Interaction of Vi antigen with proteins

Whiteside, Roberta E. (Boston University School of Medicine, Boston, Mass.), and Edgar E. Baker. Interaction of Vi antigen with proteins. J. Bacteriol. 92:1597-1603. 1966.-Purified Vi antigen (Vi) mixed in equal amounts with bovine serum albumin (BSA) or human gamma globulin (HGG) at pH values above 4.7 formed a complex which was not precipitated by trichloroacetic acid or tungstic acid. At pH values below 4.7, the interaction between Vi and either BSA or HGG produced insoluble complexes except when excess Vi antigen was present. When sufficient Vi was present at the lower pH values, the soluble complex was not precipitated by trichloroacetic acid. Other acid polysaccharides tested did not form trichloroacetic acid-soluble complexes with BSA. When subjected to immunoelectrophoresis, the Vi-BSA complex migrated in agar at a rate different from that of either BSA or Vi alone. The complex reacted with both Vi and BSA antiserum. The addition of either BSA or Vi antiserum to a Vi-BSA complex resulted in dissociation of the complex and precipitation of either Vi or BSA, depending upon the antiserum used. Vi antigen mixed with purified O antigen from Salmonella typhosa formed a complex which migrated in agar at a rate different from that of either component alone when subjected to immunoelectrophoresis.     

Inhibition of immune precipitation by complement

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.     

Whey protein fractionation: isoelectric precipitation of alpha-lactalbumin under gentle heat treatment

The selective precipitation of alpha-lactalbumin (alpha-LA) at a pH around its isoelectric point (4.2) under heat treatment is the basis for a fractionation process of whey proteins. In these conditions, beta-lactoglobulin remains soluble, whereas bovine serum albumin and immunoglobulins co-precipitate. Knowledge of the mechanism governing the alpha-LA precipitation influences the choice of operating conditions and enables optimization of the fractionation process. alpha-LA is a calcium metallo-protein and its isoelectric precipitation is governed by the protein-calcium complexation equilibrium. Citrate, a sequestrant of calcium, decreases the free calcium concentration and displaces the precipitation phenomenon to a lower temperature range. A study of the effect of citrate on the precipitation phenomena of whey proteins is presented. Whatever the citrate content, precipitation curves for bovine serum albumin (BSA) and alpha-LA intersect at a temperature around 45 degrees C. For a temperature of heat treatment lower than 40 degrees C, a selective enrichment in alpha-LA of the precipitated phase is observed. As addition of citrate leads to high alpha-LA precipitated fractions at a temperature around 35 degrees C, the precipitation step may be performed at this temperature. It results in a reduced heat denaturation of whey proteins and in a higher alpha-LA purity in the precipitated fraction. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 391-397, 1997.     

Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy

Fourier transform infrared (FT-IR) spectra were measured for an aqueous solution (pD = 5.40) of defatted monomer bovine serum albumin (BSA) over a temperature range of 25-90 degrees C to investigate temperature-induced secondary structure and conformation changes. The curve fitting method combined with the Fourier self-deconvolution technique allowed us to explore details of the secondary structure and conformation changes in defatted BSA. Particularly striking in the FT-IR spectra was an observation of the formation of an irreversible intermolecular beta-sheet of BSA on heating above 70 degrees C. A band at 1630 cm(-1) in the spectra was assigned to short-segment chains connecting alpha-helical segments. The transition temperature for the short-segment chains connecting alpha-helical segments is lower by 17-18 degrees C, when compared to those of the alpha-helix, turn, and intermolecular beta-sheet structures of BSA, suggesting that the alpha-helix and turn structures of BSA are cooperatively denatured on heating. Moreover, the results give an important feature in heat-induced denaturation of BSA that the conformation changes occur twice around both 57 and 75 degrees C. The appearance of two peaks is interpreted by the collapse of the N-terminal BSA domain due to the crevice in the vicinity between domains I and II at low-temperature transition and by the change in cooperative unit composed of the other two BSA domains at high-temperature transition.     

Humoral and cellular responses to native antigen following oral and parenteral immunization with lipid-conjugated bovine serum albumin

Humoral and cellular immune responses of rabbits to bovine serum albumin (BSA) were measured following oral and parenteral immunization with either BSA or one of two dodecanoic acid conjugates of BSA. The first consisted of a mixture of lightly and heavily conjugated BSA-molecules (L-BSA-mix), while the second (L-BSA) was a homogeneous preparation of heavily conjugated BSA with more than 95% of the 60 available amino groups covalently bound to dodecanoic acid. Animals ingesting L-BSA-mix had a similar humoral immune response but enhanced cellular reactivity to BSA in comparison to animals ingesting the native antigen. No systemic immunologic responses to BSA were detected following ingestion of L-BSA in spite of the demonstration of circulating BSA antigenic groups. This lack of a detectable immune response after oral administration was not due to masking of antigenic sites by the lipid residues since both humoral and cellular immune responses to BSA were obtained in animals injected with L-BSA. Ingestion of L-BSA did not induce tolerance since a subsequent injection of BSA elicited a normal primary immune response. The differences in immunogenicity between BSA, L-BSA and L-BSA-mix following oral administration may be related to different modes of antigen recognition by the gut-associated lymphoid tissues.     

Chronopotentiometry and Faradaic impedance spectroscopy as signal transduction methods for the biocatalytic precipitation of an insoluble product on electrode supports: routes for enzyme sensors, immunosensors and DNA sensors

The biocatalyzed precipitation of an insoluble product produced on electrode supports is used as an amplification path for biosensing. Enzyme-based electrodes, immunosensors and DNA sensors are developed using this biocatalytic precipitation route. Faradaic impedance spectroscopy and chronopotentiometry are used as transduction methods to follow the precipitation processes. While Faradaic impedance spectroscopy leads to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provides the total resistance at the interfaces of the modified electrodes. A horseradish peroxidase, HRP, monolayer-functionalized electrode is used to sense H₂O₂ by the biocatalyzed oxidation of 4-chloro-1-naphthol (1), to the insoluble product benzo-4-chlorohexadienone (2). An antigen monolayer electrode is used to sense the dinitrophenyl antibody, DNP-Ab, applying an anti-antibody–HRP conjugate as a biocatalyst for the oxidative precipitation of 1 by H₂O₂ to yield the insoluble product 2. An oligonucleotide (3) functionalized monolayer electrode is used to sense the DNA-analyte (4), that is one of the Tay–Sachs genetic disorder mutants. Association of a biotin-labeled oligonucleotide to the sensing interface, followed by the association of the avidin–HRP conjugate and the biocatalyzed precipitation of 2 leads to the amplified sensing of 4. The amount of the precipitate accumulated on the conductive support is controlled by the concentration of the respective analytes and the time intervals employed for the biocatalytic precipitation of 2. The electron-transfer resistances of the electrodes covered by the insoluble product (2) are derived from Faradaic impedance measurements, whereas the total electrode resistances are extracted from chronopotentiometric experiments. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports are found. Chronopotentiometry is suggested as a rapid transduction means (a few seconds). The precautions needed to apply chronopotentiometry in biosensors are discussed.     

Enthalpy relaxation of bovine serum albumin and implications for its storage in the glassy state

Two endothermic peaks could be observed for five commercial samples of bovine serum albumin (BSA). The smaller peak observed by differential scanning calorimetry (DSC) corresponded to enthalpy relaxation. This peak was followed on storage of BSA, in its glassy state, after it had been heated above its denaturation temperature. Enthalpy and peak temperature increased with duration of storage. On storage for one week at 60 degrees C, a sample at 8.3% moisture showed a peak at 100 degrees C with an energy value of approximately 2 J per g protein. BSA samples were heated within the DSC sufficiently to eliminate the lower enthalpy peak but without altering the denaturation enthotherm. The amount of physical aging shown by these BSA samples was similar to that of the heat-denatured samples. It was concluded that the heating endotherms of dry BSA reflect both the storage and thermal history of the sample. Possible implications of the enthalpy relaxation of BSA on the behavior of this important protein are considered.     

Phase behavior of aqueous solutions of bovine serum albumin in the presence of dextran, at rest, and under shear

The demixing conditions for aqueous solutions of bovine serum albumin (BSA, fraction V) and for joint solutions of BSA plus dextran (DEX, M(w) = 2000 kg/mol) were determined by turbidimetric measurements as a function of composition, temperature, and shear rate. Aqueous solutions of BSA phase separate upon heating. Within the region of BSA concentrations between 0.05 and 32 wt %, the demixing temperature, T1, falls from ca. 65 degrees C to an almost constant value of 45 degrees C. Adding DEX to the BSA solutions reduces the homogeneous region of the mixture drastically where the amount of DEX required to lower T1 to 25 degrees C decreases rapidly as the concentration of BSA is raised. Experiments concerning the influences of shear have been performed for the ternary system up to 500 s(-1). They demonstrate that the content of dextran determines the sign of the effect. At low DEX concentrations, the mechanical field favors the homogeneous state (shear-induced mixing), whereas the opposite effect (shear-induced demixing) is observed at high DEX concentrations. Possible reasons for this observation are discussed.     

Preparation of a new thermo-responsive adsorbent with maltose as a ligand and its application to affinity precipitation

A thermo-responsive polymer on which maltose was covalently immobilized as an affinity ligand was newly synthesized for purification of thermolabile proteins from the crude solution by affinity precipitation. Among the thermo-responsive polymers synthesized as carriers for adsorbent, poly(N-acryloylpiperidine)-cysteamine (pAP) has a lower critical solution temperature (LCST) of around 4 degrees C, at which its solubility exhibits a sharp change. Adsorbent for affinity precipitation was prepared by combining pAP with maltose using trimethylamine-borane as a reducing reagent. This adsorbent (pAPM) obtained showed a good solubility response: pAPM in the basal buffer (pH 7.0) became soluble below 4 degrees C and was completely insoluble above 8 degrees C. The affinity precipitation method using pAPM consisted of the following four steps: adsorption at 4 degrees C, precipitation of the complex at 10 degrees C, desorption by adding the desorption reagent at 4 degrees C, and recovery of a target protein at 10 degrees C. In the affinity precipitation of Con A from the crude extract of jack bean meal, 82% of Con A added was recovered with 80% purity by addition of 0.2 M methyl-alpha-D-mannopyranoside as a desorption reagent. In the repeated purification of Con A from the crude extract, pAPM could be satisfactorily reused without decrease in the affinity performance. Moreover, when pAPM was used for the purification of thermolabile alpha-glucosidase from the cell-free extract of Saccharomyces cerevisiae, 68% of total activity added was recovered and the specific activity per amount of protein of the purified solution was enhanced 206-fold higher than that of the cell-free extract without thermal deactivation of the enzyme.     

A histochemical study about the involvement of rat liver cells in the uptake of heterologous immune complexes from the circulation

Intravenously injected immune complexes (ICx) composed of bovine serum albumin (BSA) and rabbit anti-BSA were taken up by the liver. Insoluble complexes, made in antibody excess, were rapidly taken up by Kupffer cells and were metabolized within 24 h. Soluble complexes, made in antigen excess, were only partly taken up by Kupffer cells. In addition these complexes were bound, taken up and metabolized by endothelial cells. Until 2 h after injection soluble complexes could also be observed along the microvilli of hepatocytes. No signs of endocytosis in hepatocytes could be observed. It is concluded, that ICx can be taken up by Kupffer cells as well as by endothelial cells. The physical state of the complexes, soluble or insoluble, determines the cell type in which uptake occurs.     

Effect of cold on the antigen binding function of splenic cells at different periods of immunogenesis in rats

The patterns of changes in the immune response when deep cooling with different rates acted at various periods during development of the immune response, were examined in Wistar rats. Cold exposure causes not only a suppression but also stimulation of the immune response to antigen. The suppressive effect of deep cooling when it preceded immune challenge (the antigen injected at a body temperature decreased by 3-4 degrees C) waned and became stimulating with increasing time interval between antigen challenge and cold exposure. The stimulating effect on the immune response was most pronounced when cold exposure occurred in 5 days after the antigen challenge. These changes differed quantitatively also when cooling was either rapid, or slow. Thus the modulating effect of the thermal afferent signal was differently manifested depending in the cooling rate, i.e. the presence or absence of the dynamic activity of the peripheral thermosensitive afferents, and the time elapsed between antigen challenge and cold exposure.     

Rapid redistribution of clathrin onto macrophage plasma membranes in response to Fc receptor-ligand interaction during frustrated phagocytosis

We have observed increases in assembled clathrin on the plasma membrane during "frustrated phagocytosis," the spreading of macrophages on immobilized immune complexes. Resident macrophages freshly harvested from the peritoneal cavity of mice and attached to bovine serum albumin (BSA)-anti-BSA-coated surfaces at 4 degrees C had almost no clathrin basketworks on their adherent plasma membrane (less than 0.01 coated patch/micron 2), as observed by immunofluorescence, immunoperoxidase, and platinum-carbon replica techniques, although abundant assembled clathrin was observed in the perinuclear Golgi region. When the cells were warmed to 37 degrees C they started to spread by 4 min and reached their maximum extent by 20 min. Spreading preceded clathrin assembly at the plasma membrane. Clathrin-coated patches were first observed on the adherent plasma membrane at 6 min. Between 12 and 20 min assembled clathrin coats appeared on both adherent and nonadherent plasma membranes with a concomitant decrease in identifiable clathrin in the perinuclear region. A new steady state emerged by 2 h, as perinuclear clathrin began to reappear. At 20 min at 37 degrees C the adherent plasma membranes of macrophages spreading on BSA alone had 0.9 coated patch/micron 2, whereas in cells spread on immune complex-coated surfaces, the clathrin patches increased, dependent on ligand concentration, to a maximum of 2.1 coated patches/micron 2. Because frustrated phagocytosis of immune complex-coated surfaces at 37 degrees C increased the area of adherent plasma membrane, the total area coated by clathrin basket-works increased 5-fold (28 micron 2/cell) as compared with cells plated on BSA alone (5.6 micron 2/cell) and 200-fold as compared with cells adhering to immune complexes at 4 degrees C. We then determined that macrophages cultured on BSA-coated coverslips for 24 h already have abundant surface clathrin. When immune complexes were formed by the addition of anti-BSA IgG to already spread macrophages cultured on BSA-coated coverslips for 24 h, clathrin assembled at the sites of ligand-receptor interaction even at 4 degrees C, before spreading, and a 2.6-fold increase in assembled clathrin was observed on the adherent plasma membrane of cells on immune complexes as compared with cells on BSA alone. Clathrin was reversibly redistributed to the Golgi region, returning to the steady state by 2 h.(ABSTRACT TRUNCATED AT 400 WORDS)     

A histochemical study about the involvement of rat liver cells in the uptake of heterologous immune comples from the circulation

Summary Intravenously injected immune complexes (ICx) composed of bovine serum albumin (BSA) and rabbit anti-BSA were taken up by the liver. Insoluble complexes, made in antibody excess, were rapidly taken up by Kupffer cells and were metabolized within 24 h. Soluble complexes, made in antigen excess, were only partly taken up by Kupffer cells. In addition these complexes were found, taken up and metabolized by endothelial cells. Until 2 h after injection soluble complexes could also be observed along the microvilli of hepatocytes. No signs of endocytosis in hepatocytes could be observed. It is concluded, that ICx can be taken up by Kupffer cells as well as by endothelial cells. The physical state of the complexes, soluble or insoluble, determines the cell type in which uptake occurs. To Prof. Dr. H.G. Goslar on the occasion of his 65th birthday     

Selective iodination and polypeptide composition of pinocytic vesicles

We describe a method for the specific radioiodination of pinocytic vesicles (PVs) based upon the simultaneous endocytosis of lactoperoxidase (LPO) and glucose oxidase (GO). Initial experiments indicated that LPO was interiorized by the macrophage cell line J774 by fluid phase pinocytosis and without detectable binding to the plasma membrane (PM). Interiorization varied linearly with enzyme concentration and exposure time, was temperature dependent, and was undetectable at 4 degrees C. Employing EM cytochemistry, LPO activity was restricted to PVs after a 3- to 5-min pulse at 37 degrees C. These results formed the basis of the method for iodinating the luminal surface of PVs: 5-min exposure to both LPO and GO at 37 degrees C followed by washes and iodination (addition of 125I and glucose) at 4 degrees C. Enzyme-dependent incorporation of iodide into the polypeptides of both PV membrane and contents occurred. Several lines of evidence indicated that there was selective labeling of PV as opposed to PM. Iodination did not occur if the pinocytic uptake of LPO ad GO was inhibited by low temperature. EM autoradiography showed a cytoplasmic localization of grains, whereas a clear PM association was evident with surface labeling. LPO was iodinated only after PV labeling and was present within organelles demonstrating latency. After PV iodination, > 75% of several labeled membrane antigens could be immunoprecipitated by monoclonal antibodies only after cell lysis. In contrast, all labeled antigens were accessible to antibody on intact cells after surface labeling. The polypeptide compositions of PM and PV membrane were compared by SDS polyacrylamide gel electrophoresis and by quantitative immune precipitation using a panel of anti-J774 monoclonal antibodies. The electrophoretic profiles of iodinated proteins (15-20 bands) were strikingly similar in NP-40 lysates of both PV and PM iodinated cells. In addition, eight membrane antigens examined by immune precipitation, including the trypsin-resistant immunoglobulin (Fc) receptor and the H-2Dd histocompatibility antigen, were found to be iodinated to the same relative extents by both labeling procedures. We conclude that PV membrane is formed from a representative sample of PM polypeptide components.     

Chitosan–bovine serum albumin complex formation: A model to design an enzyme isolation method by polyelectrolyte precipitation

Interactions between a model protein (bovine serum albumin—BSA) and the cationic polyelectrolyte, chitosan (Chi), have been characterized by turbidimetry, circular dichroism and fluorescence spectroscopy. It has been found that the conformation of the BSA does not change significantly during the chain interaction between BSA and chitosan forming the non-covalently linked complex. The effects of pH, ionic strength and anions which modify the water structure around BSA were evaluated in the chitosan–BSA complex formation. A net coulombic interaction force between BSA and Chi was found as the insoluble complex formation decreased after the addition of NaCl. Around 80% of the BSA in solution precipitates with the Chi addition. A concentration of 0.05% (w/v) Chi was necessary to precipitate the protein, with a stoichiometry of 6.9 g BSA/g Chi. No modification of the tertiary and secondary structure of BSA was observed when the precipitate was dissolved by changing the pH of the medium. Chitosan proved to be a useful framework to isolate proteins with a slightly acid isoelectrical pH by means of precipitation.     

Stabilizing effects of caprylate and acetyltryptophanate on heat-induced aggregation of bovine serum albumin

Acetyltryptophanate (AT) and caprylate (Cap) have been used to stabilize serum albumin against heat treatment. However, the mechanism of stabilization by these additives has never been fully elucidated. Here we used thermal melting to determine the effects of these additives on the melting temperature of bovine serum albumin (BSA) and heat stress at 60 degrees C to follow degradation of the protein in the presence of varying concentrations of AT or Cap. Native polyacrylamide gel electrophoresis was used to examine degradation products generated by heat treatment. Both additives increased the melting temperature of BSA, resulting in an increase by 12 degrees C at 5 mM AT and 3 degrees C at 1 mM Cap. They also conferred stability to BSA against heat stress at 60 degrees C. Complete protection was observed at 5 mM AT and 1 mM Cap. Comparison of AT and Cap in their effects on melting temperature and heat stress-induced degradation showed that a greater protection occurs with Cap which has a weaker effect on melting temperature. Based on this observation it was concluded that the observed protection by AT may be explained by its effects on melting temperature while that of Cap should be ascribed to other mechanisms.     

Binding of antibodies in liposomes to extracellular matrix antigens

We have incorporated antibodies against fibronectin or laminin into liposomes and studied their interaction with insoluble forms of these antigens. The antibodies, after modification by palmitoylchloride, were incorporated into the lipid bilayer by the cholate dialysis method. The antibodies in the liposomes recognized their specific antigen with little reaction to the alternative attachment protein or to albumin (less than 2%). The binding of antibody-containing liposomes to insoluble antigen was inhibited by soluble antibodies to the respective antigens but not by antibodies to other antigens. The affinity constant of the liposome-antibody complex with the antigen was estimated at 1-10 X 10(-9) M liposomes. Thus, antibodies in liposomes retain their reactivity and specificity, and the reaction constant is comparable to that observed for immune complexes.     

Comparative effect on humoral response of four different proteins covalently linked on BSA-containing liposomes

The humoral response to encapsulated BSA appears to be a classical TD antigen response with a high ratio of IgG to IgM, whereas that to covalently-linked antigen is more complex, characterized by an enhanced synthesis of IgM, leading to an equal production of IgM and IgG. In a recent paper, we observed that surface-linked Con A on BSA-containing liposomes changed the isotype distribution to encapsulated BSA so as to mimic the response to surface-linked antigen. In the present study, we compared the immune response to BSA in BALB/c mice immunized with the antigen encapsulated into liposomes coated with one of four different proteins: Con A, Myo, MSA, or PWM. The humoral response was analyzed by measurements of antibody production (total Ig, IgM, and IgG isotypes) on serum samples obtained by cardiac puncture. It can be concluded from our results that any surface-linked protein may affect the interaction between liposome-associated antigen and immunocompetent cells.     

Precise localization of antigens on follicular dendritic cells

Summary Horse-spleen ferritin or bovine serum albumin conjugated to colloidal gold (BSA-gold) were injected subcutaneously in preimmunized mice. In draining lymph nodes both antigens were located in macrophages or between the cytoplasmic processes of follicular dendritic cells (FDC). Some of the antigens remained trapped on FDC until day 31 after injection. Simultaneous injection of both antigens showed that they were located between the infoldings of the same FDC. These cells are thus able to retain at least two different antigens on their surface. The peculiar arrangement of ferritin between the cytoplasmic infoldings suggests that this antigen is fixed on both cell membranes by specific antibodies. The trapped immune complexes could thus stabilize the FDC membrane system.The antigen retention requires the presence of specific antibodies since BSA-gold or ferritin injected without preimmunization were not found between FDC processes. Nonantigenic materials, such as colloidal gold or carbon particles, are not trapped by FDC, except when injected in large amounts.The antigens were trapped on the surface of FDC, however unfrequently in close contact with lymphocytes. FDC might protect lymphocytes against an excess of immune complexes and act as regulators of contacts between lymphocytes and immune complexes.Abbreviations BSA bovine serum albumin - BSA-gold BSA conjugated to colloidal gold particles - FDC follicular dendritic cells     

Purification and physico-chemical properties of collagenase synthesized by a bacterium of the type Acinetobacter sp.]

The Acinetobacter spec collagenase has been almost completely purified. This enzyme is a true collagenase the activity of which is high on collagen. The enzyme is active on insoluble collagen, gelatin and the synthetic Pz-peptide, but has no proteolytic activity on casein or bovine serum-albumin. The collagenase was obtained on a simple medium with gelatin and yeast extract. The enzyme was purified by (NH4)2SO4 precipitation. DEAE cellulose column chromatography, Sephadex G 200 gel-filtration. The molecular weight of the enzyme was found to be 102 000 daltons, and its isoelectric point was found to be 7,7 +/- 0,2. The optimum pH and temperature for insoluble collagen hydrolysis were 7.6 and 37 degrees C, respectively; so, this collagenase corresponds to true collagenase. Hydrolysis of Pz-peptide is activated by Ca2+ and inhibited by metal ions (Cu2+, Fe3+, Zn2+, Pb2+, Hg2+). EDTA and o-phenanthroline induced a very significant reduction in enzyme activity. Iodoacetate and p-CMB induced a slight reduction in enzyme activity only at high concentrations (10-2M). The collagenase is most stable for temperatures less than or equal to 50 degrees C.