin is complexed with streptavidin and used as a reporter molecule in a hybridization assay. The assay entails immobilization of an oligonucleotide probe on microtiter wells followed by hybridization with a denatured DNA target labeled with biotin through PCR. Streptavidin–biotinylated aequorin is used for quantification of the hybrids. Luminescence is measured in the presence of excess Ca2+. The analytical range extends from 80 amol of target DNA per well (with a signal-to-background ratio of 2.1) up to 40 fmol per well. The coefficient of variation is about 6%. In vivo-biotinylated aequorin produced from 1 liter of culture is sufficient for 300,000 hybridization assays.

The large subunit of the mammalian mitochondrial ribosome. Analysis of the complement of ribosomal proteins present.

Identification of all the protein components of the large subunit (39 S) of the mammalian mitochondrial ribosome has been achieved by carrying out proteolytic digestions of whole 39 S subunits followed by analysis of the resultant peptides by liquid chromatography and mass spectrometry. Peptide sequence information was used to search the human EST data bases and complete coding sequences were assembled. The human mitochondrial 39 S subunit has 48 distinct proteins. Twenty eight of these are homologs of the Escherichia coli 50 S ribosomal proteins L1, L2, L3, L4, L7/L12, L9, L10, L11, L13, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L24, L27, L28, L30, L32, L33, L34, L35, and L36. Almost all of these proteins have homologs in Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae mitochondrial ribosomes. No mitochondrial homologs to prokaryotic ribosomal proteins L5, L6, L25, L29, and L31 could be found either in the peptides obtained or by analysis of the available data bases. The remaining 20 proteins present in the 39 S subunits are specific to mitochondrial ribosomes. Proteins in this group have no apparent homologs in bacterial, chloroplast, archaebacterial, or cytosolic ribosomes. All but two of the proteins has a clear homolog in D. melanogaster while all can be found in the genome of C. elegans. Ten of the 20 mitochondrial specific 39 S proteins have homologs in S. cerevisiae. Homologs of 2 of these new classes of ribosomal proteins could be identified in the Arabidopsis thaliana genome.

Structural Characterization of Pyrrolic Cross-links in Collagen Using a Biotinylated Ehrlich's Reagent

The structures of pyrrolic forms of cross-links in collagen have been confirmed by reacting collagen peptides with a biotinylated Ehrlich's reagent. This reagent was synthesized by converting the cyano group of N-methyl-N-cyanoethyl-4-aminobenzaldehyde to a carboxylic acid, followed by conjugation with biotin pentyl-amine. Derivatization of peptides from bone collagen both stabilized the pyrroles and facilitated selective isolation of the pyrrole-containing peptides using a monomeric avidin column. Reactivity of the biotinylated reagent with collagen peptides was similar to that of the standard Ehrlich reagent, but heat denaturation of the tissue before enzyme digestion resulted in the loss of about 50% of the pyrrole cross-links. Identification of a series of peptides by mass spectrometry confirmed the presence of derivatized pyrrole structures combined with between 1 and 16 amino acid residues. Almost all of the pyrrole-containing peptides appeared to be derived from N-terminal telopeptide sequences, and the nonhydroxylated (lysine-derived) form predominated over pyrrole cross-links derived from helical hydroxylysine.

Optimization of the chiral separation of some 2-arylpropionic acids on an avidin column by modeling a combined response.

The enantiomeric separation of some nonsteroidal antiinflammatory drugs was investigated on an avidin column. An experimental design approach (central composite design) was used to evaluate the effects of three method parameters (pH, concentration of organic modifier, and buffer concentration) on the analysis time and the resolution, as well as to model these responses. This revealed that the organic modifier concentration and sometimes the pH are significant parameters to control because of their influence on both analysis time and resolution. Furthermore, the central composite design results were combined in a multicriteria decision-making approach in order to obtain a set of optimal experimental conditions leading to the most desirable compromise between resolution and analysis time.

Sugar-mediated crosslinking of alpha-biotinylated-Lys to cysteamine-agarose support: a method to isolate Maillard Lys-Lys-like crosslinks.

Advanced glycation end products (AGEs) and, specifically, protein-protein AGE crosslinks have long been studied for their potential role in aging, diabetic complications and Alzheimer disease. With few exceptions, the chemical nature of these structures remains unknown. We report here a simple approach that allows the preparation and isolation of milligram quantities of sugar-mediated AGE Lys-Lys-like crosslinks from glycation mixtures. The method is based on a sugar-dependent incorporation of N(alpha)-biotinyl-L-Lys into cysteaminyldisulfide Sepharose 6B (AE-S-S-Sepharose 6B). Glycation mixtures with six different sugars showed a time- and sugar-dependent decrease in the concentration of the support-bound primary amino groups and accounted for almost 90% loss of cysteaminyl amino groups at the end of the various incubation periods. 4-Hydroxyazobenzene-2-carboxylic acid-avidin assays indicated the incorporation of N(alpha)-biotinyl-L-Lys equal to 8% of the total support amino groups with methylglyoxal after 7 d and 1% with fructose and glucose after 1 mo of incubation. Treatment of the washed, sugar-modified supports with 2-mercaptoethanol released the bulk of the bound AGE modifications and the crosslinks. Subsequent fractionation of these preparations over a monomeric avidin column afforded a complete separation of sugar-mediated AGE modifications and the crosslinks. Depending on the sugar employed, micromolar amounts of biotinylated Lys-Lys-like crosslinks were generated by this two-step procedure from 8 mL of the original AE-S-S-Sepharose 6B.

A Labeling, Detection, and Purification System Based on 4-Hydroxyazobenzene-2-carboxylic Acid: An Extension of the Avidin–Biotin System

We introduce a new nonradioactive, chromogenic label based on 4-hydroxyazobenzene-2-carboxylic acid (HABA), which is suitable for bioanalytical application, e.g., detection, localization, isolation, and purification. The HABA label is superior to other systems where it is difficult to separate labeled from unlabeled molecules or to determine the amount of label. HABA is readily detected spectroscopically by its absorption at 350 nm or by its interaction with avidin that results in a red shift to 500 nm. The HABA reagents described can be conjugated to a variety of functional groups on biomolecules and purified thereafter by affinity chromatography on an avidin column. The interaction of the HABAylated biomolecules with their corresponding targets is detected with high-affinity anti-HABA antibodies or with avidin. The nonradioactive, chromogenic HABA-based reagents form a homogeneous system that can complement or replace systems where facile quantification of the label is desired.

26] Biotinylation of proteins in vivo and in vitro using small peptide tags

This chapter discusses the biotinylation of proteins in vivo and in vitro using small peptide tags. Proteins containing the AviTag can be efficiently biotinylated in vitro using the BirA enzyme from E. coli. This option is desirable when the expression of soluble target protein in E. coli is inefficient, as in the case of proteins that form inclusion bodies when expressed in bacteria or when using an expression system not yet developed for efficient biotinylation, e.g., in yeast or in mammalian cells. Whereas the in vitro reaction can be performed in a complex mixture of proteins, a more desirable option is to use in vitro biotinylation on purified proteins to minimize protease contamination and concentrate the substrate to achieve a more efficient reaction. Because the protein may be biotinylated poorly or not at all during expression, a protein purification scheme that does not depend on biotinylated protein is required. Most AviTag fusion proteins can also be efficiently biotinylated in vivo, saving time and eliminating the need for BirA enzyme. Biotinylated proteins can be purified efficiently by exploiting the high degree of specificity between avidin and biotin through the use of monomeric avidin columns. Regardless of the expression vector and host used, efficient in vivo biotinylation of protein- AviTag fusions requires overexpression of BirA.

Simultaneous determination of donepezil (aricept ®) enantiomers in human plasma by liquid chromatography–electrospray tandem mass spectrometry

A rapid, sensitive and enantioselective LC–MS–MS method using deuterium-labeled internal standard was developed and evaluated for the simultaneous quantitative determination of donepezil enantiomers in human plasma without interconversion during clean-up process and measurement. The use of an avidin column allowed the separation of donepezil enantiomers, which were specifically detected by MS–MS without interference from its metabolites and plasma constituents. Evaluation of this assay method shows that samples can be assayed with acceptable accuracy and precision within the range from 0.0206 ng/ml to 51.6 ng/ml for both R-donepezil and S-donepezil. This analytical method was applied to the simultaneous quantitation of donepezil enantiomers in human plasma.

Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator.

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) chloride channel give rise to the most common lethal genetic disease of Caucasian populations, CF. Although the function of CFTR is primarily related to the regulation of apical membrane chloride permeability, biochemical, immunocytochemical, and functional studies indicate that CFTR is also present in endosomal and trans Golgi compartments. The molecular pathways by which CFTR is internalized into intracellular compartments are not fully understood. To define the pathways for CFTR internalization, we investigated the association of CFTR with two specialized domains of the plasma membrane, clathrin-coated pits and caveolae. Internalization of CFTR was monitored after cell surface biotinylation and quantitation of cell surface CFTR levels after elution of cell lysates from a monomeric avidin column. Cell surface levels of CFTR were determined after disruption of caveolae or clathrin-coated vesicle formation. Biochemical assays revealed that disrupting the formation of clathrin-coated vesicles inhibited the internalization of CFTR from the plasma membrane, resulting in a threefold increase in the steady-state levels of cell surface CFTR. In contrast, the levels of cell surface CFTR after disruption of caveolae were not different from those in control cells. In addition, although our studies show the presence of caveolin at the apical membrane domain of human airway epithelial cells, we were unable to detect CFTR in purified caveolae. These results suggest that CFTR is constitutively internalized from the apical plasma membrane via clathrin-coated pits and that CFTR is excluded from caveolae.

ISOCRATIC DETERMINATION OF DACARBAZINE AND RELATED IMPURITIES 2-AZAHYPOXANTHINE AND 5-AMINO-IMIDAZOLE-4-CARBOXAMIDE BY HPLC ON AN AVIDIN PROTEIN COLUMN

The separation of dacarbazine and its two related impurities, 5-amino-imidazole-4 carboxamide (AIC) and 2-azahypoxanthine (AHX) was investigated by capillary electrophoresis and high performance liquid chromatography. Baseline separation of dacarbazine and its related impurities was achieved by capillary electrophoresis (CE) on a fused silica capillary (70 cm × 50 μm, i.d) with an electrolyte buffer of methanol: phosphate buffer (0.025 M, pH 4) - 2:98 v/v at an applied voltage of 15 kV and detection at 220 nm. The CE method could not be used for the quantitation of impurities because at high concentration, the dacarbazine peak overlapped the nearest impurity peak. The initial HPLC method developed on an octadecylsilane (ODS) column successfully separated the impurities from the dacarbazine, but required a longer time of analysis (>50 min). Another HPLC method developed on an avidin protein column separated the drug and its impurities with a run time of less than 27 min and was chosen for method development and validation. Dacarbazine and its two impurities were separated isocratically on avidin column using a mobile phase of 2-propanol: phosphate buffer (pH 7, 0.02 M) 4:96 v/v at a flow rate of 0.6 mL/min and detection at 230 nm. Calibration curves were prepared for each impurity from 100–1000 ng/mL. The detector response was linear (r2 >0.997) for each individual impurity down to 100 ng/mL which represented 0.5% w/w of the dacarbazine concentration. Accuracy and precision of analysis for each impurity were less than 3 % at 100 ng/mL. The limits of quantitation and detection (s/n >3) for both impurities were 100 ng/mL and 50 ng/mL, respectively. Linearity was also demonstrated for dacarbazine in the 15–25 μg/mL range with a correlation coefficient greater than 0.998.

Chiral Separations of Selected Pharmaceuticals on Avidin Column

An unexplored commercially available avidin protein column was investigated for chiral separation of selected pharmaceuticals. Mobile phase compositions such as pH, buffer strength, and organic modifier were varied to affect separation of racemic mixtures. Baseline resolution of enantiomers were successfully achieved for thalidomide, glutethimide, primaquine, aminoglutethimide, hydroxyzine, chlorthalidone, and pyridoglutethimide. Partial separation of enantiomers were achieved for oxazepam, lorazepam, verapamil, and hexobarbital. Separation conditions of each racemate were optimized so that the methods can be used for the analysis of racemic pharmaceuticals. The pH and organic modifier of mobile phase played a greater role than buffer strength in the separation of individual racemate. The presence of aromatic rings and carbonyl functions directly attached to the chiral center of an analyte may play an important role in recognizing a specific site in the protein bonded phase.

Determination of Racemic Thalidomide in Human Plasma by use of an Avidin Column and Solid Phase Extraction

A HPLC method was developed to determine racemic thalidomide in plasma on a avidin protein column using solid phase extraction. The enantiomers were separated isocratically using a mobile phase of 2:98 v/v 2-propanol: phosphate buffer (0.1M, pH 4) at a flow rate of 0.6 mL/min at ambient temperature with detection at 300 nm. The chromatographic run time was less than 13 min. Calibration curves were prepared for each enantiomer in the range 100–5000ng/mL. The correlation coefficients for each enantiomer were greater than 0.999. The method is sensitive and can be used to measure plasma blood levels (100–1000ng/mL) of each enantiomer. The % RSD for quality control samples for both enantiomers were less than 10%. A C-18 SPE cartridge was used to extract the drug from plasma and recoveries of the enantiomers were greater than 95%. The limits of quantitation and detection were 100ng/mL and 50 ng/mL (s/n <3), respectively, for each enantiomer. Two of the commercially available metabolites of thalidomide were found not to interfere with the assay procedure.

Affinity Purification of Yeast Cytochrome Oxidase with Biotinylated Subunits 4, 5, or 6

Null mutants inCOX4, COX5a, orCOX6, which encode subunits 4, 5, and 6 of yeast cytochrome oxidase are blocked in assembly of the enzyme. The mutants are complemented by gene constructs expressing cytochrome oxidase subunits with a carboxyl terminal extension containing a biotinylation signal sequence. Spectra and enzyme activities of mitochondria from transformants expressing a biotinylated subunit indicate restoration of a functional cytochrome oxidase. Biotinylated cytochrome oxidase can be affinity-purified from mitochondrial extracts by fractionation on a monomeric avidin column. This method can be used to purify the enzyme from small amounts of starting material.

Separation of Immunoglobulin G from Cheddar Cheese Whey by Avidin‐Biotinylated IgY Chromatography

ABSTRACTHigh‐purity immunoglobulins (Ig), which may be useful for immunologic supplementation of food products, were isolated from Cheddar cheese whey in a one‐step process using avidin‐biotinylated yolk immunoglobulin (IgY) column chromatography. Yolk antibodies specific to bovine IgG (IgYIgG) were biotinylated with biotinyl amido‐hexanoic acid‐N‐hydroxy‐sulfo‐succinimide ester without any notable effect on antigen‐binding activity, and coupled to immobilized avidin columns. The resulting avidin‐biotinylated IgYIgG columns, with binding capacity of 50–55% (w/w percent ratio of IgG to immobilized IgYIgG), were used for specific binding of IgG from cheese whey. Elution with a commercially available eluent (Actisep) or 0.1 M glycine‐HCl buffer at pH 2.8 yielded IgG with purity of 99% by radial immunodiffusion.

Affinity Chromatographic Purification of Antibodies to a Biotinylated Fusion Protein Expressed inEscherichia coli

HOP1,a protein component of the synaptonemal complex inSaccharomyces cerevisiaewhich is believed to play an important role in meiotic synapsis, was expressed inEscherichia colias a fusion protein incorporating a “tag” polypeptide which is biotinylated naturally in the bacteria. TheHOP1fusion protein was produced in an insoluble form within the bacteria; once solubilized using a denaturing agent, the protein was purified by avidin monomer affinity chromatography. The recombinant protein was used to immunize rabbits and produce polyclonal antibodies. Procedures for affinity purification of antibodies using the recombinant protein attached to the avidin column and a magnetic method for concentration of antibodies are described. Antibody elution conditions in these procedures do not affect the affinity of the column for the recombinant protein, which can be recovered afterward. Affinity-purified antibodies show high binding capacity toHOP1recombinant protein in immunoblotting experiments, but reduced background compared with crude antiserum or purified IgG fraction. The affinity-purified antibodies recognize a major band around 70 kDa in Western blots of yeast protein extracts following meiotic induction.

Identification of Protein Phosphatase-1-binding Proteins by Microcystin-Biotin Affinity Chromatography

Biotinylated microcystin was used to affinity purify over avidin-Sepharose the entire cellular content of active forms of protein phosphatase (PP) 1 and 2A holoenzymes present in three subcellular fractions of skeletal muscle. Biotinylated microcystin displayed IC50 values in the nM range against PP-1C (1.58 +/- 0.6 nM S.E., n = 3), PP-2AC (0.63 +/- 0.2 nM S.E., n = 3) and SMPP-1M (5.9 +/- 1.3 S.E., n = 3). Subsequent anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis of the microcystin-biotin eluates of the three fractions revealed a complex pattern of proteins associated with PP-1C and PP-2AC. Far Western analysis and the rebinding interaction with recombinant PP-1C distinguished proteins in the eluates that bound PP-1C from those that bound PP-2AC. In Far Western analysis, 29 distinct proteins were identified to bind PP-1C. Significantly, these same proteins, plus seven others, were also recovered from the isothiocyanate eluates from microcystin-Sepharose by a rebinding interaction with PP-1C-microcystin-biotin. The number of proteins and range of novel molecular masses (18-125 kDa) identified to interact with PP-1C by these two techniques cannot be accounted for by the previously characterized subunits of PP-1. Our findings further support the concept that PP-1C is regulated in vivo by multiple and distinct substrate-targeting subunits.