Production of an antibody against guinea pig MIF. I. Specificity of the anti-MIF antibody.

An antibody was produced in rabbits against partially purified MIF which was released from the specifically stimulated lymphocytes of tuberculin-hypersensitive guinea pigs. The MIF used as an antigen was fractionated by polyacrylamide gel electrophoresis. The antibody thus prepared was then examined for its specificity for several lymphokines by affinity column chromatography. It was observed that the antibody column adsorbed MIF, but not the other three lymphokines, MCF, NCF, and SRF, indicating a keen specificity of the antibody against MIF.     

Lymphokines secreted from sodium periodate-treated lymphocytes

This study was designed to determine if sodium metaperiodate (NaIO₄)-treated lymphocytes secrete lymphokines and if these lymphokines are similar to those obtained from mitogen- or antigen-stimulated lymphocytes. A brief exposure of CBA spleen cells to NaIO₄ induced the secretion of significant amounts of migration inhibitory factor (MIF). This MIF had a molecular weight range between 30,000 and 58,000, and was stable when heated at 56 °C for 30 min, but unstable at 80 °C. These characteristics are similar to those previously reported for mitogen- and antigen-induced MIF. In addition, NaIO₄ induced the secretion of lymphotoxin (LT) from CBA and Balb/c spleen cells, as well as from guinea pig lymph node cells. NaIO₄ was compared to the other inducers in regard to the quantity of LT secreted. Supernatant derived from NaIO₄-treated mouse spleen cells contained less LT than supernatants derived from concanavalin A- or phytohemagglutinin-treated cells, but contained more activity than those supernatants derived from lipopolysaccharide-treated cells. CBA spleen cells secreted significantly more LT than Balb/c spleen cells after NaIO₄ stimulation. NaIO₄-stimulated CBA spleen cells secreted LT in cultures with or without serum, but stimulated Balb/c spleen cells secreted LT only in serum-containing cultures. The advantages of NaIO₄ as an inducer of lymphokines, as opposed to other mitogens or antigens, is the brief exposure of this agent to the cells after which the NaIO₄ is removed, and the lymphokines can be obtained free from the inducer.     

Lectin induction of lymphokines in cultured murine leukocytes

Antigens induce sensitized lymphocytes to undergo mitosis and to secrete soluble products, termed lymphokines, which modulate the immune response. Plant lectins are known to act as polyclonal lymphocyte mitogens and, in some cases, stimulate lymphocytes to produce lymphokines. In an effort to explore the relationship of specific cell surface glycoconjugates to the induction of mitosis and the production of lymphokine activities we have examined the ability of the mitogenic lectins, concanavalin A and Wistaria floribunda mitogen, and the nonmitogenic hemagglutinin from Wistaria floribunda seeds to stimulate the production of macrophage migration inhibition factor (MIF), macrophage chemotactic factor (CF), and lymphotoxin (LT). Concanavalin A causes lymphocytes to produce MIF and LT but no detectable CF activities. W. floribunda mitogen induces lymphocytes to produce soluble substances which exhibit all three lymphokine activities. The nonmitogenic W. floribunda agglutinin causes lymphocytes to produce MIF and CF but no observable LT activity. Within the sensitivity of the assays employed, the results indicate that mitogenesis is not a corequisite of the expression of either macrophage migration inhibition factor or lymphocyte-derived chemotactic factor but it may be associated with the induction of lymphotoxin. It is also apparent that the expression of each lymphokine activity is independent of the expression of the other lymphokines studied.     

Cell surface receptors for lymphokines. I. The possible role of glycolipids as receptors for macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF).

Incubation of culture supernatants from concanavalin A-stimulated guinea pig and rat lymphocytes with protein-free preparations of bovine brain gangliosides abolished their macrophage migration inhibitory factor (MIF) and macrophage activation factor (MAF) activity. The identity of the MIF/MAF-binding component(s) present in these glycolipid mixtures has yet to be established, but adsorption experiments using purified preparations of mono- (GM₁, GM₂, and GM₃), di- (GD_1a), and trisialogangliosides (GT₁) were negative. Since these gangliosides account for over 90% of the glycolipid content in brain ganglioside mixtures it appears that the MIF-binding component(s) is present only in very small amounts. Treatment of guinea pig peritoneal macrophages with liposomes containing similar brain gangliosides or water-soluble glycolipids extracted from guinea pig macrophages enhanced their responsiveness to MIF. The enhanced response to MIF of liposome-treated macrophages was abolished by incubation of the treated macrophages with fucose-binding lectins (Lotus agglutinin and Ulex europaeus agglutinin I) before exposure to MIF, suggesting that the MIF-binding component donated by the liposomes may be a fucose-containing glycolipid. The possible role of glycolipids as surface receptors for MIF and MAF is discussed.     

Mechanisms in the in vivo release of lymphokines. 1. Comparative kinetics in the release of six lymphokines in inbred strains of mice

Lymphokines were detected in the sera of 16 strains of inbred mice sensitized intravenously with cell walls of Mycobacterium bovis strain BCG and challenged subsequently intravenously with old tuberculin (OT). Variations occurred between the strains in the types and quantities of six lymphokines studied, namely, chemotactic factor (CF), type II interferon (IF II), lymphotoxin (LT), migration inhibitory factor (MIF), mitogenic factor (MF), and skin reactive factor (SRF). The times for maximum release of the lymphokines in the different strains were similar for MIF, IF II, SRF, and MF, but differed for CF and LT. The degree of activity of MIF and IF II generally paralleled one another in the different strains but such parallelism did not occur for the other four lymphokines. Each of the 16 strains was a high responder for at least one of the lymphokines, indicating that in sensitized mice the release or presence in the circulation of lymphokines in response to a specific antigen is a selective process. Thus, each strain may have an individual combination of lymphokines, interactions of which may determine the types of pathway utilized in an immunological response.     

The biochemistry and in vitro activity of soluble factors of activated lymphocytes

Summary Activated lymphocytes release numerous products which are either synthesized de novo or in increased amounts; some of these products play a role in the regulation of the immune response and are designated as mediators of cellular immune reactions or lymphokines. The first lymphokine described was the macrophage migration inhibitory factor (MIF) which has been studied most extensively with regard to its chemical and biological properties. Using sensitive radiolabelling techniques and an antiserum against highly purified fractions of MIF we were able to identify several products of activated guinea pig lymphocytes with different molecular weights of 15.000, 30.000, 45.000, 60.000 which all had an isoelectric point of 5.2 and were all inhibitory to macrophage migration. It is suggested, that these molecules are oligomers of a common subunit of molecular weight 15.000. It was further shown, that molecules of the same physical-chemical and serological characteristics are produced by activated B-cells, L₂C leukemia cells and growing fibroblasts, thus further substantiating earlier reports on the production of MIF by lymphoid and non-lymphoid cells. The described molecules were also shown not to contain determinants of the major histocompatibility complex and to be distinct from lymphotoxin, another lymphocyte activation product. It is concluded, that MIF is not sa single molecule but rather a system of structurally related molecules. Thier interaction with macrophages and possible relationship to macrophage activating factor is discussed.     

Multiple forms of glucocorticosteroid receptors in the neural retina of chick embryo,revealed by polyacrylamide gel electrophoresis

The glucocortiocoid receptors in the cytosol of neural retina of the 15-day chick embryo were analyzed by quantitative polyacrylamide gel electrophoresis. Maintenance of the triamcinolone acetonide (TA)-receptor complexes under conditions of electrophoretic analysis is dependent on temperatures not exceeding ?2 °C and is favored by low ionic strength, but is relatively insensitive to changes in pH between 5 and 10. Polyacrylamide gel electrophoresis in highly crosslinked Resolving Gels (15% crosslinking with N,N′-diallyltartardiamide) at low wattage and under temperature control at ?2 °C, allowed for detection and partial characterization of over 80% of the specific TA-binding activity of the tissue. One form of the glucocorticoid receptor, designated as complex II, was found to have a molecular weight (M_r) of 175,000. In addition, specifically bound TA was found in a multimillion M_r aggregate which was unable to enter gels of any concentration investigated and has been designated TA-complex I. The ratio of complex I/complex II increased with increasing gel concentration, indicating physical or chemical interaction between II and I. A polyacrylamide gel electrophoresis rerun of isolated TA-complex II gave rise to two smaller TA-binding species: Component B, of M_r 108,000 and component A, a relatively fast migrating molecule which could not be characterized under the conditions used. The ratio of $\text{B}\text{A}$ appeared constant and close to 2, suggesting that A and B may be significant structural elements of complex II. Polyacrylamide gel electrophoresis of isolated TA-complex I gave rise to component C of M_r 60,000, but not to components A or B. Components A and B associated to a large M_r complex, designated as I′, which was revealed to an extent directly proportional to gel concentration. Similarly, component C aggregated to I″, as evidenced at elevated gel concentrations. In conclusion, it has been possible to define by gel electrophoresis three of the molecular species (A, B, and C) that comprise the glucocorticoid receptor, and the possible relationships between them.     

Purification and properties of dipeptidyl peptidase IV from Streptococcus mitis ATCC 9811

Dipeptidyl peptidase IV (EC 3.4.14.—) from Streptococcus mitis ATCC 9811 was purified to a specific activity of 56.2 units/mg protein by a series of column chromatographic techniques. The purified enzyme was apparently homogeneous as judged by disc gel electrophoresis. Gel filtration on a calibrated column indicated an apparent molecular weight of 120,000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate in a constant acrylamide concentration resulted in the appearance of a single component for which a molecular weight of 53,000 was calculated. The purified enzyme has an optimum pH between 6.0 and 8.7 and an isoelectric point of 4.0. The K_m value toward glycylprolyl-p-nitroanilide is about 6.0 × 10^?5m. Substrate specificity studies indicated that the purified enzyme hydrolyzes specifically N-terminal X-proline from X-Pro-p-nitroanilides. Inhibition of this enzyme was achieved with Hg²⁺, Pb²⁺, Zn²⁺, EDTA, and diisopropyl phosphorofluoridate, but not with N-ethyl-maleimide and sulfhydryl inhibitors.     

Role of lymphokines in regulation of macrophage differentiation

The regulatory mechanism of guinea pig lymphokines was investigated in regard to differentiation of myeloid cells to macrophages. The Ml-cell line, established from a myeloid leukemia of an SL-strain mouse, was induced to differentiate in vitro into mature macrophages possessing Fc receptors and the ability to phagocytize latex particles by treatment with crude lymphokines. Both concanavalin A- and antigen-induced lymphokines showed the differentiation-inducing factor (D factor) activity. However, macrophage migration inhibitory factor/ macrophage activation factor (MIF/MAF) purified by an immunoadsorbent column with anti-MIF antibody had no such an activity. The D-factor activity was detected in the lymphokine preparation that was not retained on the immunoadsorbent column. In contrast, colony-stimulating factor (CSF) was adsorbed to the immunoadsorbent column, and could be recovered in the purified MIF/MAF preparation. These findings suggest that the molecular entity of D factor is distinct from MIF/ MAF and CSF. A culture supernatant of guinea pig peritoneal macrophages activated with MIF/ MAF (CSF) exhibited strong D-factor activity. However, the supernatant possessed rather reduced CSF activity as compared to that of the original MIF/MAF (CSF) preparation. Thus, MIF/MAF may play an important role in macrophage differentiation by regulating the production of D factor or CSF from macrophages.     

Macrophage migration inhibitory factor (MIF): Genetic evidence for participation in early onset and early stage rheumatoid arthritis

Macrophage migration inhibitory factor (MIF) is an upstream pro-inflammatory cytokine that is associated with the pathogenesis of autoimmune inflammatory diseases including rheumatoid arthritis (RA). Two polymorphisms in the upstream region exist in the MIF gene and are associated with RA susceptibility or severity in different populations. In this case-control study, we investigated whether MIF polymorphisms are associated with RA susceptibility or activity in a western Mexican population .The relationship of MIF levels with clinical features of disease also was assessed. Genotyping of the ?794 CATT_5–8 (rs5844572) and the ?173 G > C (rs755622) polymorphisms was performed by PCR and PCR-RFLP respectively on 226 RA patients and 210 healthy subjects. Serum MIF levels were determined by ELISA. We found a significant association between the ?794 CATT_5–8 6,7 MIF genotype with RA. Moreover, we detected an association between the ?794 CATT₇ allele with early onset RA. The ?794 CATT₇ and ?173^*C alleles, which are in linkage disequilibrium, were associated with high disease activity on RA patients. A positive correlation between circulating MIF levels and C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor, anti-citrullinated protein/peptides antibodies and TNFα was detected. MIF levels appear to be associated with disease progression rather than disease activity, which is distinct from the established relationship between disease activity and TNFα levels. In conclusion, the MIF gene and protein are associated with RA in a western Mexican population, with a main contribution onto early onset and early stages of disease.     

Production of macrophage migration inhibitory factor and lymphotoxin by leukocytes from normal and Wiskott-Aldrich syndrome patients

The production of macrophage migration inhibitory factor (MIF) and lymphotoxin (LT) by cultured leukocytes from patients with Wiskott-Aldrich syndrome (WAS) and normal controls was studied. The presence of these lymphokines in leukocyte culture supernatants usually correlated directly with the dose of stimulant used. Doses of nonspecific mitogens and specific antigens, which produced maximal in vitro lymphocyte transformation, stimulated maximal production of these mediators. When the incorporation of tritiated thymidine by stimulated leukocyte cultures from patients with Wiskott-Aldrich syndrome (WAS) was deficient, they usually produced less MIF and lymphotoxin than normal. However, when their in vitro lymphoproliferative responses were normal, the lymphotoxin activity in supernatants of WAS leukocyte cultures was normal.     

A defect of in vitro monocyte leukotaxis induced by cutaneous allograft rejection

Previous studies have demonstrated defective monocyte leukotaxis in human sarcoidosis and tuberculosis, diseases associated with chronic cellular immune activation. In the present study, we sought to determine if acute cellular immune activation might also alter this monocyte function, using a primate model of skin allograft rejection. A profound, short-lived depression of monocyte leukotactic responsiveness occurred during the time of allograft rejection. The leukotactic defect was mediated by a cell-directed inhibitor in plasma, a heat-stable protein of 230,000 daltons molecular weight. Neither monocyte leukotactic responsiveness nor plasma inhibitory activity was affected by autografting or nonspecific cutaneous inflammation. A physicochemically and functionally similar inhibitor was produced in vitro during the course of one-way primed mixed lymphocyte cultures. The cell-directed inhibitor appears to be a lymphocyte product which is clearly distinguishable from MIF and leukotactic lymphokines.     

Purification and some properties of guanidinoacetate amidinohydrolase produced by Corynebacterium sp.

Guanidinoacetate amidinohydrolase (EC 3.5.3.2) was purified from Cornebacterium sp. grown in a medium supplemented with guanidinoacetate, and some of its properties were investigated.The molecular weight of the enzyme was estimated to be 150,000 by gel filtration. SDS-polyacrylamide gel electrophoresis showed a single subunit component with a molecular weight of 38,000, suggesting that the enzyme is composed of four identical subunits. The isoelectric point of the enzyme was pH 5.8.The enzyme showed optimum activity at pH 9.0–9.5 and was stable at pH 6.0–10.5. 3-Guanidinopropionate and 4-guanidinobutyrate were respectively hydrolyzed 32% and 5% as fast as guanidinoacetate. The apparent K_m for guanidinoacetate was 16 mM. Incubation of the enzyme by o-phenanthroline or 8-hydroxyquinoline resulted in almost complete inactivation. The activity of the inactivated enzyme was restored by incubation with Zn²⁺. p-Chloromercuribenzoic acid and iodine effectively inhibited the enzyme activity. Glycine was a competitive inhibitor, and n-alkyl amines such as n-octylamine, n-decylamine and n-dodecylamine were uncompetitive inhibitors.     

P(700) Chlorophyll a-Protein : Purification, Characterization, and Antibody Preparation

The P₇₀₀ chlorophyll α-protein was purified by preparative sodium dodecyl sulfate (SDS) gel electrophoresis from SDS-solubilized barley (Hordeum vulgare L., cv Himalaya) chloroplast membranes. After elution from the gel in the presence of 0.05 to 0.1% Triton X-100, the recovered protein had a chlorophyll/P₇₀₀ ratio of 50 to 60/1 and contained no chlorophyll b or cytochromes. Analysis of the polypeptide composition of the chlorophyll-protein revealed a 58 to 62 kilodalton (kD) polypeptide component but no lower molecular weight polypeptides. The 58 to 62 kD component was further resolved into two distinct polypeptide bands which were subsequently mapped by partial cyanogen bromide digestion and Staphylococcus aureus proteolysis. Based on results from the mapping experiments and other data, we suggest that the two components are conformational variants of a single polypeptide. Measurement of the chlorophyll to protein ratio by quantitative amino acid analysis and consideration of the yield of P₇₀₀ in the protein isolate suggest that, contrary to previous models (Bengis and Nelson, 1975, 1977), P₇₀₀in vivo is associated with a minimum of four subunits of approximately 60 kD.

Antibodies raised against the photochemically active chlorophyll-protein complex from barley reacted specifically with the 58 to 62 kD apoprotein. The same preparative electrophoresis procedure was used to isolate photochemically active P₇₀₀ chlorophyll a-protein from soybean (Glycine max L.), tobacco (Nicotiana tobacum L.), petunia (Petunia × hybrida), tomato (Lycopersicum esculentum), and Chlamydomonas reinhardti. The isolated complex from all species exhibited identical polypeptide compositions and chlorophyll/P₇₀₀ ratios. Antibodies to the barley protein cross reacted with all species tested demonstrating the highly conserved structure of the apoprotein.

     

Purification and subunit structure of mouse liver cystathionase

Cystathionase has been purified from mouse liver by ammonium sulfate precipitation, ethanol precipitation, column chromatography on DEAE-cellulose and on hydrox-ylapatite, as well as Sephadex G-200 gel filtration. These procedures yielded a chromatographically homogeneous enzyme which was purified more than 1000-fold relative to whole liver extract. Overall recovery was approximately 4%. The purified enzyme does not contain detectable carbohydrate and migrates as a single protein component on analytical disc gel electrophoresis. A sedimentation coefficient of 8.3 S has been determined for the active enzyme by rate zonal centrifugation in glycerol gradients. This value suggests a molecular weight for the native enzyme of approximately 160,000 g/mol, a value similar to that estimated by gel filtration. Following sodium dodecyl sulfate gel electrophoresis in the presence of reducing agent and at different gel concentrations, a single protein component with a molecular weight of 40,000 g/mol was obtained. Thus, the enzyme appears to consist of four subunits of equal size. The K_m value for cystathionine at pH 8.1, 37 °C, and in the presence of 1 mm dithioerythritol is approximately 1 mm.     

Trehalase from the cellular slime mold Dictyostelium discoideum: Purification and characterization of the homogeneous enzyme from myxamoebae

Trehalase (α-α′-trehalose 1-d-glucohydrolase, EC 3.2.1.28) was solubilized from myxamoebae of the cellular slime mold Dictyostelium discoideum by a freeze-thaw cycle and was subsequently purified to homogeneity using the techniques of ethanol fractionation, molecular sieve chromatography, DEAE-cellulose ion-exchange chromatography, chromatofocusing, and preparative polyacrylamide disc gel electrophoresis. The 1000-fold purified enzyme had a specific activity of about 104 units/mg, which was accompanied by a net recovery of 5 to 7% of the original activity. The purified enzyme was maximally active at pH 5.5, showed high specificity for trehalose, and exhibited a typical hyperbolic response as a function of trehalose concentration with a K_m of 1.2 mm. The enzyme was maximally active at 50 °C and had an energy of activation of 12–13 kcal/mol. Thermal stability studies demonstrated that full enzymatic activity was recovered following a 5-min incubation of trehalase at temperatures up to 45–50 °C. Analysis of various compounds for inhibitory effects indicated that Tris and urea were slightly effective, reducing enzymatic activity by 28 and 6% at concentrations of 100 and 10 mm, respectively. Of five heavy metals tested, HgCl₂ was the most inhibitory, reducing activity by 58% when present at a final concentration of 1.0 mm. Enzymatic activity was not affected by any adenine derivative examined (e.g., ATP, ADP, AMP, cAMP, adenosine, and adenine). The molecular weight of the native enzyme was determined by molecular sieve chromatography, pore gradient electrophoresis, and electrophoresis as a function of acrylamide concentration. All three methods yielded a value of about 10⁵ ± 5 × 10³. Estimation of the subunit or monomer molecular weight by sodium dodecyl sulfate-gel electrophoresis indicated a value of 95–100 × 10³. The isoelectric point as determined in 7.5% polyacrylamide gels with pH 3–10 ampholytes was 7.2–7.3. The purified enzyme adsorbed to concanavalin A-Sepharose in the presence of KCl (0.1 m) and was eluted with α-methylmannoside, thereby suggesting an association between trehalase and carbohydrate. In agreement with this conclusion was the observation that trehalase could be specifically stained for carbohydrate with the Alcian blue and periodic acid-Schiff's reagents following polyacrylamide disc gel electrophoresis.     

An integrative in silico methodology for the identification of modulators of macrophage migration inhibitory factor (MIF) tautomerase activity

Macrophage migration inhibitory factor (MIF) is a major proinflammatory cytokine that has been increasingly implicated in the pathogenesis of several inflammatory, autoimmune, infectious and oncogenic diseases. Accumulating evidence suggests that the tautomerase activity of MIF plays a role in modulating some of its intra- and extra-cellular activities. Therefore, the identification and development of small-molecule inhibitors targeting the catalytic activity of MIF has emerged as an attractive and viable therapeutic strategy to attenuate its function in health and disease. Herein we report a novel virtual screening protocol for the discovery of new inhibitors of MIF’s tautomerase activity. Our protocol takes into account the flexibility and dynamics of the catalytic site by coupling molecular dynamics (MD) simulations aimed at modeling the protein’s flexibility in solution to (i) docking with FlexX, or (ii) docking with FlexX and pharmacophoric filtering with Unity. In addition, we applied in parallel a standalone docking using the new version of Surflex software. The three approaches were used to screen the ChemBridge chemical library and the inhibitory activity of the top-ranked 333 compound obtained from each approach (1000 compound in total) was assessed in vitro using the tautomerase assay. This biochemical validation process resulted in the identification of 12 novel MIF inhibitors corresponding to a 1.2% hit rate. Six of these hits came from Surflex docking; two from FlexX docking with MD simulations and four hits were identified with MDS and pharmacophore filtering with minimal overlap between the hits from each approach. Six hits were identified with IC₅₀ values lower than 10 μM (three hits with IC₅₀ lower than 1 μM); four were shown to be suicide inhibitors and act via covalent modification of the N-terminal catalytic residues Pro1. One additional inhibitor, N-phenyl-N-1,3,4-thiadiazol-2-yl-thiourea, (IC₅₀ = 300 nM) was obtained from FlexX docking combined to pharmacophoric filtering on one of the eight MD structures. These results demonstrate the power of integrative in silico approaches in the discovery of new modulator of MIF’s tautomerase activity. The chemical diversity and mode of action of these compounds suggest that they could be used as molecular probes to elucidate the functions and biology of MIF and as lead candidates in drug developments of anti-MIF drugs.     

Purification and some properties of an Mg2+-, Ca2+- and calmodulin-stimulated ATPase from spinach chloroplast envelope membranes

Spinach chloroplasts display an ATPase activity which is associated with the envelope. This envelope-bound activity is stimulated by Ca²⁺, Mg²⁺ and calmodulin (Nguyen, T.D. and Siegenthaler, P.A. (1983) FEBS Lett. 164, 67–70). The Triton X-100-solubilized enzyme was retained specifically on a calmodulin-Sepharose affinity column in the presence of calcium. The fractions eluted by EGTA contained two proteins characterized by pI values of 7.3 and 6.0 (isoelectric focusing). Both proteins, separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis), were resolved into a single polypeptide having and identical apparent M_rmr of 65 000. This suggests that the two initial proteins might be isoelectric variants. However, the amount of the enzyme fraction obtained by the calmodulin-Sepharose column was small and the ATPase activity was very labile. A linear glycerol gradient allowed the recovery of a greater amount of the enzyme which was, however, only partially purified, but the activity of which was much more stable. Electrophoresis of the ATPase-containing fractions in a native polyacrylamide gradient gel permitted the separation of a 260 kDa protein which was resolved by SDS-polyacrylamide gel electrophoresis into a single polypeptide of 65 kDa. Thus, the chloroplast envelope-bound ATPase might be a tetramer (260 kDa) consisting of 4 identical monomers (65 kDa). The purified ATPase had properties similar to that of the envelope-bound enzyme. TheK_m value for ATP was 0.45 mM. The activity was stimulated by Ca²⁺ and Mg²⁺, and further enhanced by calmodulin. The physiological significance of the chloroplast envelope-bound ATPase is discussed.     

Trypanosoma cruzi: Isolation and characterization of a proteinase

Lysates of Trypanosoma cruzi epimastigotes were able to hydrolyze casein (K_m = 2.5 mg/ml) as well as bovine and human hemoglobins (K_m = 12.2 mg/ml); there was optimum activity was around pH 7.0. The proteinase activity detected with these substrates was enhanced by sodium diaminotetraacetate (EDTA) and reducing agents (SO^2?₃, mercaptoethanol, cysteine) and was inhibited by sulfhydryl reagents, thus suggesting an SH-dependent enzyme. Purification (60×) of the proteinase was carried out as follows: (1) precipitation at ?20 C, pH 4.5, with 80% acetone, (2) gel filtration on Sephadex G-200, (3) affinity chromatography on Sepharose 4B covalently linked to p-aminophenyl mercuric acetate. Only a single component (with an estimated molecular weight of 60,000) was detected in purified preparations by polyacrylamide gel electrophoresis. However, in addition to the major component identified as a proteinase, crossed immunoelectrophoresis experiments indicated the presence of at least three other antigens that apparently were devoid of proteinase activity. Optimum pH activity of the purified preparations was around pH 6.0 for casein and pH 3.0 for hemoglobins, but these activities probably are due to the one enzyme since they were altered identically by the same agents.