FPLC Anion-exchange Chromatography Research Articles

Production, purification and biochemical characterization of a novel detergent-stable serine alkaline protease from Bacillus safensis strain RH12

A novel extracellular protease (SAPRH) was hyper-produced (9000 U/mL) from Bacillus safensis RH12, a newly isolated enzyme from a Tunisian offshore oil field. The enzyme was purified to homogeneity, using salt-precipitation, heat-treatment and FPLC anion-exchange chromatography. The purified enzyme was a monomer of molecular mass of ~28 kDa. The NH2-terminal 23 amino-acid sequence of SAPRH showed high homology with those of Bacillus-proteases. SAPRH displayed optimal activity at pH 9 and 60 °C. It was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), indicating that it belongs to the serine-proteases family. Moreover, SAPRH was extremely stable at a broad range of temperature and pH retaining 85% of its activity at 50 °C and 75% at pH 11. The enzyme exhibited excellent stability and compatibility with surfactants and commercial detergents, revealing 90% stability with SDS and 100% stability with Class commercial laundry detergent. One of the most distinctive properties is its catalytic efficiency, which is higher than that of Alcalase 2.5 L, typeDX (commercial enzyme) and SAPB from B. pumilus CBS. Interestingly, the results of the wash performance analysis demonstrated considerably good de-staining at 40 °C for 30 min with low supplementation (500 U/mL). Accordingly, such a protease could be considered as a good detergent-additive in detergent industry.

Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest

The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22h of incubation at 45°C was 3000U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40–60%)-dialysis followed by heat-treatment (70°C for 30min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29kDa. The sequence of the 25 NH2-terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60°C, respectively. Its half-life times at 70 and 80°C were 8 and 4h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis.

Novel serine keratinase from Caldicoprobacter algeriensis exhibiting outstanding hide dehairing abilities

The current paper reports on the purification of an extracellular thermostable keratinase (KERCA) produced from Caldicoprobacter algeriensis strain TH7C1T, a thermophilic, anaerobic bacterium isolated from a hydrothermal hot spring in Algeria. The maximum keratinase activity recorded after 24-h of incubation at 50°C was 21000U/ml. The enzyme was purified by ammonium sulfate precipitation-dialysis and heat treatment (2h at 50°C) followed by UNO Q-6 FPLC anion exchange chromatography, and submitted to biochemical characterization assays. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI–TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 33246.10Da. The sequence of the 23 N-terminal residues of KERCA showed high homology with those of bacterial keratinases. Optimal activity was achieved at pH 7 and 50°C. The enzyme was completely inhibited by phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP), which suggests that it belongs to the serine keratinase family. KERCA displayed higher levels of hydrolysis and catalytic efficiency than keratinase KERQ7 from Bacillus tequilensis strain Q7. These properties make KERCA a potential promising and eco-friendly alternative to the conventional chemicals used for the dehairing of goat, sheep, and bovine hides in the leather processing industry.

A “heavy metal sponge” for selective removal and recovery of toxic or precious metals from water (844.8)

Metallothionein (MT) is a low molecular weight metal binding protein. It has the unique ability to selective bind trace amounts of toxic heavy metals such as lead and cadmium. It does not bind essential metals such as sodium or calcium. We are developing a novel patented “heavy metal sponge” technology for the removal and recovery of toxic, e.g., lead and mercury, and precious metal, e.g., gold and platinum, from water. MT also has the ability to bind the lanthanides lanthanum and europium. Recombinant MT was cloned into pET vectors with and without a Pel secretory sequence, pPMpel, and pCODAmt, respectively. The protein is expressed in bacteria and purified to near homogeneity from cell lysates by a simple and cost effective method using FPLC anion exchange chromatography. The purity of the preparation was confirmed by SDS‐PAGE. Expression of MT and secretion of the protein using the pPMpel construct is pH dependent. The level of expression from both constructs was the same for IPTG ranging from 0.5 to 2.0 mM. The purified MT was used to determine the potential for removing trace amounts of heavy metal from seawater. The protein was mixed with seawater containing 100 ppt (trillion) of 109Cd. The protein/metal complex was then collected using a membrane filter centrifugal device and the flow through assayed for radioactivity. The efficiency of metal removal was determined from the radioactivity in the flow through. Under these conditions, 88% of the metal was removed from the seawater after a single centrifugation.Grant Funding Source: Funded by a grant from CSUPERB

A Lectin with Highly Potent Inhibitory Activity toward Breast Cancer Cells from Edible Tubers of Dioscorea opposita cv. Nagaimo

A 70-kDa galactose-specific lectin was purified from the tubers of Dioscorea opposita cv. nagaimo. The purification involved three chromatographic steps: anion exchange chromatography on a Q-Sepharose column, FPLC-anion exchange chromatography on a Mono Q column, and FPLC-gel filtration on a Superdex 75 column. The purified nagaimo lectin presented as a single 35-kDa band in reducing SDS-PAGE while it exhibited a 70-kDa single band in non-reducing SDS-PAGE suggesting its dimeric nature. Nagaimo lectin displayed moderate thermostability, retaining full hemagglutinating activity after heating up to 62°C for 30 minutes. It also manifested stability over a wide pH range from pH 2 to 13. Nagaimo lectin was a galactose-specific lectin, as evidenced by binding with galactose and galactose-containing sugars such as lactose and raffinose. The minimum concentration of galactose, lactose and raffinose required to exert an inhibitory effect on hemagglutinating activity of nagaimo lectin was 20 mM, 5 mM and 40 mM, respectively. Nagaimo lectin inhibited the growth of some cancer cell lines including breast cancer MCF7 cells, hepatoma HepG2 cells and nasopharyngeal carcinoma CNE2 cells, with IC50 values of 3.71 µM, 7.12 µM and 19.79 µM, respectively, after 24 hour treatment with nagaimo lectin. The induction of phosphatidylserine externalization and mitochondrial depolarization indicated that nagaimo lectin evoked apoptosis in MCF7 cells. However, the anti-proliferative activity of nagaimo lectin was not blocked by application of galactose, signifying that the activity was not related to the carbohydrate binding specificity of the lectin.

Purification, physico-chemical and kinetic properties of the deglycosylated Talaromyces thermophilus lipase

The Talaromyces thermophilus strain produces only one form of lipase called TTLI. When the culture medium was concentrated and stored at 4°C during a few days, we noticed the appearance of a second short form of lipase named TTLII. This second form was purified to homogeneity using gel filtration and FPLC-Anion exchange chromatography. The NH2-terminal 24 amino acid residues were found to be identical to those of TTLI. The treatment of the TTLI with endoglycosidase H decreased its apparent molecular weight from 39 to 30kDa which corresponds to the molecular weight of TTLII. This difference was mostly attributed to the N-glycosylation of the enzyme. In fact, the glycan chain content and concavaline A–Sepharose affinity column confirmed that the TTLII was completely deglycosylated. Compared to TTLI, the TTLII activity was completely decreased over a broad range of temperature and pH. Furthermore, the deglycosylation of the enzyme reduced its specific activity by 50% toward different substrates; strongly suggest that the N-glycans are determinants for optimal catalytic activity and thermal stability of this enzyme. Covalent immobilization of the enzymes on supports suggests the involvement of the glycan moiety in enzyme–polymer interactions. In the case of TTLI the glycan moiety can constitute an extra site for the covalent linkage of the enzyme on the carrier.

Purification and properties of a phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279

An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The enzyme was purified about 66-fold with a recovery of 27%. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Michaelis constant (KM) and turnover number (kcat ) for sodium phytate at pH 5.0 and 50°C were calculated from the Lineweaver-Burk plot to be 760 µM and 4.14s-1, respectively. The enzyme showed narrow substrate specificity and not phytate, but GTP was dephosphorylated with the highest relative rate of hydrolysis. However, according to the kcat/KM values, phytate was concluded to be the in vivo substrate of the enzyme. Optimal activity was determined at pH 4.5 and 45-55°C. The enzyme was strongly inhibited by Fe3+, Cu2+, Zn2+, molybdate, vanadate, fluoride and phosphate (1 mM).

A simple purification procedure of biologically active recombinant human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of 80.1% by ammonium sulfate precipitation and a single chromatographic step involving FPLC-anion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from 21.5∼29 kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.

Functional characterization of the purified holo form of hemoglobin I from Lucina pectinata overexpressed in Escherichia coli.

The clam Lucina pectinata inhabits the sulfide-rich west coast of the island of Puerto Rico. It contains three different hemoglobins. Hemoglobin I (HbI), which is monomeric at all concentrations, carries H2S in its ferric state. Overexpression of recombinant HbI from Lucina pectinata in BL21STAR(DE3) Escherichia coli cells was performed in the presence of delta-aminolevulinic acid (delta-ALA). Purification of the protein was achieved using FPLC anion exchange and size exclusion chromatography. Functional characterization of the recombinant holo-protein was assessed by detection of the protein heme O2, CO, and H2S derivatives by UV-Vis spectroscopy, with Soret maxima at 416, 421, and 425 nm, respectively. The results indicated that the recombinant HbI binds H2S and forms a heme sulfide complex like the HbI wild-type hemoglobin. Kinetic measurements were performed to determine the H2S affinity of the recombinant HbI. The H2S dissociation and association rate constants were 0.055 x 10(-3)s(-1) and 0.068 x 10(5) M(-1)s(-1), respectively. The H2S affinity constant of the recombinant HbI (0.124 x 10(9) M(-1)) is eightfold lower than that of the native clam HbI reported earlier. This effect is attributed mostly to the first of two missense mutations [Met 61 (E4)-->Val 61 and Ile101 (FG4)-->Val 101] and additional amino acids present in our construct as demonstrated by measurements of the association rate with a new construct lacking most of the additional residues and the missense mutations. The elimination of these residues restores the similarity between the expressed and wild-type hemoglobins, as evidenced by H2S association kinetics. A pH dependence on the H2S association rate was also contributing to the overall affinity constant and was taken into account in the measurements of the functional properties of the new HbI construct.

Biochemical characterization of partially purified gaba:pyruvate transaminase from Nicotiana tabacum

Pyruvate-dependent 4-aminobutyrate transaminase (EC 2.6.1.19) activity in crude extracts or lysed mitochondrial preparations from tobacco (Nicotiana tabacum L. cv Samsun N.N.) leaf was separated from 2-oxoglutarate-dependent GABA-T activity by FPLC anion exchange chromatography. Pyruvate-dependent GABA-T was partially purified 1530-fold by a combination of mitochondrial isolation and FPLC anion-exchange chromatography. This enzyme preparation had an apparent Km of 1.220.2 mM for GABA and 0.2420.05 mM for pyruvate. Two-oxoglutarate-dependent GABA-T activity was not detected in the partially purified preparation. Our data indicate the existence of a pyruvate-specific mitochondrial GABA-T. # 1999 Elsevier Science Ltd. All rights reserved.

Protein binding of mercury in milk and plasma from mice and man — a comparison between methylmercury and inorganic mercury

Inorganic mercury has previously been shown to be excreted to milk from plasma to a higher extent than methylmercury. Protein binding of mercury as methylmercury and inorganic mercury in whey and plasma from mouse and man was studied in order to get a better understanding of the transport of mercury into milk. Mice were administered a single i.v. dose of 0.25 mg Hg/kg body weight labelled with CH 3 203HgCl or 203HgCl 2, resulting in 11 ng Hg/g milk and 38 ng Hg/g milk after 1 h, respectively. Milk and plasma from mice and man were also incubated with the respective radiolabelled compound (150 ng Hg/g milk or plasma). Casein, fat and whey fractions in milk from methylmercury treated mice were found to contain 11, 39 and 34%, respectively, and from inorganic mercury treated mice 31, 15 and 41%, respectively, of the total amount of mercury in milk. Serum albumin was a major mercury binding protein in whey and plasma from mice for both methylmercury and inorganic mercury, as demonstrated by FPLC gel filtration and anion-exchange chromatography and further characterised by SDS-PAGE for whey. In addition, anion-exchange chromatography indicated that inorganic mercury, but not methylmercury, in whey from mouse milk formed a dimer of serum albumin. The unbound fraction of mercury in whey and plasma from mice was very small (<0.7%), and somewhat higher in plasma and whey from man. It is concluded, that the unbound fraction in plasma cannot be a determining factor for the observed differences in milk excretion between the two mercury compounds. Instead, it is suggested that methylmercury and to some extent inorganic mercury are transferred from plasma into milk using albumin as a passive carrier.

Expression and Mutagenesis of Mammalian Cytosolic NADP+-Specific Isocitrate Dehydrogenase

Rat liver cytosolic NADP+-specific isocitrate dehydrogenase (IDP2) was expressed in bacteria as a fusion protein with maltose binding protein (MBP). High levels of expression were obtained. The fusion protein was purified from bacterial lysates by affinity chromatography with an amylose resin and found to be catalytically active. IDP2 was separated from MBP by cleavage with protease Xa and purified to homogeneity by FPLC anion-exchange chromatography. A specific activity of 56.3 units/mg and respective apparent Km values for dl-isocitrate and NADP+ of 9.7 +/- 2.9 microM and 11.5 +/- 0.2 microM were obtained for the purified enzyme. These values are similar to those previously reported for cytosolic isocitrate dehydrogenase isolated from a variety of tissues. Evolutionarily conserved arginine residues implicated in substrate binding were changed to glutamate residues using PCR based site-directed mutagenesis of the bacterial fusion plasmid. Mutant enzymes containing residue changes of R100E, R109E, R119E, or R132E were expressed, purified, and characterized by initial rate kinetic analyses. The R119E and R109E mutant enzymes exhibited respective 15- and 31-fold increases in Km values for dl-isocitrate relative to the wild-type enzyme. In contrast, Km values for NADP+ were, respectively, unchanged and increased 9-fold. The most significant reductions in kcat/Km values were obtained for the R100E, R109E, and R132E enzymes. These results suggest that substrate binding residues are highly conserved between bacterial and mammalian enzymes despite low overall homology.

Interrelation between pH and availability of α-lactalbumin and β-lactoglobulin for proteolysis by papain

Abstract The enzymic hydrolysis of α-lactalbumin and β-lactoglobulin by papain was studied on pure whey protein fractions and on a commercial whey protein concentrate (Lacprodan 80). The effect of pH on substrate specificity of papain was indicated by the increase in proteolytic fragments as free α-amino nitrogen, by the decrease in proteinaceous matter using the BIO-RAD protein assay, by gel permeation chromatography (Sephadex G-100) and FPLC anion-exchange chromatography. Enzymic availability of β-lactoglobulin was determined in view of its pH-dependent molecular association status and was best at pH > 7.5, favouring the monomeric structure, whereas α-lactalbumin was hydrolysed without limitation only at acidic pH. The binding of calcium protected completely native α-lactalbumin against proteolytic degradation at pH 6.0 and 8.0. This protective effect was lower for α-lactalbumin in the commercial whey protein concentrate and was absent in calcium-depleted α-lactalbumin.

The major N-linked carbohydrate chains from human urokinase. The occurrence of 4-O-sulfated, (alpha 2-6)-sialylated or (alpha 1-3)-fucosylated N-acetylgalactosamine(beta 1-4)-N-acetylglucosamine elements.

The primary structure of the major N-linked carbohydrate chains attached to Asn302 of urinary-type plasminogen activator (urokinase) have been determined. Urokinase was completely deglycosylated with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F from Flavobacterium meningosepticum. Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100, and then on Bio-Gel P-6. Fractionation of the oligosaccharides was achieved by a combination of FPLC anion-exchange chromatography on Mono Q HR 5/5 and amine-adsorption HPLC on LiChrospher 100-NH2. Analysis by 1H-NMR spectroscopy demonstrated that the collection of N-glycans comprises di-, tri-, and tri'-antennary structures. The glycans contain predominantly GalNAc beta 1-4GlcNAc beta instead of Gal beta 1-4GlcNAc beta elements. The GalNAc residue is mainly sulfated at O4, or to a lesser extent it bears N-acetylneuraminic acid at O6; alternatively the GlcNAc residue can be fucosylated at O3. The major component, which accounts for more than 30 mol/100 mol of the total oligosaccharide pool, consists of an (alpha 1-6)-fucosylated diantennary N-linked carbohydrate chain with (SO4-)-4GalNAc beta 1-4GlcNAc beta 1-2 antennae.

The Major N-Linked Carbohydrate Chains from Human Urokinase. The Occurrence of 4-O-sulfated, (alpha2-6)-sialylated or (alpha1-3)-fucosylated N-acetylgalactosamine(beta1-4)-N-acetylglucosamine Elements

The primary structure of the major N-linked carbohydrate chains attached to Asn302 of urinary-type plasminogen activator (urokinase) have been determined. Urokinase was completely deglycosylated with peptide-N4-(N-acetyl-β-glucosaminy)asparagine amidase F from Flavobacterium meningosepticum. Released oligosaccharides were separated from the remaining protein using gel-permeation chromatography on Bio-Gel P-100, and then on Bio-Gel P-6. Fractionation of the oligosaccharides was achieved by a combination of FPLC anion-exchange chromatography on Mono Q HR 5/5 and amine-adsorption HPLC on LiChrospher 100-NH2. Analysis by 1H-NMR spectroscopy demonstrated that the collection of N-glycans comprises di-, tri-, and tri′-antennary structures. The glycans contain predominantly GalNAcβ1-4GlcNAcβ instead of Galβ1-4GlcNAcβ elements. The GalNAc residue is mainly sulfated at O4, or to a lesser extent it bears N-acetylneuraminic acid at O6; alternatively the GlcNAc residue can be fucosylated at O3. The major component, which accounts for more than 30 mol/100 mol of the total oligosaccharide pool, consists of an (α1-6)-fucosylated diantennary N-linked carbohydrate chain with (SO4−)-4GalNAcβ1-4GlcNAcβ1-2 antennae.

Isolation and characterization of a 21 kDa whey protein in Rhesus monkey ( Macaca mulatta) milk

A soluble protein in Rhesus monkey milk was isolated to apparent homogeneity by FPLC gel filtration, anion-exchange and reverse-phase chromatography. It is a major milk protein and is present at 2.5—3.0 mg/ml milk throughout lactation. It is only found in the whey fraction of milk; acid precipitation of casein does not result in any significant change in its concentration. A molecular weight (MW) of about 21.6 kDa was estimated from gel filtration and SDS gel electrophoresis and also calculated from its amino acid composition. The amino acid composition of this protein is similar to that of bovine β-lactoglobulin (β-Lg), but it is larger in size, possibly representing a family of primate β-Lgs.

The ubiquinol cytochrome c oxidoreductase complex of spinach leaf mitochondria is involved in both respiration and protein processing

Nuclear encoded mitochondrial precursor proteins are cleaved to mature size products by the general mitochondrial processing peptidase (MPP). In contrast to non-plant tissues where MPP is located in the matrix, the general processing activity of potato tuber (storage tissue) mitochondria has been shown to constitute an integral part of the isolated cytochrome c reductase complex of the respiratory chain. Here we show isolation of MPP from photosynthetic tissue, spinach leaf mitochondria, starting from the total membrane processing extract using extraction with dodecyl-β-maltoside followed by FPLC anion-exchange and gel filtration chromatography. The total spinach leaf MPP is found in the fractions containing the cytochrome c reductase complex and is shown to be an integral part of the complex. No processing activity has been found in any other fractions. The isolated cytochrome c reductase complex is shown to process three precursor proteins of different intramitochondrial localisation, the F 1β subunit of ATP synthase (extrinsic membrane protein on matrix side), the Rieske FeS protein (integral membrane protein facing intermembrane space) and the malate dehydrogenase (matrix protein). The processing activity is totally inhibited by EDTA and orthophenanthroline. Our results together with the results in potato mitochondria show that integration of MPP into the cytochrome c reductase is a general phenomenon for plants. The complex consists of ten protein bands on SDS-PAGE of 61, 54, 52, 34, 32, 26, 15, 12, 11 and 10 kDa. The 61, 54 and 52 kDa bands correspond to Core proteins, the 32 kDa band to cytochrome b and the 26 kDa band to Rieske FeS protein as estimated by immunological methods. The three Core proteins are shown to be immunologically related to MPP from other sources, the Core 1 protein corresponding to β-MPP and the Core 2 and Core 3 proteins corresponding to α-MPP, which in comparison to MPP in potato mitochondria indicates species-dependent differences in the appearence of the processing components. Furthermore, the processing activity of the isolated and membrane-bound spinach cytochrome c reductase complex is shown to be inhibited by antimycin A and myxothiazol, electron transfer inhibitors of the complex. The inhibition of processing is, however, not correlated to the inhibition of electron transfer through the complex or to the redox state of the complex.

Partial purification and characterization of ‘injurin-like’ factor which stimulates production of hepatocyte growth factor

We have previously reported the evidence for presence of a humoral factor ‘injurin’, which induces expression of the hepatocyte growth factor (HGF) gene in MRC-5 human embryonic lung fibroblasts. We have now purified a factor from porcine liver which stimulates HGF production but differs from injurin. When injurin activity was measured as a stimulatory effect on HGF production by MRC-5 cells, this activity was found in various acid extracts from porcine tissues, including liver, kidney, brain, and lung, and acid extracts from the liver was used for purification. When the acid extract was applied to Q-Sepharose anion-exchange chromatography, 50–60% of the total injurin activity was adsorbed to the column and the remaining activity was detected in the flow through fractions. Injurin activity was eluted from the Q-Sepharose column by NaCl concentration gradient with four peaks at 0.5–0.6 M, 0.7–0.8 M, 0.9–1.2 M. 1.5–2.0 M NaCl, thereby suggesting that the factor exists in heterogenous or various forms in tissues. The major active fractions were combined and applied to Mono-Q FPLC anion-exchange chromatography. Injurin activity eluted with a single peak at 0.9–1.5 M NaCl and this activity was 4286 fold purified from the starting extract. Addition of this fraction to MRC-5 cells increased the amount of HGF pulse-labeled with [ 35S]methionine to a 3–4-fold higher level than that seen in control cells, whereas it had no significant effect on HGF mRNA levels. Therefore, this factor seems to stimulate HGF synthesis affecting translational processes and is distinct from the previously characterized injurin which stimulates HGF gene expression. Chemical treatments and SDS-polyacrylamide gel electrophoresis of this injurin-like factor indicated that injurin-like factor is a acid- and heat-stable non-proteinous factor with an apparent M r of 8–15 kDa. Since the injurin activity of the factor was decreased by heparinase treatment, the factor may be a polysulfated glycosaminoglycan related to heparin or to heparan sulfate. These results suggest that HGF production may be regulated by this non-proteinous injurin-like factor and that this factor may also play an important role in the regeneration of organs, through translationally enhancing HGF production.

Purification and characterization of a new aminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus B14

Abstract An aminopeptidase was purified from crude extracts of Lactobacillus delbrueckii subsp. bulgaricus B14 by ammonium sulphate precipitation, anion exchange chromatography over DEAE-Sepharose, hydroxy apatite chromatography, and FPLC anion exchange chromatography on Mono Q . By HPLC gel filtration, a molecular mass of ∼260 kDa was estimated. SDS polyacrylamide gel electrophoresis resulted in a single protein band with a molecular mass of ∼32 kDa. Optimal aminopeptidase activity was obtained at pH 6·5 and 45°C. The enzyme was activated by Mg 2+ , Co 2+ and Zn 2+ at concentrations up to 0·1 mM. Higher concentrations of these metal ions inhibited peptidase activity. Ca 2+ increased enzyme activity even at concentrations of 10 mM. Metal chelating agents inhibited enzyme activity strongly whereas thiol group- and serine enzyme inhibitors as well as reducing agents had no remarkable effect on peptidase activity. The EDTA-inhibited enzyme could be reactivated by the divalent cations Co 2+ and Zn 2+ . Best substrates among the hydrolyzed di- and tripeptides were Leu-Leu-Leu, Leu-Gly-Gly, Leu-Ala-Pro and Leu-Leu. Amino acid- and peptide- nitroanilides were cleaved at a lower rate. For the substrates Leu-Leu-Leu, Leu-Gly-Gly, and Leu-Leu, K m -values of 0·14, 1·37 and 1·52 mM were calculated, respectively.

Stoichiometry of the Human Lysosomal Carboxypeptidase-β-Galactosidase Complex

The understanding of the lysosomal β-galactosidase-carboxypeptidase-neuraminidase multienzymatic complex structure and function requires an efficient system for dissociation and association of its isolated protein components under controlled conditions. In this paper such a system was used to determine the stoichiometry of the two main components of this complex - β-galactosidase and carboxypeptidase. The complex, after affinity purification from human placenta, was dissociated at pH 7.5 and β-galactosidase and carboxypeptidase were separated and purified to homogeneity by FPLC anion-exchange chromatography. The 680 kD complex of β-galactosidase and carboxypeptidase was reconstituted in vitro by mixing the isolated enzymes in a 1:2 molar ratio at pH 7.5 and then progressively acidifying the medium towards the intra-lysosomal pH value of 4.75. Under the same conditions, β-galactosidase and carboxypeptidase independently existed as 306 kDa tetramer and 98 k.Da dimer, respectively. Reconstitution experiments with various ratios of purified β-galactosidase and carboxypeptidase allowed us to conclude that the whole complex is made of 4 β-galactosidase and 8 carboxypeptidase monomers. Cross-linking of the native and reconstituted complexes with dimethylsuberimidate or glutaric dialdehyde suggested that the native and the reconstituted complexes have the same supramolecular structure.