Stepwise dissociation of subcomponents of C1, the first component of human complement, upon activation on an affinity sorbent

An affinity sorbent comprising macroporous glass coated with the polymer with the polymer with immobilized immunoglobulin IgG was used for the isolation from human serum of the first component of the complement and for its separation into subcomponents C1r, C1s and C1q by the one-step procedure. Serum C1 was quantitatively bound to the sorbent at 0 degrees C. The unbound part of the serum can be used as a R1 reagent for determining the hemolytic activity of C1. After activation of bound C1 by heating (30 degrees C, 40 min) the activated subcomponent C1r is eluted from the sorbent. Stepwise elution with EDTA at pH 7.4 or with EDTA + 1 M NaCl at pH 8.5 results in a selective and quantitative elution of the activated subcomponent C1s and subcomponent C1q. Stepwise elution of C1 subcomponents from the affinity sorbent after activation reflects the process of C1 breakdown following its activation on immune complexes.     

Soybean Bowman–Birk Inhibitor Conjugates with Clinical Dextran. Synthesis and Antiproteolytic Activity

Conjugates of the classical soybean Bowman-Birk inhibitor (BBI) with clinical dextran were synthesized. Clinical dextran was preliminarily oxidized with periodate to dialdehydedextran (DAD). The effect of the degree of oxidation of DAD on coupling of the inhibitor was evaluated. The binding of the protein was shown to increase with increasing degree of DAD oxidation (5, 10, 20%). Total coupling of the inhibitor occurred when the degree of oxidation of the dextran was 20%. The BBI-DAD (20%) conjugate contained 13% protein with BBI/DAD molar ratio 1 : 1. The conjugates retained the ability to inhibit trypsin (Ki = 0.2-0.3 nM) and alpha-chymotrypsin (Ki = 15-30 nM). Thus, the coupling of BBI with the polymeric carrier caused practically no decrease in the antiproteolytic activity of the inhibitor.     

Oxidation of methionine residues in lutropin

Bovine lutropin and its subunits were submitted to oxidation by sodium periodate or chloramine T. Methionine residues were easily oxidized but partial destruction of fucose was observed. After oxidation treatment most of the lutropin exhibits the same elution volume in gel filtration as the native hormone. Sucrose gradient sedimentation or gel filtration experiments show however that the oxidation of isolated subunits is accompanied by aggregation of conformational changes even in the case of porcine beta subunit which contains only one methionine residue, unit and intact alpha subunit exhibit very low residual activity.     

A kinetic study of the participation of zinc in human spermatozoa metabolism

Incubation of washed human spermatozoa in the presence of 6 mM concentrations of EDTA, histidine and cysteine induces a release of about 75% of the zinc bound to the cells. No zinc is released by human spermatozoa when incubation is done in the absence of the mentioned reagents. No detectable amounts of calcium or magnesium were found to be released by the sperm cells under any of the experimental conditions tested. Zinc release induced by the presence of EDTA, histidine and cysteine is accompanied: 1) by a significant increase in oxygen uptake, both under basal conditions and in the presence of some substrates (glucose, pyrubate and succinate) and 2) by a significant increase in motility. This increase was greater with cysteine than with histidine, and greater with the latter than with EDTA. This behavior of human spermatozoa resembles that previously described for invertebrate spermatozoa and is related to the regulation of energy metabolism and probably to sperm capacitation.     

Endogenous inhibition of cyclic nucleotide phosphodiesterase in cultured human epithelial cells

We have identified an endogenous inhibitor of cyclic nucleotide phosphodiesterase (PDE) activity in cultured human epithelial cells. The inhibitor was non-dialyzable, inactivated by trypsin and boiling, but stable to a 60° C, 30 min. treatment. Separation of inhibitor from PDE was achieved by blue dextran affinity chromatography. PDE was eluted from this column by EDTA, while the inhibitor remained bound and was subsequently eluted with buffer containing cyclic GMP. The inhibitor was active against PDE from several sources including both Ca⁺⁺ dependent and Ca⁺⁺ independent forms from bovine brain and retina respectively. These characteristics differentiate the PDE inhibitor from human epithelial cells from those previously described from various bovine tissues.     

Purification and characterization of acetohydroxyacid reductoisomerase from spinach chloroplasts.

Acetohydroxyacid reductoisomerase was purified over 400-fold to a specific activity of 62 nkat.mg-1, with 2-aceto-2-hydroxybutyrate as substrate, from the stroma of spinach leaf chloroplasts. The enzyme was not intrinsically membrane bound. The native enzyme was a tetramer with a subunit Mr of 59,000. The activity was optimum between pH 7.5 and 8.5. The apparent Km for 2-acetolactate was 25 microM and for 2-aceto-2-hydroxybutyrate was 37 microM. The enzyme required Mg2+ and the Vmax. was attained at physiological Mg2+ concentrations. NADP+ competitively inhibited the reaction when NADPH was the varied substrate. The native enzyme eluted from Mono-Q ion-exchange resins as three distinct peaks of activity. This elution pattern was preserved when the peaks were combined, dialysed and re-chromatographed. Each form exhibited identical Mr of 59,000 after SDS/polyacrylamide gel electrophoresis (PAGE), whereas they were easily distinguishable from each other after PAGE under non-denaturing conditions. These results provide evidence for the existence of multiple forms of acetohydroxyacid reductoisomerase in chloroplasts isolated from spinach leaves.     

Modification of brain fructose-1,6-bisphosphatase activity by chelators: "induction" of 5'-AMP sensitivity

Brain fructose-1,6-bisphosphatase (EC 3.1.3.11) from various sources are ordinarily insensitive to 5'-AMP. In addition to stimulation and conferring a "neutral" behaviour, prior treatment with histidine, EDTA or imidazole renders the brain enzyme sensitive to 5'-AMP. The histidine treated enzyme(s) bind to Blue-Sepharose CL-6B column and are specifically eluted by 5'-AMP in contrast to the untreated enzyme(s) which do not bind to the affinity column at all. The histidine effect in inducing 5'-AMP sensitivity was abolished by treatment of the native enzyme by subtilisin or by a number of divalent cations including Zn++.     

Specific and non-specific affinities of the extracellular glucosyltransferase complex of Streptococcus mutans 6715

Glucosyltransferases (GTF) from different strains of streptococci exhibited different elution profiles when fractionated on insoluble-dextran affinity columns. The proportions of unadsorbed and adsorbed GTF were not related to their extent of stimulation by exogenous dextran, and GTF preparations exposed to, and freed from, clinical dextran prior to fractionation lost their ability to bind to the dextran columns. Different proportions of bound GTF were released by irrigation of columns with different concentrations of salt and clinical dextran, and the “specific” binding and release of GTF exhibited by a column possessing covalently linked, clinical dextran ligands was duplicated on a control column that did not possess the dextran ligands. These results, and the high affinity of GTF for hydrophobic alkyl (Shaltiel) ligands, demonstrate that ionic and hydrophobic properties of impure GTF aggregates may lead to erroneous characterization of the dextran affinity of some protein fractions. Fractionations on DEAE-Sepharose and on hydroxylapatite showed that the two dextran-dependant GTF activities (GTF-S and GTF-I) were present in the major enzyme fraction (Streptococcus mutans 6715) recovered from a Sephacryl S-200 affinity column. A minor, dextran-independent GTF was not adsorbed onto the Sephacryl column. The presence of SDS (0.005%) and Triton X100 (0.01%) stabilized GTF activity during gel filtration and improved the separation of GTF-S and GTF-I in hydroxylapatite fractionation of the highly aggregated enzyme. A comparable separation of the two enzyme forms on DEAE-Sepharose was achieved only if T10 dextran (10 mg/mL) was included with the detergent mixture in the column irrigant.     

Immunoaffinity purification of the calpains

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

Role of integrin beta1-like protein in proliferation and differentiation of cultured stem cells from midgut of Heliothis virescens

Cultured midgut cells from Heliothis virescens larvae were incubated with anti-human integrin beta1 made in rabbit and then passed over a column of magnetic beads bound to anti-rabbit IgG (MACS, Miltenyi Bergisch Gladbach, Germany). Cells bound to integrin beta1 antibody also bound to the anti-rabbit IgG on the magnetic beads (MACS) and were retained in the column while it remained in the magnetic field. Non-bound cells were eluted at this time. They did not stain with anti-integrin antibody just after elution. Removing the column from the magnetic field allowed cells bound to the beads-integrin beta1 antibody to be eluted. All of these cells stained with human anti-integrin beta1 upon elution. Each cell fraction was cultured in medium for 3 days. During this time, the populations of cells tended to return to heterogeneous staining patterns characteristic of control populations. However, cells that did not stain immediately with anti-integrin beta1 antibody exhibited double the rate of multiplication and 8 times more differentiation than the integrin-antibody positive cells that eluted later, as well as the non-treated control cells. In a second experiment, midgut cells were incubated for 4 days with various titers of human anti-integrin beta1 to block surface integrin beta1-like reactive sites. Stem cells blocked with anti-integrin beta1 antibody during incubation exhibited double the rate of differentiation than non-treated control cells and those showing anti-integrin beta1-positive stain upon elution.     

High-performance liquid chromatography with concanavalin A immobilized by metal interactions on the stationary phase

Concanavalin A (Con A) was immobilized via metal interactions on macroporous, microparticulate silica support having covalently bound iminodiacetic acid functions (IDA-silica) chelated with Cu(II) at the surface. The amount of copper and of Con A in the column could readily be controlled by the conditions used for chelating the metal by IDA-silica and for immobilization of the lectin. The retention behavior of columns packed with the stationary phase did not change under a wide range of elution conditions, indicating no loss of immobilized lectin. However, the Con A proper could readily be removed from the column at pH 3.0 or together with Cu(II) by perfusion with EDTA at neutral pH. Columns containing Con A immobilized by this technique exhibited dual retention behavior for proteins, glycoproteins, and carbohydrates according to the pertinent glycan-lectin or protein-metal interactions. The glycoproteins, peroxidase and alpha 1-acid glycoprotein, were retained by the Con A moiety and eluted with eluents containing competing sugars, whereas the proteins, beta-lactoglobulin, alpha-chymotrypsinogen A, and ribonuclease A and B were retained by the chelated copper and were eluted and separated with eluents containing sodium chloride or borate. Binding constants of glycosides on the immobilized Con A were evaluated chromatographically and found to be one-third to two-thirds those reported in the literature on the basis of experiments in free solution.     

Structural requirements for heparan sulphate self-association

To investigate heparan sulphate self-association, various sub-fractions of beef-lung heparan sulphate have been subjected to affinity chromatography on heparan sulphate-agarose. A particular variant of heparan sulphate was chiefly bound to matrices substituted with the same or cognate heparan sulphates. N-desulphation and N-acetylation abolished the chain-chain interaction. Also, dermatan sulphates and chondroitin sulphates showed affinity for heparan sulphate-agarose. [3H]Heparan sulphates that were bound to a heparan sulphate-agarose were desorbed by elution with the corresponding heparan sulphate chains and also with unrelated heparan sulphates, heparin, and the galactosaminoglycans to various degrees. However, the corresponding heparan sulphate species was the most efficient at low concentrations. Dextran sulphate was unable to desorb bound heparan sulphate. When the corresponding heparan sulphate was N-desulphated/N-acetylated, carboxyl-reduced, or periodate-oxidised (D-glucuronate), the modified polymer was unable to displace [3H]heparan sulphate from heparan sulphate-agarose. The displacing ability of heparin was also destroyed by periodate oxidation. It is concluded that self-interaction between heparan sulphate chains is strongly dependent on the overall molecular conformation. The N-sulphate and carboxylate groups as well as the integrity of the D-glucuronate residue are all essential for maintaining the proper secondary structure.     

Separation of putrescine oxidase and spermidine oxidase in foetal bovine serum with the aid of a specific radioactive assay of spermidine oxidase.

1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate.     

Inactivation of aminated horseradish peroxidase by interaction with S-Sepharose

Horseradish peroxidase which had been aminated by periodate oxidation and reductive amination was purified by cation-exchange chromatography on S-Sepharose. Instead of the expected single peak of aminated enzyme, two distinct peaks of protein were eluted from the column. Evaluation of the protein in each of the two distributions showed that peak number 1 had spectral properties and specific activity similar to those of native enzyme. Distribution number 2 had a threefold reduction in the extinction in the Soret region at 404 nm and was completely devoid of enzymatic activity. This inactivation was caused by a specific interaction between the aminated peroxidase and the S-Sepharose matrix, resulting in a displacement of the heme prosthetic group out of its native orientation. The inactivation of the aminated peroxidase was found to be dependent on time, pH, and the support matrix itself. These results indicate that the S-Sepharose and Mono-S resins are not interchangeable, despite the chemical similarities of the two resins.     

Estimation of the percolation thresholds in lobenzarit disodium native dextran matrix tablets

The objective of the present work is to estimate for the first time the percolation threshold of a new series of dextran (native dextran of high molecular weight [B110-1-2, Mw = 2 x 10(6)]), in matrices of lobenzarit disodium (LBD) and to apply the obtained result to the design of hydrophilic matrices for the controlled delivery of this drug. The formulations studied were prepared with different amounts of excipient in the range of 20% to 70% wt/wt. Dissolution studies were performed using the paddle method (100 rpm) and one face water uptake measurements were performed using a modified Enslin apparatus. The Higuchi, zero-order, and Hixson-Crowell models as well as the nonlinear regression model were employed as empiric methods to study the release data. Values of diffusion exponent 0.563 < n < 0.786 (Korsmeyer equation) for dissolution profile and water uptake mechanism 0.715 < n < 1 (Davidson and Peppas equation) suggested anomalous or complex mechanisms. On the other hand, the contribution of the relaxation or erosion and of the diffusive mechanism in Peppas-Sahlin equation indicated that the main mechanism for drug delivery from tablets is swelling controlled delivery (K(r)/K(d) < 1). The critical points observed in kinetic parameters above 58.63% vol/vol of native dextran B110-1-2 plus initial porosity in the LBD-dextran matrices with a relative polymer/drug particle size of 4.17 were attributed to the existence of an excipient percolation threshold.     

Interaction of ox heart mitochondrial F1-ATPase with immobilized ADP and ATP.

The reaction of mitochondrial F1-ATPase with immobilized substrate was studied by using columns of agarose-hexane-ATP. Mg2+ was required for binding of the enzyme to the column matrix. The column-bound enzyme could be eluted fully by ATP and other nucleoside triphosphates. Nucleoside di- and mono-phosphates were less effective. At a fixed concentration of nucleotide the effectiveness of elution was proportional to the charge on the eluting molecule. The ATP of the column matrix was hydrolysed by the bound F1-ATPase to release phosphate, probably by a uni-site reaction mechanism. Thus the F1-ATPase was bound to the immobilized ATP by a catalytic site. Treatment of the bound F1-ATPase with 4-chloro-7-nitrobenzofurazan prevented complete release of the enzyme by ATP. Only one-third of the bound enzyme was now eluted by the nucleotide. The inhibition of release could be due either to the inhibitor blocking co-operative interactions between sites or to its increasing the tightness of binding of immobilized ADP at the catalytic site.     

Role of dextran-specific suppressor T cells in the regulation of the immune response by a reactive form of dextran

Reactive forms of antigens or haptens have been shown to induce a state of hyporesponsiveness mediated in part by suppressor T cells. Injection of Balb/c x C57B16 F1 (CB6F1) mice with a reactive form of dextran B1355S (periodate oxidized dextran, dex-P) specifically reduced responses to dextran immunization within 1 day after dex-P treatment. This unresponsiveness lasted at least 23 days and required a reactive form of dextran for its induction since native dextran and oxidized/reduced dextran failed to induce tolerance. Furthermore, hyporesponsiveness could be induced by iv injection of dextran-coupled cells, especially peripheral blood lymphocytes, a result which suggests that in vivo coupling to cellular antigens is involved in dex-P-induced hyporesponsiveness. Suppression of the anti-dextran response could be transferred to normal mice with T-cell-enriched spleen cell populations from dex-P-injected mice. Interestingly, the presence of B cells in the transferred cell preparations interfered with detection of suppression. Both Lyt 1+2- and Lyt 1-2+ cells were involved in the dex-P-induced suppression; indeed, mixtures of these types of T cells led to the most profound degree of suppression. The suppressive activity of spleen cells from dex-P-injected mice could be removed by passage over dextran-coated plates. Moreover, cells eluted from the plates specifically suppressed anti-dextran responses of normal mice, indicating that dex-P injection induces a population of antigen-binding suppressor cells. This system will allow the study of the suppressor-T-cell receptors in a well-defined idiotypic system.     

Immobilization of laccase from Cerrena unicolor on controlled porosity glass

White-rot basidiomycete Cerrena unicolor grown in non-induced and induced conditions was tested for production of laccase, lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP). A typical correlation between the concentration of phenolic compounds in the culture fluid and the extracellular laccase activity was observed. The heterogeneous crude laccase preparation obtained after the non-induced fermentor cultivation was immobilized both on controlled porosity glass (CPG) activated by γ-aminopropyltriethoxysilane (APTES) and on CPG with its surface covered by dextran layers. The laccase activities were tested in the aqueous solution for the native and immobilized preparations using different pH and temperature conditions. Laccase activities were additionally examined for native and immobilized forms of laccase preparations in the aqueous solution containing organic solvents. The greatest activity toward the substrate used in the presence of organic solvents was shown by the laccase preparation coupled with the CPG covered by a dextran layer. Potential inhibitors such as thioglycolic acid, thiourea and EDTA used in 1-mM concentration did not show inhibiting properties towards the laccase preparations.     

An affinity adsorbent, 5'-adenylate-aminohexyl-sepharose. I. Purification and properties of two forms of RNase U2

An affinity adsorbent, 5'-adenylate-aminohexyl-Sepharose 4B, was prepared by the periodate oxidation of AMp followed by coupling and condensation with amino-hexyl-Sepharose 4B. RNase U2, a purine-specific RNase, was specifically bound to this adsorbent at pH 4.5 and eluted critically at pH 5.9 in the presence of 1 M NaCl, corresponding to the pH dependence of the binding of 2'-AMP to RNase U2. By using this affinity chromatography as a main tool, a simplified and effective purification method for RNase U2 was established with a high yield of 58%. Another form of RNase U2 with low specific activity, named RNase U2-B, was eluted at a slightly higher pH from this adsorbent. RNase U2-B was indistinguishable from the original enzyme (RNase U2-A) in base specificity, affinity for ApA, molecular weight and amino acid composition, but was clearly different in specific activity, molecular activity for ApA, isoelectric point and conformation of molecule. This affinity adsorbent is also effective for the detection or isolation of small amounts of base-specific RNases in crude cell extract.     

Periodate oxidation of sperm-whale myoglobin and the role of the methionine residues in the antigen–antibody reaction

1. Oxidation of sperm-whale metmyoglobin and its apoprotein with periodate has been investigated under various conditions of pH and temperature to find those under which the reagent acted with specificity. 2. At pH6.8 and 22 degrees consumption of periodate ceased in 3(1/2)hr. at 43 moles of periodate/mole of myoglobin. The two methionine residues, the two tryptophan residues, the three tyrosine residues and two histidine residues were oxidized; serine increased in the hydrolysates from 6 to 9 residues/mol. 3. At pH5.0 and 22 degrees , consumption levelled off in 4(1/2)hr. at 26 moles of periodate/mole of myoglobin and resulted in the modification of the two methionine residues, the two tryptophan residues, the three tyrosine residues and two histidine residues; serine increased from 6 to 7 residues/mol. and, also, ferrihaem suffered considerable oxidation. 4. Oxidation at pH5.0 and 0 degrees resulted at completion (4hr.) in the consumption of 22 moles of periodate/mole of myoglobin and in the modification of the methionine, tyrosine and tryptophan residues. Spectral studies indicated oxidation of the haem group. This derivative reacted very poorly with rabbit antisera to MbX (the major component no. 10 obtained by CM-cellulose chromatography; Atassi, 1964). 5. Oxidation of apomyoglobin at pH5.0 and 0 degrees was complete in 4hr. with the consumption of 7.23 moles of periodate/mole of apoprotein. The rate of oxidation in decreasing order was: methionine; tryptophan; tyrosine; and after 7hr. of reaction the following residues/mol. were oxidized: methionine, 2.0; tryptophan, 1.6; tyrosine, 0.99. No peptide bonds were cleaved. Metmyoglobin prepared from the 7hr.-oxidized apoprotein showed that the reactivity with antisera to MbX had diminished considerably. 6. Milder oxidation of apoprotein (2 molar excess of periodate, pH5.0, 0 degrees , 2hr.) resulted in the modification of 1.66 residues of methionine/mol. Metmyoglobin prepared from this apoprotein was identical with native MbX spectrally, electrophoretically and immunochemically. It was concluded that the methionine residues at positions 55 and 131 were not essential parts of the antigenic sites of metmyoglobin.