Protein Band Of Molecular Mass Research Articles

A novel diglycosidase for the transformation of naringin to naringenin and neohesperidose

A novel fungal diglycosidase that transforms naringin into naringenin and neohesperidose, a rare biotransformation, has been purified to homogeneity using a simple procedure involving precipitation of the enzyme from the culture filtrate of the fungal strain using 80 % saturation of ammonium sulphate, dissolving the precipitate in minimum volume of the buffer and dialysing that against the buffer. The purified enzyme gives single protein bands of molecular mass 64.6 kDa in SDS-PAGE analysis. The purity of the enzyme has been further confirmed by the appearance of single protein band in native page analysis. Using naringin as the substrate, the diglycosidase has Km and kcat values of 0.20 m mol L−1 and 0.66 s−1, respectively, at pH 4.0 and 313 K. The specific activity of the purified enzyme using naringin as the natural substrate is 1.018 katal/kg. The diglycosidase also transforms rutin into quercetin and rutinose but has no effect on hesperidin. The feasibilities of preparing neohesperidose from naringin and rutinose from rutin on milligram scales using the pure enzyme have been demonstrated. These results open the way for developing an enzymatic process for preparation of neohesperidose from naringin. The reported diglycosidase has immense future applications in food and pharmaceutical industries.

Production of a murine mAb against Bothrops alternatus and B. neuwiedi snake venoms and its use to isolate a thrombin-like serine protease fraction

Accidents with snakes from the genus Bothrops represent ~90 % of all snakebites in Brazil. Monoclonal antibodies (mAbs) targeting venom components can be important assets for treating envenoming syndromes, for developing diagnostic tests and for research purposes. Therefore, in this study, we aimed to generate murine mAbs against the antigenic mixture of Bothropic venoms traditionally used as immunogen to produce Bothropic antivenoms in Brazil. ELISA showed that one of the produced mAbs recognizes B. alternatus and B. neuwiedi venoms (mAb anti-Ba/Bn) specifically and Western Blot revealed that this mAb binds to a single protein band of molecular mass of ≈50 kDa. MAb anti-Ba/Bn inhibited the coagulant activity but was unable to neutralize hemorrhagic and phospholipase A2 activities caused by the B. neuwiedi venom. MAb anti-Ba/Bn was immobilized to Sepharose beads and used for immunoaffinity chromatography of B. neuwiedi venom. Proteolytic activity assays indicated that the immunoaffinity-purified fraction (BnF-Bothrops neuwiedi fraction) has a serine protease thrombin-like profile, which was supported by coagulability assays in mice. Bottom-up proteomic analysis confirmed the prevalence of serine proteases in BnF using label-free quantification. In conclusion, this work characterized a mAb with neutralizing properties against B. neuwiedi coagulant activity and demonstrates that immunoaffinity chromatography using mAbs can be a useful technique for purification of bioactive toxic proteins from Bothrops spp. snake venoms.

Role of Musa paradisiaca ascorbate peroxidase in the transformation of methyl phenyl sulfide to its sulfoxide

An ascorbate peroxidase from a new source Musa paradisiaca leaf juice has been purified to homogeneity using a simple procedure involving concentration by ultra filtration and anion exchange chromatography on diethyl amino ethyl [DEAE] cellulose column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis [SDS-PAGE] analysis of the purified enzyme has shown a single protein band of molecular mass 208.9 kDa which has been confirmed by native-PAGE and intact mass analysis by mass spectrometry. The Km and kcat values of the enzyme using ascorbate and H2O2 as the variable substrates were 0.13 m mol L−1, 40.42 s−1 and 0.23 m mol L−1, 27.24 s−1, respectively. The pH and temperature optima of the enzyme were 7.0 and 298 K, respectively. The enzyme transformed approximately 97% methyl phenyl sulfide to its sulfoxide. The product was racemic mixture.

Purification and Characterization of Glutathione Binding Protein GsiB from Escherichia coli.

Objectives To purify and characterize the glutathione binding protein GsiB of glutathione importer (GSI) in Escherichia coli (E. coli). Results The coding sequence of GsiB was cloned from E. coli MG1655 and expressed in BL21(DE3). GsiB protein was expressed and purified to homogeneity using Ni-affinity and gel filtration chromatography. SDS-PAGE of purified GsiB showed a single protein band of molecular mass 56 kDa, while native gel showed two bands around 56 kDa and 110 kDa. Gene knockout showed that GsiB was essential for GSI mediated glutathione import. Interactions of GsiA, B, C, and D were determined using bacterial two-hybrid method. Without glutathione, GsiB showed no direct interaction with the other three proteins. However, GsiB could interact with GsiC and GsiD when using glutathione as sole sulfur source. Conclusions GsiB functions in E. coli was characterized which could help elucidate the glutathione import mechanism in gram-negative bacteria.

Vaccine Potential and Diversity of the Putative Cell Binding Factor (CBF, NMB0345/NEIS1825) Protein of Neisseria meningitidis.

The cbf gene from Neisseria meningitidis strain MC58 encoding the putative Cell Binding Factor (CBF, NMB0345/NEIS1825) protein was cloned into the pRSETA system and a ~36-kDa recombinant (r)CBF protein expressed in Escherichia coli and purified by metal affinity chromatography. High titres of rCBF antibodies were induced in mice following immunization with rCBF-saline, rCBF-Al(OH)3, rCBF-Liposomes or rCBF-Zwittergent (Zw) 3–14 micelles, both with and without incorporated monophosphoryl lipid A (MPLA) adjuvant. Anti-rCBF sera reacted in western blots of meningococcal lysates with a single protein band of molecular mass ~29.5 kDa, indicative of mature CBF protein, but did not react with a lysate of a Δnmb0345 mutant (CBF-), demonstrating specificity of the murine immune responses. CBF protein was produced by all strains of meningococci studied thus far and the protein was present on the surface of MC58 (CBF+) bacteria, but absent on Δnmb0345 mutant (CBF-) bacteria, as judged by FACS reactivity of anti-rCBF sera. Analysis of the NEIS1825 amino acid sequences from 6644 N. meningitidis isolates with defined Alleles in the pubmlst.org/Neisseria database showed that there were 141 ST types represented and there were 136 different allelic loci encoding 49 non-redundant protein sequences. Only 6/6644 (<0.1%) of N. meningitidis isolates lacked the nmb0345 gene. Amongst serogroup B isolates worldwide, ~68% and ~20% expressed CBF encoded by Allele 1 and 18 respectively, with the proteins sharing >99% amino acid identity. Murine antisera to rCBF in Zw 3–14 micelles + MPLA induced significant serum bactericidal activity (SBA) against homologous Allele 1 and heterologous Allele 18 strains, using both baby rabbit serum complement and human serum complement (h)SBA assays, but did not kill strains expressing heterologous protein encoded by Alelle 2 or 3. Furthermore, variable bactericidal activity was induced by murine antisera against different meningococcal strains in the hSBA assay, which may correlate with variable surface exposure of CBF. Regardless, the attributes of amino acid sequence conservation and protein expression amongst different strains and the ability to induce cross-strain bactericidal antibodies indicates that rCBF could be a potential meningococcal vaccine antigen and merits further testing.

IgE response to two new allergen proteins of Solanum melongena L. (eggplant)

A number of allergens from eggplant (Solanum melongena L.) have been previously identified. In this study, we could detect IgE reactivity of two allergic subjects' sera towards two protein bands of molecular mass of about 35 and 15kDa. As IgE were reactive to both raw and cooked eggplant extracts, a heat-stable nature of these novel allergens is apparent.

Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65°C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.

PURIFICATION AND CHARACTERIZATION OF MUTANASE PRODUCED BY Paenibacillus curdlanolyticus MP-1

Mutanases are enzymes that catalyze hydrolysis of α-1,3-glucosidic bonds in various α-glucans. One of such glucans, mutan, which is synthesized by cariogenic streptococci, is a major virulence factor for induction of dental caries. This means that mutan-degrading enzymes have potential in caries prophylaxis. In this study, we report the purification, characterization, and partial amino acid sequence of extracellular mutanase produced by the MP-1 strain of Paenibacillus curdlanolyticus, bacterium isolated from soil. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular mass 134 kD, while native gel filtration chromatography confirmed that the enzyme was a monomer of 142 kD. Mutanase showed a pH optimum in the range from pH 5.5 to 6.5 and a temperature optimum around 40–45°C. It was thermostable up to 45°C, and retained 50% activity after 1 hr at 50°C. The enzyme was fully stable at a pH range of 4 to 10. The enzyme activity was stimulated by the addition of Tween 20, Tween 80, and Ca2+, but it was significantly inhibited by Hg2+, Ag+, and Fe2+, and also by p-chloromercuribenzoate, iodoacetamide, and ethylenediamine tetraacetic acid (EDTA). Mutanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-1,3-glucans. It also showed binding activity to insoluble α-1,3-glucans. The N-terminal amino acid sequence was NH2-Ala-Gly-Gly-Thr-Asn-Leu-Ala-Leu-Gly-Lys-Asn-Val-Thr-Ala-Ser-Gly-Gln. This sequence indicated an analogy of the enzyme to α-1,3-glucanases from other Paenibacillus and Bacillus species.

Purification of a 19-kDa pore-forming cytolysin from the sea anemone Heteractis magnifica

Pore-forming cytolysins of 19 kDa from sea anemones present a remarkable cytolytic property. In the present work, a purified 19-kDa cytolysin was obtained from the sea anemone Heteractis magnifica. The purification steps involved ammonium sulfate precipitation and subsequently desalting by dialysis against 10 mM sodium phosphate buffer (pH 7.4), followed by anion exchange chromatography in DEAE-Sepharose® column (GE Healthcare, Sweden) and gel filtration chromatography using Sephadex® G-50 matrix (GE Healthcare, Sweden). The active fractions from the gel filtration chromatography were pooled and rechromatographed in the same column. The final active fraction showed a prominent protein band of molecular mass of 19 kDa when analyzed by SDS-PAGE.

Proteinase inhibitory activity of sarcoplasmic proteins from threadfin bream (Nemipterus spp.)

Thailand is the second largest surimi producer in the world and 50% of surimi is produced from threadfin bream. During surimi processing, sarcoplasmic proteins are removed through water washing and discarded in the waste stream. This study was aimed at investigating the proteinase inhibitory activity of sarcoplasmic proteins. Sarcoplasmic proteins from threadfin bream (TBSP) exhibited inhibitory activity toward trypsin but did not inhibit papain and chymotrypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition stained by trypsin inhibitory activity revealed three protein bands of molecular mass of 95, 41 and 37 kDa. Inhibitory activity of TBSP reached a maximum when subjected to 45 degrees C and completely disappeared at 60 degrees C. The breaking force and deformation of lizardfish surimi gel with added TBSP and pre-incubated at 37 degrees for 20 min increased with additional levels of TBSP (P < 0.05). Trichloroacetic acid-oligopeptide content of lizardfish surimi gel with added TBSP decreased with the addition of 4 g kg(-1) TBSP (P < 0.05). Retention of myosin heavy chain (MHC) increased when TBSP concentration was increased. TBSP effectively protected MHC from proteolysis at 37 degrees C to a similar extent as egg white powder, but efficacy of TBSP was not observed at 65 degrees C. TBSP could be applied to reduce proteolytic degradation of lizardfish surimi or other surimi associated with trypsin-like proteinase, rendering an improvement in surimi gelation set at 37-40 degrees C.

Purification and characterization of endo-beta-1,4 mannanase from Aspergillus niger gr for application in food processing industry.

A thermostable extracellular beta-mannanase from the culture supernatant of a fungus Aspergillus niger gr was purified to homogeneity. SDS-PAGE of the purified enzyme showed a single protein band of molecular mass 66 kDa. The beta- mannanase exhibited optimum catalytic activity at pH 5.5 and 55 degrees C. It was thermostable at 55 degrees C, and retained 50% activity after 6 h at 55 degrees C. The enzyme was stable at a pH range of 3.0 to 7.0. The metal ions Hg(2+), Cu(2+), and Ag(2+) inhibited complete enzyme activity. The inhibitors tested, EDTA, PMSF, and 1,10-phenanthroline, did not inhibit the enzyme activity. N-Bromosuccinimide completely inhibited enzyme activity. The relative substrate specificity of enzyme towards the various mannans is in the order of locust bean gum>guar gum>copra mannan, with K(m) of 0.11, 0.28, and 0.33 mg/ml, respectively. Since the enzyme is active over a wide range of pH and temperature, it could find potential use in the food-processing industry.

Differential subcellular distribution and colocalization of microsomal and soluble epoxide hydrolases in rat cortical astrocytes.

The purpose of this study was to determine the expression, localization and subcellular distribution of microsomal (mEH) and soluble (sEH) epoxide hydrolases in cultured rat cortical astrocytes. Cultures of astrocytes were probed for mEH and sEH by immunocytochemistry, immunoblotting and enzymatic assays. Our results showed immunofluorescence for mEH and sEH, which colocalized with the astroglial cytoskeletal marker, glial fibrillary acidic protein (GFAP). Western blotting analyses for mEH revealed a protein band of molecular mass ∼50 kDa, the intensity of which was increased about 1.5‐fold, especially in the microsomal fraction. Whereas the polyclonal sEH rabbit serum recognized a protein band of molecular mass ∼62 kDa, which increased 2.0 fold in the 105,000xg supernatant over the cell lysate. The sEH rabbit serum also recognized similar protein bands in mitochondrial and nuclear fractions. The enzymatic analyses, using mEH‐ and sEH‐selective substrates further corroborated these findings. Our results suggest that astroglial cells, by expressing both mEH and sEH, may play a role in the cerebrovascular functions affected by epoxides (Funding Support: G12RR03045‐17 (ACS) and R37ES02710 (BDH)).

Active bovine selenophosphate synthetase 2, not having selenocysteine

During the course of studying selenocysteine (Sec) synthesis mechanisms in mammals, we prepared selenophosphate synthetase (SPS) from bovine liver by 4-step chromatography. In the last step of chromatography of hydroxyapatite, we found a protein band of molecular mass 33 kDa on SDS-PAGE, consistent with the pattern of SPS activity that was indirectly manifested by [(75)Se]Sec production activity; however, we could not detect significant Se content in this active fraction. We also found a clear band of 33 kDa by Western blotting with antibody against a common peptide (387-401) in SPS2. We detected selenophosphate as the product of this active enzyme in the reaction mixture, composed of ATP, [(75)Se]H(2)Se and SPS. Chemically synthesized selenophosphate plays a role in Sec synthesis, not the addition of this enzyme. These results support that the product of SPS2 is selenophosphate itself. During this investigation, the probable sequence of bovine SPS2 not having Sec was reported in the blast information and the molecular mass was near with the protein in this report. Thus, bovine active SPS2 of molecular mass 33 kDa does not contain Sec.

CDNA for phytoene desaturase in Dunaliella salina and its expressed protein as indicators of phylogenetic position of the β‐carotene biosynthetic pathway

AbstractThe desaturations of phytoene are indispensable reaction phases to carotenogenesis. Full‐length cDNA for phytoene desaturase (PDS) in the chlorophyte Dunaliella salina was cloned by reverse transcriptase–polymerase chain reaction and rapid amplification of cDNA ends. The cDNA was expressed in Escherichia coli to produce an expected protein band of molecular mass 65 kDa, as well being expressed in another color complementary E. coli system in which the original phytoene desaturase coding gene crtI of the modified plasmid pACCAR16 ▵ IX was inactivated/complemented. But the colony of the E. coli system did not display the orange color which in the positive control colony appeared to be due to the accumulation of β‐carotene. It is deduced that the expressed enzyme from the green algae could only partially functionally complement the deleted bacterium CrtI, which means another carotene desaturase (ζ‐carotene desaturase) is required for formation of lycopene in D. salina, and the β‐carotene biosynthesis pathway in this alga is similar with that in cyanobacteria and higher plants. The presence of the cloned cDNA and its expressed phytoene desaturase indicated the phylogenetic position of the β‐carotene biosynthesis pathway of D. salina in the molecular evolution of carotenoid biosynthesis from bacteria to plants. Copyright © 2007 Society of Chemical Industry

Biochemical analysis of neomycin-resistance in the methanoarchaeon Methanothermobacter thermautotrophicus and some implications for energetic processes in this strain

Methanogenesis-driven ATP synthesis in a neomycin-resistant mutant of Methanothermobacter thermautotrophicus (formerly Methanobacterium thermoautotrophicum strain ΔH) was strongly inhibited at both pH 6.8 and pH 8.5 by the uncoupler 3,3′,4′,5 -tetrachorosalicylanilide (TCS) in the presence of either 1 or 10 mM NaCl. The generation of a membrane potential in the mutant cells at pH 6.8 was also strongly inhibited by TCS in the presence of 1 or 10 mM NaCl. On the other hand, at pH 8.5 in the presence of 10 mM NaCl, a protonophore-resistant membrane potential of approximately 150 mV was found. These results indicate that in the mutant cells the process of energy transduction between methanogenesis and membrane potential generation is not impaired. In contrast to the wild-type strain, ATP synthesis in the mutant cells was driven by an electrochemical gradient of H + under alkaline conditions. Unlike wild-type cells, the mutant lacks the capacity to transduce an uncoupler-resistant membrane potential energy at pH 8.5 into ATP synthesis. Na +/H + exchange was comparable in the wild type and the mutant cells. Western blots of sub-cellular fractions with polyclonal antiserum reactive to the B-subunit of the halobacterial A-type H +-translocating ATPase confirmed the presence of A-type ATP synthase in the mutant cells. Furthermore, in the mutant cells a protein band of molecular mass about 45 kDa is absent but there was an abundant protein band at about 67 kDa. Based on the observed bioenergetic features of the mutant cells, neither the A 1A o ATP synthase alone nor together with the Na +/H + antiporter seems to be responsible for ATP synthesis driven by sodium motive force. Rather, some other links between neomycin-resistance and failure of sodium motive force-dependent ATP synthesis in the neomycin resistant mutant are discussed.

ISOLATION AND PARTIAL CHARACTERIZATION OF CATALYTIC ANTIBODIES WITH OLIGONUCLEASE ACTIVITY FROM BOVINE COLOSTRUM

ABSTRACT Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA fromEscherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75°C for 52 min.

Ca2+/calmodulin dependent protein kinase from Mycobacterium smegmatis ATCC 607.

A soluble Ca2+/calmodulin dependent protein kinase has been partially purified (approximately 400 fold) from Mycobacterium smegmatis ATCC 607 using several purification steps like ammonium sulphate precipitation (30-60%), Sepharose CL-6B gel filtration, DEAE-cellulose and finally calmodulin-agarose affinity chromatography. On SDS-PAGE, this enzyme preparation showed a major protein band of molecular mass 35 kD and its activity was dependent on calcium, calmodulin and ATP when measured under saturating histone IIs (exogenous substrate) concentration. Phosphorylation of histone IIs was inhibited by W-7 (calmodulin inhibitor) and KN-62 (CaM-kinase inhibitor) with IC50 of 1.5 and 0.25 microm respectively, but was not affected by inhibitors of PKA (Sigma P5015) and PKC (H-7). All these results confirm that purified enzyme is Ca2+/calmodulin dependent protein kinase of M. smegmatis. The protein kinase of M. smegmatis demonstrated a narrow substrate specificity for both exogenous as well as endogenous substrates. These results suggest that purified CaM-kinase must be involved in regulating specific function(s) in this organism.

Microsomal Alcohol Oxygenase: Purification and Characterization of a Cytochrome P450 Responsible for Oxidation of 7-Hydroxy-Δ8-tetrahydrocannabinol to 7-Oxo-Δ8-tetrahydrocannabinol in Guinea Pig Liver

Guinea pig hepatic enzyme, microsomal alcohol oxygenase, was able to oxidize both 7α- and 7β-hydroxyΔ8-tetrahydrocannabinol (7α- and 7β-hydroxy-Δ8-THC) to 7-oxo-Δ8-THC. A cytochrome P450, named P450GPF-B, which mediates this oxidative metabolism was purified from hepatic microsomes of untreated female guinea pigs. The purified enzyme showed a single protein band of molecular mass 50,000 on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequence of P450GPF-B is highly homologous with those of several cytochrome P450s belonging to the CYP3A subfamily.18O derived from atmospheric oxygen was incorporated into 31 and 6%, respectively, of 7-oxo-Δ8-THC formed from 7α- and 7β-hydroxy-Δ8-THC when the substrates were incubated with P450GPF-B under18O2. The antibody against P450GPF-B significantly suppressed the oxidative activities of 7α- and 7β-hydroxy-Δ8-THC to 7-oxo-Δ8-THC in hepatic microsomes of guinea pig. These results indicate that P450GPF-B is a major enzyme responsible for the hepatic microsomal alcohol oxygenase activities in the guinea pig.

Evidence for the involvement of a 66 kDa membrane protein in the synthesis of sterolglucoside in Saccharomyces cerevisiae.

The membrane-bound sterolglucoside synthase from the yeast Saccharomyces cerevisiae has been solubilized by nonionic detergent, Nonidet P-40, Triton X-100, and partially purified by DEAE-cellulose column chromatography and ammonium sulfate fractionation. SDS/PAGE of the purified fraction revealed the presence of two protein bands of molecular mass 66 kDa and 54 kDa. In an attempt to identify further the polypeptide chain of sterolglucoside synthase, the partially purified enzyme was treated with [di-125I]-5-[3-(p-azidosalicylamide)]allyl-UDPglucose, a photoactive analogue of UDP glucose, which is a substrate for this enzyme. Upon photolysis the 125I-labeled probe was shown to link covalently to the 66 kDa protein. The photoinsertion was competed out by the presence of unlabeled UDPglucose thus suggesting that this protein contains substrate binding site for UDPglucose. Since photoinsertion of the probe to protein of 66 kDa correlates with the molecular mass of the protein visualized upon enzyme purification we postulate that the 66 kDa protein is involved in sterolglucoside synthesis in yeast.

Identification and partial purification of (Ca2+ or Mg2+)-ATPase in renal brush-border membranes.

The protein responsible for the (Ca2+ or Mg2+)-ATPase activity in brush-border membranes from pig kidney tubular cells was characterized to distinguish this enzyme from the N-ethylmaleimide-sensitive Mg(2+)-ATPase, also present in renal brush borders. Both enzymes are clearly different in their pH optimum and their sensitivity to divalent cations, nucleoside 5'-triphosphates and inhibitors. Solubilization of the (Ca2+ or Mg2+)-ATPase from brush-border membrane vesicles was accomplished with Nonidet P-40 or dodecylmaltoside. However, simultaneous inactivation of the enzyme was inevitable. A tenfold enrichment of the ATPase activity was obtained by chromatofocusing of Nonidet-P-40-solubilized brush borders. A similar degree of purification was achieved by ion-exchange chromatography of dodecylmaltoside-solubilized preparations. From the SDS/polyacrylamide gels of partially purified (Ca2+ or Mg2+)-ATPase, a few protein bands could still be tentatively identified as responsible for the enzyme activity. Labeling of solubilized brush-border preparations with several radioactive ATP analogues also revealed that a protein band of molecular mass 90 kDa is the most probable candidate for the catalytic peptide of the (Ca2+ or Mg2+)-ATPase. Finally, immunoprecipitation as well as semi-dry blotting with antibodies generated against partially purified enzyme preparations, confirmed that a 90-kDa component is a reasonable candidate for the (Ca2+ or Mg2+)-ATPase in renal brush-border membranes.