Immunoaffinity purification of type I protein kinase C

We designed a simple procedure for the purification of type I protein kinase C, using immunoaffinity chromatography with a monoclonal antibody, MC-1b, obtained by rescreening hybridoma cells available for an affinity ligand. Western blotting demonstrated that MC-1b specifically reacted with type I protein kinase C, and the enzyme molecule dissociated from MC-1b-coupled Sepharose 4B with mild eluants such as thiocyanate retained the kinase activity. A 1148-fold purification was achieved and 210 micrograms of type I protein kinase C was obtained from three rabbit brains, by means of a two-step procedure, using DEAE-cellulose and immunoaffinity chromatography. The resultant preparation was homogeneous, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis hydroxylapatite chromatography, and immunological analysis using MC-1a, MC-2a, and MC-3a.     

Immunoaffinity purification of human thromboxane synthase

A recently produced monoclonal antibody against human thromboxane synthase was used to purify the enzyme from platelets in a one-step procedure with good yields. The isolated protein exhibited a single band of about 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained one heme/mol. Although the visible spectrum of the oxidized enzyme displayed a peak at 418 nm like the previously isolated enzyme after dithionite reduction and CO addition, it shifted to 419 nm but not to 450 nm where only a small shoulder could be detected. Its catalytic activity was only 1-5% of the previous preparations, but with the same Km of about 10 microM and a ratio of thromboxane B2: 12-hydroxyheptadecatrienoic acid of 1:1. Studies with EPR spectrometry and inhibitors confirmed that only a minor part of the enzyme was in its native heme-thiolate conformation, whereas the major part had been converted to the inactive P420 form by the elution procedure. The amino acid analysis revealed 46% hydrophobic residues. According to the sequence of 26 amino acids from the N-terminus and two tryptic peptides no homology to one of the cytochrome P450 monooxygenases, or to cyclooxygenase, or to prostacyclin synthase was detected.     

Immunoaffinity purification of monoclonal antibodies: In search of an elution buffer of general applicability

Summary Of a number of elution buffers tested, 4MgCl2 in 25% ethylene glycol was the most eficient for recovering active monoclonal antibodies from immunoaffinity columns. This solution may be useful as a general purpose eluant for monoclonal antibody purification.     

Immunoaffinity purification and immunoassay determination of human erythrocyte acylphosphatase

Specific anti-human erythrocyte acylphosphatase antibodies were raised in rabbits, purified by affinity chromatography, and used to develop an enzyme purification procedure based on an immunoaffinity chromatography step. This procedure permitted the rapid purification of the enzyme, with a high final yield and with a specific activity very similar to that found for the enzyme purified by the standard procedure. The noncompetitive enzyme-linked immunoadsorbent assay developed with the affinity-purified antibodies was very specific and sensitive in that a positive reaction could be detected in the presence of antigen amounts of as little as 0.01 ng/ml. By this assay the enzyme content was determined in normal cells, tissues, and organs as well as in blood samples from hemopathy-affected patients. This test could possibly have clinical applications.     

Immunoaffinity purification and fluorescence studies of human adenosine deaminase

Human thymus adenosine deaminase was isolated by using a monoclonal antibody affinity column. The highly purified enzyme produced by this rapid, efficient procedure had a molecular weight of 44,000. Quenching of the intrinsic protein fluorescence by small molecules was used to probe the accessibility of tryptophan residues in the enzyme and enzyme-inhibitor complexes. The fluorescence emission spectrum of human adenosine deaminase at 295-nm excitation had a maximum at about 335 nm and a quantum yield of 0.03. Addition of polar fluorescence quenchers, iodide and acrylamide, shifted the peak to the blue, and the hydrophobic quencher trichloroethanol shifted the peak to the red, indicating that the emission spectrum is heterogeneous. The fluorescence quenching parameters obtained for these quenchers reveal that the tryptophan environments in the protein are relatively hydrophobic. Binding of both ground-state and transition-state analogue inhibitors caused decreases in the fluorescence intensity of the enzyme, suggesting that one or more tryptophans may be near the active site. The kinetics of the fluorescence decrease were consistent with a slow conformational alteration in the transition-state inhibitor complexes. Fluorescence quenching experiments using polar and nonpolar quenchers were also carried out for the enzyme-inhibitor complexes. The quenching parameters for all enzyme-inhibitor complexes differed from those for the uncomplexed enzyme, suggesting that inhibitor binding causes changes in the conformation of adenosine deaminase. For comparison, parallel quenching studies were performed for calf adenosine deaminase in the absence and presence of inhibitors. While significant structural differences between adenosine deaminase from the two sources were evident, our data indicate that both enzymes undergo conformational changes on binding ground-state and transition-state inhibitors.     

Overexpression, purification, and preliminary X-ray crystallographic analysis of human brain-type creatine kinase

Creatine kinase (CK; E.C. 2.7.3.2) is an important enzyme that catalyzes the reversible transfer of a phosphoryl group from ATP to creatine in energy homeostasis. The brain-type cytosolic isoform of creatine kinase (BB-CK), which is found mainly in the brain and retina, is a key enzyme in brain energy metabolism, because high-energy phosphates are transferred through the creatine kinase/phosphocreatine shuttle system. The recombinant human BB-CK protein was overexpressed as a soluble form in Escherichia coli and crystallized at 22 degrees C using PEG 4000 as a precipitant. Native X-ray diffraction data were collected to 2.2 A resolution using synchrotron radiation. The crystals belonged to the tetragonal space group P43212, with cell parameters of a=b=97.963, c= 164.312 A, and alpha=beta=gamma=90 degrees. The asymmetric unit contained two molecules of CK, giving a crystal volume per protein mass (Vm) of 1.80 A3 Da-1 and a solvent content of 31.6%.     

Immunoaffinity purification of peptide hormones prior to liquid chromatography-mass spectrometry in doping controls

For most peptide hormones prohibited in elite sports the concentrations in plasma or urine are very low (pg/mL). Accordingly, hyphenated purification and enrichment steps prior to mass spectrometric detection are required to obtain sufficient doping control assays. Immunoaffinity purification in combination with nano-scale liquid chromatography coupled to high resolution/high accuracy mass spectrometry was found to have the potential of providing the necessary sensitivity and unambiguous specificity to produce reliable results. With the presented methodology 12 prohibited peptides (porcine insulin, Novolog, Apidra, Lantus DesB30-32 metabolite, Humalog and human insulin, Synacthen (synthetic ACTH analogue), luteinizing hormone-releasing hormone (LH-RH), growth hormone releasing hormone (GH-RH(1-29)) and CJC-1295 (GH-RH analogue), LongR(3)-IGF-1 and IFG-1) were simultaneously purified from plasma/serum or urine. With limits of detection for each target compound ranging in the low pg/mL level (urine), the method enables the determination of urinary peptides at physiologically relevant concentrations. For each class of peptides an appropriate antibody and a respective internal standard was implemented ensuring robust analysis conditions. Due to the fast and simple sample preparation procedure (～25 samples per day) and the fact that all materials are commercial available, the implementation of the methodology to laboratories from other analytical fields (forensics, pharmacokinetic sciences, etc.) is enabled.     

Immunoaffinity purification of the calpains

A monoclonal antibody to the small subunit common to both mu- and m-calpains can be used in an immunoaffinity column to purify either mu- or m-calpain in a proteolytically active form. Extracts in 150 mM NaCl, pH 7.5, are loaded onto a column containing the anti-28-kDa antibody; the column is washed with 500 mM NaCl, pH 7.5, and the bound calpain is eluted with 150 mM NaCl, 50 mM Tris-HCl, pH 9.5, and 1 mM EDTA. These elution conditions do not affect the proteolytic activity of either mu- or m-calpain. It is most efficient to reduce the volume and to remove any proteolytic activity from crude extracts by using successive phenyl Sepharose and ion-exchange columns before loading onto the immunoaffinity column. The column purifies m-calpain more effectively than mu-calpain; m-calpain is greater than 90% pure after a single pass through this column, whereas mu-calpain can be purified to >70% purity. The epitope for the monoclonal antibody is between amino acids 92 and 104 (numbers for human calpain) in the 28-kDa subunit. Evidently, this area is shielded in the calpain molecule in a way that affects binding of the antibody to the native molecule.     

Immunoaffinity purification and characterization of nucleotide pyrophosphatase from human placenta

Nucleotide pyrophosphatase [EC 3.6.1.9] was purified to homogeneity from human placenta using a monoclonal antibody affinity column. By sodium dodecylsulfate--polyacrylamide gel electrophoresis, the purified enzyme showed a major band at a molecular size of 130 K. The enzyme was a glycoprotein with N-linked oligosaccharides consisting of both complex- and oligomannoside-types. Substrate specificity to hydrolyze phosphodiester and phosphosulfate linkages as well as other properties were similar to those of nucleotide pyrophosphatase and phosphodiesterase from other sources.     

Immunoaffinity purification and characterization of leucine aminopeptidase from human liver

Leucine aminopeptidase was purified from human liver cytosol to homogeneity, 1538-fold, with a yield of 84.4% by immunoaffinity chromatography. Increases in the activity and the stability of the enzyme were simultaneously observed during the purification procedure, suggesting the presence of some endogenous inhibitor in cytosol. The specific activity and Km value of the enzyme for L-leucine amide were found to be 58.00 mumol/min/mg of protein and 4.02 mM, respectively, at pH 8.0. The molecular weight of the enzyme was determined to be 360,000 by both polyacrylamide gradient gel electrophoresis and Sephadex G-200 gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of native and dimethyl suberimidate cross-linked enzyme indicate that the native enzyme has two subunits of Mr 53,000 (a) and 65,000 (b) and is a hexamer arranged as a trimer of dimers (3 X (a X b)). The optimum pH was 10.5, and the enzyme was stable in the pH range from 7.5-8.5. The enzyme was activated by divalent metal ions, especially by Mg2+ and Mn2+, with no change in Km value. The enzyme was inhibited by metal-chelating agents, indicating it to be a metalloenzyme. Amastatin and bestatin strongly inhibited the enzyme, but leupeptin did not. The enzyme had a broad substrate specificity toward oligopeptides and amino acid amides but had little or no activity toward chromogenic substrates. The enzyme also could hydrolyze natural substrates contained in liver cytosol and accordingly produce many kinds of amino acids commonly found in proteins.     

Immunoaffinity purification of human prorenin produced in Chinese hamster ovary cells

A simple immunoaffinity column chromatographic procedure is described whereby recombinant human prorenin secreted from Chinese hamster ovary cells may be isolated in a high state of purity from serum-free culture medium. Prorenin thus purified has been characterized by SDS-polyacrylamide gel electrophoresis and by partial sequence analysis which has revealed the expected N-terminal sequence. Trypsin treatment gives rise to renin, and reversible acid activation has also been demonstrated for the recombinant zymogen.     

Properties of human creatine kinase isoenzymes]

Properties of human creatine kinase isoenzymes (MM, MB and BB) are investigated. The most pronounced differences in properties of these isoenzymes are found under their urea inactivation, heat denaturation and the inhibition by rabbit antisera to isoenzymes. Differences in values of the Mikhaelis constant and substrate and pH dependencies are much less pronounced. The presence of ADP stabilizes creatine kinase isoenzymes under conditions of urea and heat inactivation. Properties of hybrid MB isoenzymes are found to be intermediate with respect to MM and BB isoenzymes. A mode of the interaction of M and B subunits in dimeric molecules of creatine kinase isoenzymes is discussed.     

Characterization of isoforms of human mitochondrial creatine kinase by isoelectric focusing

High enzyme activity of mitochondrial creatine kinase (creatine-N-phosphotransferase, mCK, EC 2.7.3.2) was detected in serum from a patient with advanced carcinoma of the rectum and its isoforms were characterized by means of isoelectric focusing (IEF). Three forms of mCK, membrane-bound (pI 6.9–7.0), octameric (pI 7.0–7.9) and dimeric (pI 6.7, 6.8, 6.9 and 7.0), were detected in the fresh serum. These three forms of mCK were converted to five dimeric isoforms, and these were characterized as one reduced form (pI 7.0) and four oxidized (pI 6.6, 6.7, 6.8 and 6.9) forms upon treatment with urea, hydrogen peroxide or 2-mercaptoethanol (2-ME). The C-terminal of the mCKs was concluded to be a lysine residue because the mCKs treated with carboxypeptidase B migrated to positions closer to the anode than did those not treated with carboxypeptidase B. Therefore, four bands were concluded to represent one reduced-delysined isoform (pI 6.4) and three oxidized-delysined isoforms (pI 6.1, 6.2 and 6.3). The broad octameric mCK band disappeared and a narrow band focused at pI 6.8–6.9 appeared upon probable delysination of the mCKs. Thus, the number of lysine residues at the C-terminal of the octamer was concluded to be variable due to variable catalysis by carboxypeptidase N in the plasma. mCKs seemed to be inactivated during conversion from a membrane-bound form to dimeric oxidized-delysined forms via the octameric, dimeric reduced and oxidized forms.     

Immunoaffinity purification and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocytes

A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase （GAPDH, EC 1.2.1.12） from human erythrocytes. The comparison between this rapid method （one step） and the tra- ditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of -150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide （NAD＋）. Western blot analysis using purified monospecific polyclonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43℃ and 8.5, respectively. The kinetic parameters were also calculated： the Vmax was 4.3 U/mg and the Km values against G3P and NAD＋ were 20.7 and 17.8 μM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.     

Immunoaffinity purification of the lipid transfer protein complex directly from human plasma

The human cholesteryl ester (CE) and triglyceride (TG) exchange protein (denoted LTC or lipid transfer complex) was isolated in a single step from plasma using immunoaffinity batch extraction. Antibodies were raised against two preparations of conventionally purified LTC. LTC-I and LTC-II (purified 20,000-fold and 3500-fold, respectively) were used as immunogens. The antiLTC antibodies were isolated by anion-exchange chromatography and coupled to Affi-Gel 10. Chromatography of plasma on antiLTC Affi-Gel removed all of the CE and TG transfer activity. Moreover, LTC prepared from both antiLTC-I and antiLTC-II-Affi-Gel matrices were identical when analyzed by sodium dodecyl sulfate-polyacrylamide gel LTC electrophoresis. LTC exhibited two protein bands of Mr (apparent) 67,000 and 58,000 and a broad, faintly staining region at greater than 150,000. Analysis of LTC by immunoblotting indicated that both antiLTC-I and antiLTC-II antibodies recognized the same LTC proteins. Isoelectric focussing of LTC gave two pI values, 5.2 and 8.7. These data suggest that LTC is a complex of specific proteins and perhaps lipid. Specific CE and TG exchange activities of immunoaffinity-purified LTC were comparable, although the activities were low with respect to that of the antigen used to generate antiLTC-I. This is not due to contamination of LTC by albumin, lecithin:cholesterol acyltransferase, or apolipoproteins AI, AII, B, CIII, D, or E.     

Crystal structure of human ubiquitous mitochondrial creatine kinase

Creatine kinase (CK), catalyzing the reversible trans-phosphorylation between ATP and creatine, plays a key role in the energy metabolism of cells with high and fluctuating energy requirements. We have solved the X-ray structure of octameric human ubiquitous mitochondrial CK (uMtCK) at 2.7 A resolution, representing the first human CK structure. The structure is very similar to the previously determined structure of sarcomeric mitochondrial CK (sMtCK). The cuboidal octamer has 422 point group symmetry with four dimers arranged along the fourfold axis and a central channel of approximately 20 A diameter, which extends through the whole octamer. Structural differences with respect to sMtCK are found in isoform-specific regions important for octamer formation and membrane binding. Octameric uMtCK is stabilized by numerous additional polar interactions between the N-termini of neighboring dimers, which extend into the central channel and form clamp-like structures, and by a pair of salt bridges in the hydrophobic interaction patch. The five C-terminal residues of uMtCK, carrying positive charges likely to be involved in phospholipid-binding, are poorly defined by electron density, indicating a more flexible region than the corresponding one in sMtCK. The structural differences between uMtCK and sMtCK are consistent with biochemical studies on octamer stability and membrane binding of the two isoforms.     

Aggregation and folding of recombinant human creatine kinase

The processes of aggregation and refolding of recombinant human creatine kinase (rHCK) were studied. Most of the rHCK expressed in E. coli was present in the insoluble traction and it could be solubilized in 6 M urea solution. Unfolding of rHCK in 6 M urea showed biphasic kinetic courses (kappa1 = 6.5 x 10(-3) s(-1); kappa2 = 0.54 x 10(-3) s(-1)) as observed by maximum fluorescence wavelength change. During refolding of the rHCK dissolved in urea, significant aggregation was noticed following first-order kinetics. Aggregation rate constants were influenced by the concentration of NaCl, which increased the difference in transition-free energy (deltadeltaG), showing that stabilization of folding intermediates by NaCl could efficiently reduce the formation of insoluble aggregates. Formations of aggregate were also reduced by adjusting temperature, pH, and concentration of rHCK. Refolding of rHCK under the optimized condition which prevented the aggregation also showed multi-kinetic phases (kappa1 = 3.0 x 10(-3) s(-1); kappa2 = 0.64 x 10(-3) s(-1)). Under optimized conditions applied in this study, rHCK could correctly refold retrieving the high specific enzymatic activity.