Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage

The presence of a glycoinositol phospholipid anchor in Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The [125I]TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence [Hall, L.M.C., & Spierer, P. (1986) EMBO J. 5, 2949-2954] confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine.     

An Immunological Study of Rat Acetylcholinesterase: Comparison with Acetylcholinesterases from Other Vertebrates

We have examined the immunoreactivity of acetylcholinesterase from different vertebrate species with a rabbit antiserum raised against the purified rat brain hydrophobic enzyme (G4 form). We found no significant interaction with enzymes from Electrophorus, Torpedo, chicken, and rabbit. The antiserum reacted with acetylcholinesterases from the brains of the other mammalian species studied, with titers decreasing in the following order: rat = mouse greater than human greater than bovine. The serum was inhibitory with murine and human acetylcholinesterases, but not with the bovine enzyme. The inhibition was partially depressed in the presence of salt (e.g., 1 M NaCl). In those species whose acetylcholinesterase was recognized by the antiserum, both soluble and detergent-soluble fractions behaved in essentially the same manner, interacting with the same antibodies. The apparent immunoprecipitation titer was decreased in the presence of salt, and it did not make any difference whether NaCl was included in the solubilization procedure or added to the extracts. Both G1 and G4 forms of acetylcholinesterase in the soluble and detergent-soluble fractions were recognized by the antiserum, and in the case of the human enzyme, by monoclonal antibodies produced against human erythrocyte acetylcholinesterase. However, the monomer G1 showed a clear tendency to form smaller complexes and precipitate less readily than the tetramer G4. Although we cannot exclude the existence of significant differences between the various molecular forms of acetylcholinesterase, our results are consistent with the hypothesis that they all derive from the same gene or set of genes by posttranslational modifications.     

Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues

Acetylcholinesterase extracted with Triton X-100 from bovine brain caudate nuclei was purified by affinity chromatography to apparent homogeneity. The purified enzyme was labeled with [3H]diisopropyl fluorophosphate at the active sites and with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a compound which has been shown to be selective for the hydrophobic membrane-binding domains of several other proteins. The subunit structure was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate before and after disulfide reduction. After reduction, a single 3H-labeled band at 70 kDa was stained by silver, but most of the 125I label corresponded to a 20-kDa species. Prior to reduction, five 3H-labeled and silver-stained bands were apparent at 70, 140, 160, 260, and greater than 360 kDa. These species were presumed to represent monomer and disulfide-linked oligomers of 70-kDa catalytic subunits. 125I label was selectively associated with the 160-, 260-, greater than 360-, and a 90-kDa species. Quantitative gel slicing of 3H- and 125I-labeled nonreduced enzyme supported a structural model in which the tetrameric enzyme is a dimer of nonidentical catalytic subunit dimers, one of which involves a direct intersubunit disulfide linkage between two 70-kDa catalytic subunit monomers and the second of which contains two disulfide linkages through an intervening 125I-labeled 20-kDa noncatalytic subunit. This 20-kDa subunit is proposed to contain the membrane attachment site. The brain enzyme did not contain components characteristic of the glycolipid anchors of erythrocyte acetylcholinesterases. However, part of the 125I label was associated with fatty acids, indicating that at least a portion of the brain enzyme membrane anchor is composed of nonamino acid components.     

A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi

Epimastigotes of different stocks of Trypanosoma cruzi contain similar levels of proteinase activity on azocasein; amastigotes and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.     

Partial purification and reconstitution of the (Ca2+ + Mg2+)-ATPase of erythrocyte membranes.

Benzenemethane Sulfonylfluoride (329-98-6) is an irreversible inactivator of many esterases including mammalian acetylcholinesterases. However, previous reports indicated that acetylcholinesterase from the electric eel, Electrophorus electricus (EC 3.1.1.7) failed to react with benzenemethane sulfonylfluoride at measurable rates. We report here that eel acetylcholinesterase reacts with this inactivator at a low rate. Hydrolysis of the sulfonylating agent is so much faster than enzyme inactivation that, under most conditions, there will be only slight inactivation. Like the reaction of other active site acylating agents with this enzyme, inactivation can be accelerated in the presence of certain organic cations. We introduce a rate equation for enzyme sulfonylation which incorporates both the hydrolysis of the inactivator and the complication that fluoride resulting from hydrolysis of the inactivator is a potent competitive inhibitor of this enzyme. This rate equation accurately describes the time course of enzyme inactivation.     

Monoclonal Antibodies to Rat Brain Acetylcholinesterase: Comparative Affinity for Soluble and Membrane-Associated Enzyme and for Enzyme from Different Vertebrate Species

Seven unique monoclonal antibodies were generated to rat brain acetylcholinesterase. Upon density gradient ultracentrifugation, immunoglobulin complexes with the monomeric enzyme appeared as single peaks of acetylcholinesterase activity with a sedimentation coefficient approximately 3S greater than that of the free enzyme. This behavior is consistent with the assumption of one binding site per enzyme molecule. Apparent dissociation constants of these antibodies for rat brain acetylcholinesterase calculated on the basis of this assumption ranged from about 10 nM to more than 1,000 nM. Some of the antibodies were less able to bind the membrane-associated enzyme that required detergent for solubilization than the naturally soluble acetylcholinesterase of detergent-free brain extracts. Species cross-reactivity was investigated with crude brain extracts from mammals (human, mouse, rabbit, guinea pig, cow, and cat) and from other vertebrates (chicken, frog, and electric eel). Three antibodies bound rat acetylcholinesterase exclusively; one had nearly the same affinity for all mammalian acetylcholinesterases investigated; the remaining three showed irregular binding patterns. None of the antibodies recognized frog and electric eel enzyme. Pooled antibody was found to be suitable for specific immunofluorescence staining of large neurons in the ventral horn of the rat spinal cord and smaller cells in the caudate nucleus. Other potential applications of these antibodies are discussed.     

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.     

On the molecular weight and subunit composition of calf thymus ribonuclease H1

We have reinvestigated the molecular weight and subunit composition of calf thymus ribonuclease H1. Earlier studies suggested a variety of molecular weights for the enzyme in the range of 64K-84K and reported that the enzyme either was a single polypeptide of 74 kDa or consisted of from two to four subunits in the range of 21-34 kDa. Although we too find bands in this lower molecular weight range in our highly purified preparations following SDS-PAGE, our data suggest that the native structure of RNase H1 is a dimer of 68-kDa subunits. The evidence includes the following: (1) Western blot analysis of fractions taken at various stages of the purification indicates that the predominant antigenic form of the enzyme in crude extracts has a molecular weight of 68K but that during purification in the absence of sufficient protease inhibitors a variety of lower molecular weight forms appear concomitant with the disappearance of the 68-kDa band. (2) Activity gel analysis of the highly purified enzyme prepared in the presence of a battery of protease inhibitors reveals that the 68-kDa band (as well as several bands of lower molecular weight) possesses RNase H activity. (3) The 68-kDa band recognized by Western blotting with anti-RNase H immune sera is not detected by using preimmune sera. Furthermore, when immune sera are used, a trace of a 140-150-kDa antigenic form can sometimes be detected, consistent with the existence of a dimeric form of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and some properties of a cholinesterase from bush bean (Phaseolus vulgaris L.) roots.

A cholinesterase was partially purified from bush bean (Phaseolus vulgaris L.) roots by using acridinium-based ligand affinity chromatography. The procedure gave a 78-fold increase in specific activity, although at least three inactive contaminants remained. The enzyme activity was maximal against acetyl esters of choline and was inhibited by neostigmine. Di-isopropyl phosphorofluoridate completely inhibited activity at concentrations greater than 0.1 mM. The catalytic centre activity was 2 X 10(-4) times that of electric eel acetylcholinesterase. Cholinesterase activity appeared as a peak (s = 4.2 +/- 0.1 S) after isokinetic sedimentation. The Stokes radius was 4.00 nm and the apparent molecular weight was 72700 +/- 1900. The smallest active and native form of the enzyme appeared to be a monomer. This contrasts with animal acetylcholinesterases, in which the smallest active and native forms are multimeric.     

Yolk vitronectin. Purification and differences from its blood homologue in molecular size, heparin binding, collagen binding, and bound carbohydrate.

This is the first report on a unique vitronectin molecule, yolk vitronectin, which is similar to its blood homologue in cell spreading activity but different in molecular size, bound carbohydrate, and heparin and collagen binding activity. Yolk vitronectin was purified 2,500-fold from chick egg yolk by a combination of hydroxylapatite, DEAE-cellulose, and anti-vitronectin-Sepharose column chromatographies. In SDS-polyacrylamide gel electrophoresis under reducing conditions, yolk vitronectin was separated into 54- and 45-kDa bands, which are 16 and 25 kDa smaller, respectively, than the 70-kDa major band of chick blood vitronectin. The 54-kDa band shares the same NH2-terminal sequence as chick blood vitronectin. In contrast, the NH2-terminal sequence of the 45-kDa band is somewhat homologous with the internal sequences of mammalian vitronectins beginning at the 50th amino acid from the NH2 terminus. The bound carbohydrate of the 54- and 45-kDa species of yolk vitronectin is similar to, but distinct from, that of blood vitronectin. Unlike blood vitronectin, yolk vitronectin cannot bind to either heparin or collagen.     

Properties of the partially purified tonoplast H+-pumping ATPase from oat roots

Higher plant cells have one or more vacuoles important for maintaining cell turgor and for the transport and storage of ions and metabolites. One driving force for solute transport across the vacuolar membrane (tonoplast) is provided by an ATP-dependent electrogenic H+ pump. The tonoplast H+-pumping ATPase from oat roots has been solubilized with Triton X-100 and purified 16-fold by Sepharose 4B chromatography. The partially purified enzyme was sensitive to the same inhibitors (N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid, and NO-3) as the native membrane-bound enzyme. The partially purified enzyme was stimulated by Cl- (Km(app) = 1.0 mM) and hydrolyzed ATP with a Km(app) of 0.25 mM. Thus, the partially purified tonoplast ATPase has retained the properties of the native membrane-bound enzyme. [14C]DCCD labeled a single polypeptide (14-18 kDa) in the purified tonoplast ATPase preparation. Two major polypeptides, 72 and 60 kDa, that copurified with the ATPase activity and the 14-18-kDa DCCD-binding peptide are postulated to be subunits of a holoenzyme of 300-600 kDa (estimated by gel filtration). Despite several catalytic similarities with the mitochondrial H+-ATPase, the major polypeptides of the tonoplast ATPase differed in mass from the alpha and beta subunits (58 and 55 kDa) and the [14C] DCCD-binding proteolipid (8 kDa) of the oat F1F0-ATPase.     

Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.     

The Drosophila acetylcholine receptor subunit D alpha5 is part of an alpha-bungarotoxin binding acetylcholine receptor

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.     

Peptidasic Activities Associated with Acetylcholinesterase Are Due to Contaminating Enzymes

The esterasic and peptidasic activities of two different sources of acetylcholinesterase purified from electric eel were examined. Hydrolyses of leucine-enkephalin and neurotensin indicated that both sources exhibited exopeptidasic and tryptic-like activities. However, the enzyme preparation which appeared 10-fold enriched with regard to the esterasic activity was found to display a 50- and 185-fold lower tryptic-like and exopeptidasic function, respectively. This lack of parallelism in the enrichment of the various activities seemed to indicate that they were not co-purified. Immunoprecipitation experiments performed with monoclonal antibodies directed towards the catalytic subunit of globular or asymmetric forms of electric eel acetylcholinesterase allowed the physical dissociation of esterasic and peptidasic functions and therefore confirmed that the ability of acetylcholinesterase to hydrolyze various neuropeptides was likely due to contaminating peptidases.     

Purification of nitrate reductase from Nicotiana plumbaginifolia by affinity chromatography using 5'AMP-sepharose and monoclonal antibodies

Nitrate reductase was purified from leaves of Nicotiana plumbaginifolia using either 5'AMP-Sepharose chromatography or two steps of immunoaffinity chromatography involving monoclonal antibodies directed against nitrate reductase from maize and against ribulose-1,5-bisphosphate carboxylase from N. plumbaginifolia. Nitrate reductase obtained by the first method was purified 1000-fold to a specific activity of 9 units/mg protein. The second method produced an homogenous enzyme, purified 21,000-fold to a specific activity of 80 units/mg protein. SDS/PAGE of nitrate reductase always resulted in two bands of 107 and 99.5 kDa. The 107-kDa band was the nitrate reductase subunit of N. plumbaginifolia; the smaller one of 99.5 kDa is thought, as commonly reported, to result from proteolysis of the larger protein. The molecular mass of 107 kDa is close to the values calculated from the coding sequences of the two nitrate reductase genes recently cloned from tobacco (Nicotiana tabacum cv Xanthi).     

Comparison of asymmetric forms of acetylcholinesterase from the electric organ of Narke japonica and Torpedo californica

The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared. Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8. The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme from N. japonica contained more A8 than A12, while that of T. californica contained more A12. After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen-like tails. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140 000, 100 000, 70 000 and 60 000, while that from T. californica gave bands of Mr 140 000, 100 000, 70 000 and 55 000. The bands of Mr 70 000 and 140 000 were monomers and non-reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60 000 and 55 000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components. The Mr 100 000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6-8% of the two preparations. The catalytic subunits and the Mr 100 000 subunits bound concanavalin A, indicating that they are glycoproteins. The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen-like tails. The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity.     

Antigenic relatedness between phospholipases A2 from Naja naja venom and from mammalian cells

Polyclonal antiserum was prepared against phospholipase A2 from Naja naja and used to prepare a purified antibody. It cross-reacted with the antigen, and with intracellular mammalian PLA2. This antibody was immunoreactive and inhibited the PLA2 activity of Naja naja and of guinea pig alveolar macrophages or rat lymphocytes. By immunoblotting, this antiserum revealed one band of PLA2 from Naja naja (14 kDa) and 3 bands for guinea pig alveolar macrophages and rat lymphocytes (30, 45 kDa and a minor band of 14 kDa). These results show an antigenic relatedness between an extracellular PLA2 and membrane-bound PLA2 from two different mammalian species and cell types.     

Purification, characterization, and amino-terminal sequence of rat ovarian receptor for luteinizing hormone/human choriogonadotropin

The luteinizing hormone (LH)/human choriogonadotropin (hCG) receptor of rat ovary was solubilized with Lubrol PX in the presence of 20% glycerol and protease inhibitors, and purified by one-step affinity chromatography. Purified receptor had a specific hCG binding capacity of 4900 pmol/mg protein, and displayed a single class of high affinity binding sites (Ka = 6.20 X 10(9) M-1). An 11,200-fold purification over the starting crude homogenate was achieved. The purified LH/hCG receptor was identified by sodium dodecyl sulfate-gel electrophoresis and silver staining as a single protein of 92 kDa. The ability of the purified 92-kDa protein to specifically bind hormone was demonstrated by electroblotting onto Immobilon P membrane, incubation with 125I-labeled hCG, and autoradiography of the blot. In addition to a 92-kDa band, ligand blotting also yielded a 170-kDa band representing receptor dimer. Covalent cross-linking of hCG, with isotope in either the alpha- or beta-subunit, to membrane-bound receptor produced complexes that contained a single receptor component of approximately 92 kDa. The cross-linking studies indicated that both subunits interact with receptor and also suggested receptor dimer formation. Following sodium dodecyl sulfate-electrophoresis, purified receptor was electroblotted onto polyethylenimine-treated glass fiber filters for direct microsequencing in a gas-phase sequenator. Eleven cycles of sequence analysis yielded the unique sequence: NH2-Arg-Glu-Leu-Ser-Gly-Ser-Leu-XXX-Pro-Glu-Pro-COOH. These results indicate that the rat ovarian LH/hCG receptor is a protein of 92 kDa which can be easily purified in microgram amounts. This study also describes a relatively simple technique for electroblotting and microsequencing that should be applicable to other membrane-bound hormone receptors.     

Rat brain N-acetylated alpha-linked acidic dipeptidase activity. Purification and immunologic characterization

N-Acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase) is a membrane-bound metallopeptidase that cleaves glutamate from the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate. In this report, we have solubilized NAALA dipeptidase activity from synaptosomal membranes with Triton X-100 and purified it to apparent homogeneity by sequential column chromatography on DEAE-Sepharose, CM-Sepharose, and lentil lectin-Sepharose. This procedure resulted in a 720-fold purification with 1.6% yield. The purified ezyme migrated as a single silver-stained band on a sodium dodecyl sulfate gel with an apparent molecular weight of 94 kDa. Using an enzymatic stain to visualize NAALA dipeptidase activity within a gel matrix, we have confirmed that the 94-kDa band is, indeed, NAALA dipeptidase. The purified enzyme was characterized and found to be pharmacologically similar to NAALA dipeptidase activity described previously in synaptosomal membrane extracts. Using the purified NAALA dipeptidase as antigen, we have raised specific and high titer polyclonal antibodies in guinea pig. Immunocytochemical studies show intense NAALA dipeptidase immunoreactivity in the cerebellar and renal cortices.     

The leukotoxin of Pasteurella haemolytica binds to β2 integrins on bovine leukocytes

The putative receptor proteins of Pasteurella haemolytica leukotoxin were isolated from bovine polymorphonuclear neutrophil lysate by affinity chromatography on a leukotoxin-specific monoclonal antibody column to which the leukotoxin was pre-bound. SDS-PAGE of the purified proteins showed four bands at 180 kDa, 170 kDa, 150 kDa and 95 kDa, in addition to the expected 102-kDa leukotoxin band and a series of bands with molecular masses lower than 102 kDa representing the disintegrated leukotoxin. N-terminal amino acid sequencing of the 170-kDa band showed homology with human and murine CD11b. The purified proteins reacted specifically with monoclonal antibodies specific for CD11a, CD11b, CD11c (the alpha chains of beta(2) integrins), and CD18 (the beta chain of beta(2) integrins). Pre-incubation of polymorphonuclear neutrophils with a monoclonal antibody specific for CD18 reduced the cytotoxicity of the leukotoxin to the cells. These results indicate that the leukotoxin binds to the beta(2) integrins on bovine leukocytes, very likely via CD18.