Inhibition, by lanthanides, of neutral proteinases secreted by human, rheumatoid synovium

Fragments of human, rheumatoid synovium were maintained on organ culture for three days under serum-less conditions. Their conditioned media contained collagenolytic, gelatinolytic and caseinolytic activities, which were susceptible to inhibition by lanthanide ions. Of the four lanthanides tested, Sm3+ proved the best inhibitor of gelatinase and caseinase, while La3+ inhibited collagenase the most strongly. Inhibition of collagenase by La3+ was uncompetitive. A direct binding assay confirmed the greater association between collagen fibrils and collagenase in the presence of La3+. Ca2+ was not required for binding of the uninhibited enzyme to collagen, but acted to stabilize collagenase against thermoinactivation.     

Potato tuber succinate semialdehyde dehydrogenase: purification and characterization

Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass (Mr) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of Mr 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mM sodium pyrophosphate buffer. In 100 mM Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD+ as cofactor and NADP+ and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a Km of 4.6 microM for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 microM. The Km for NAD+ was found to be 31 microM. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethyl-maleimide, by heavy metal ions Hg2+, Cu2+, Cd2+, and Zn2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.     

Intracellular hexose-6-phosphate:phosphohydrolase from Streptococcus lactis: purification, properties, and function.

An intracellular hexose 6-phosphate:phosphohydrolase (EC 3.1.3.2) has been purified from Streptococcus lactis K1. Polyacrylamide disc gel electrophoresis of the purified enzyme revealed one major activity staining protein and one minor inactive band. The Mr determined by gel permeation chromatography was 36,500, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single polypeptide of apparent Mr 60,000. The enzyme exhibited a marked preference for hexose 6-phosphates, and the rate of substrate hydrolysis (at 5 mM concentration) decreased in the order, galactose 6-phosphate greater than 2-deoxy-D-glucose 6-phosphate greater than fructose 6-phosphate greater than mannose 6-phosphate greater than glucose 6-phosphate. Hexose 1-phosphates, p-nitrophenylphosphate, pyrophosphate, and nucleotides were not hydrolyzed at a significant rate. In addition, the glycolytic intermediates comprising the intracellular phosphoenolpyruvate potential in the starved cells (phosphoenolpyruvate and 2- and 3-phosphoglyceric acids) were not substrates for the phosphatase. Throughout the isolation, the hexose 6-phosphate:phosphohydrolase was stabilized by Mn2+ ion, and the purified enzyme was dependent upon Mn2+, Mg2+, Fe2+, or Co2+ for activation. Other divalent metal ions including Pb2+, Cu2+, Zn2+, Cd2+, Ca2+, Ba2+, Sr2+, and Ni2+ were unable to activate the enzyme, and the first four cations were potent inhibitors. Enzymatic hydrolysis of 2-deoxy-D-glucose 6-phosphate was inhibited by fluoride when Mg2+ was included in the assay, but only slight inhibition occurred in the presence of Mn2+, Fe2+, or Co2+. The inhibitory effect of Mg2+ plus fluoride was specifically and completely reversed by Fe2+ ion. The hexose 6-phosphate:phosphohydrolase catalyzes the in vivo hydrolysis of 2-deoxy-D-glucose 6-phosphate in stage II of the phosphoenolpyruvate-dependent futile cycle in S. lactis (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982).     

Purification and properties of a peptidase acting on a synthetic substrate for collagenase from monkey kidney.

A peptidase cleaving a synthetic substrate for collagenase, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (designated as PZ-peptide) has been purified extensively (about 5200-fold) from a soluble extract of monkey kidney with a view of carrying out studies on its possible physiological role. The purified PZ-peptidase appeared essentially free of collagenase, nonspecific protease and di- and tri-peptidase activities. The properties of the purified PZ-peptidase resemble very much the granuloma enzyme. It is optimally active around pH 7.0. Its apparent Km value for PZ-peptide is 0.72 mM and V is 10.1 mumol/mg protein/min. It is reversibly inhibited by p-hydroxymercuribenzoate and HgCl2, whereas iodoactetamide does not affect the enzyme activity. N-Ethylmaleimide inhibited the enzyme partially (50%). Heavy metals like Cu-2+, Cd-2+, Ag+, Pb-2+, Ni-2+, and Zn-2+ completely inhibited the enzyme activity, while the inhibition by Co-2+ was only partial. Fe-2+ did not exert any effect on the activity. The enzyme activity was completely inhibited by EDTA and was restored almost to the original value by metal ions like Mn-2+, Mg-2+, Ca-2+ and Ba-2+. The approximate molecular weight of the purified enzyme was estimated to be 56 000.     

Evidence for mammalian collagenases as zinc ion metalloenzymes.

Collagenases (EC 3.4.24.3) from human skin, rat skin and rat uterus were inhibited by the chelating agents EDTA, 1,10-phenanthroline and tetraethylene pentamine in the presence of excess Ca2+, suggesting that a second metal ion participates in the activity of the enzyme. Collagenase inhibition by 1,10-phenanthroline could be both prevented and reversed by a number of transition metal ions, specifically Zn2+, Co2+, Fe2+ and Cu2+. However, Zn2+ is effective in five-fold lower molar concentrations (1-10(-4) M) than the other ions. Furthermore, Zn2+ was the only ion tested able to prevent and reverse the inhibition of collagenase by EDTA in the presence of excess Ca2+. Atomic absorption analysis of purified collagenase for Zn2+ showed that Zn2+ was present in the enzyme preparations, and that the metal co-purifies with collagenase during column chromatography.     

Phosphorylase phosphatase complex from skeletal muscle. Activation of one of two catalytic subunits by manganese ions

Sarcoplasmic phosphorylase phosphatase extracted from ground skeletal muscle was recovered in a high molecular weight from (Mr = 250000). This enzyme has been purified from extracts by anion-exchange and gel chromatography to yield a preparation with three major protein components of Mr 83000, 72000, and 32000 by sodium dodecyl sulfate gel electrophoresis. The phosphorylase phosphatase activity of the complex form was activated more than 10-fold by Mn2+, with a K0.5 of 10(-5) M, but not by Mg2+ or Ca2+. Manganese activation occurred over a period of several minutes and resulted primarily in an increase in Vmax of a phosphatase that was sensitive to trypsin. Activation persisted after gel filtration, and the active form of the enzyme did not contain bound manganese measured by using 54Mn2+. A contaminating p-nitrophenylphosphatase was activated by either Mn2+ (K0.5 of 10(-4) M) or Mg2+ (K0.5 of 10(-3) M). Unlike the protein phosphatase this enzyme was inactive following removal of the metal ions by gel filtration. The phosphatase complex could be dissociated into its component subunits by precipitation with 50% acetone at 20 degrees C in the presence of an inert divalent cation, reducing agent, and bovine serum albumin. Two catalytic subunits were quantitatively recovered; one of Mr 83000 was a trypsin-sensitive manganese-activated phosphatase and the second of Mr 32000 was trypsin-stable and metal ion dependent. Both enzymes were effective in catalyzing the dephosphorylation of either phosphorylase a or the regulatory subunit of adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase, but neither subunit possessed p-nitrophenylphosphatase activity.     

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.     

Comparison of asymmetric forms of acetylcholinesterase from the electric organ of Narke japonica and Torpedo californica

The asymmetric forms of acetylcholinesterase were purified from the electric organs of the electric rays Narke japonica and Torpedo californica, and their properties were compared. Asymmetric acetylcholinesterase was purified by immunoaffinity chromatography with a monoclonal antibody (Nj-601) to acetylcholinesterase. The MgCl2 extracts of these electric organs were applied to a column of Nj-601-Sepharose, and the bound acetylcholinesterase was eluted by lowering the pH of the eluent to 2.8. The purified asymmetric acetylcholinesterases gave peaks of 17 S (A12) and 13 S (A8) on sucrose density gradients. The enzyme from N. japonica contained more A8 than A12, while that of T. californica contained more A12. After treatment with collagenase, the enzymes gave three peaks on sedimentation; 20 S, 16 S and 11 S for N. japonica, and 19 S, 15 S and 11 S for T. californica, indicating the presence of collagen-like tails. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, the asymmetric acetylcholinesterase from N. japonica gave bands of Mr 140 000, 100 000, 70 000 and 60 000, while that from T. californica gave bands of Mr 140 000, 100 000, 70 000 and 55 000. The bands of Mr 70 000 and 140 000 were monomers and non-reducible dimers, respectively, of the catalytic subunits. The bands of Mr 60 000 and 55 000 were the tail subunits, since collagenase treatment of the purified enzymes markedly decreased the amounts of these components. The Mr 100 000 subunit constituted less than 3% of the total asymmetric acetylcholinesterase from N. japonica but 18% of that from T. californica. The tail subunits constituted 6-8% of the two preparations. The catalytic subunits and the Mr 100 000 subunits bound concanavalin A, indicating that they are glycoproteins. The amino acid compositions of the enzymes from N. japonica and T. californica were very similar. Both contained hydroxyproline and hydroxylysine, characteristic of the collagen-like tails. The enzyme required divalent metal ions for activity, but only Mn2+, Mg2+ and Ca2+ were effective. Mn2+ was effective at the lowest concentrations, while Mg2+ gave the highest activity.     

Purification and properties of the MboII, a class-IIS restriction endonuclease.

After five purification steps a homogeneous preparation of endonuclease MboII was obtained, and several properties of the enzyme were determined. MboII is a monomer, with Mr under native and denaturing conditions being 47-49 x 10(3) Da. Endonuclease MboII is a basic protein (pI 8.3) which remains active when Mg2+ is replaced by Mn2+, Co2+, Ca2+, or Fe2+. MboII exhibits a star activity in the presence of some of the following reagents or ions: DMSO, glycerol, ethanol (and Co2+ or Mn2+ at pH 6). MboII does not bend DNA and is heat sensitive, losing activity after 15 min at 50 degrees C.     

Probing the metal-binding sites of cod parvalbumin using europium(III) ion luminescence and diffusion-enhanced energy transfer.

Excitation spectroscopy of the 7F0----5D0 transition of Eu3+ and diffusion-enhanced energy transfer are used to study metal-binding characteristics of the calcium-binding protein parvalbumin from codfish. Energy is transferred from Eu3+ ions occupying the CD- and EF-binding sites to the freely-diffusing Co(III) coordination complex energy acceptors: [Co(NH3)6]3+, [Co(NH3)5H2O]3+, [CoF(NH3)5]2+, [CoCl(NH3)5]2+, [Co(NO2)3(NH3)3], and [Co(ox)3]3-. In the absence of these inorganic energy acceptors, the excited-state lifetimes of Eu3+ bound to the CD and EF sites are indistinguishable, even in D2O; however, in the presence of the positively charged energy acceptor complexes, the Eu3+ probes in the cod parvalbumin have different excited-state lifetimes due to a greater energy-transfer site from Eu3+ in the CD site than from this ion in the EF site. The observation of distinct lifetimes for Eu3+ in the two sites allows the study of the relative binding site affinities and selectivity, using other members of the lanthanide ion series. Our results indicate that during the course of a titration of the metal-free protein, Eu3+ fills the two sites simultaneously. Eu3+ is competitively displaced by other Ln3+ ions, with the CD site showing a preference for the larger Ln3+ ions while the EF site shows little, if any, competitive selectivity across the Ln3+ ion series.     

Monoclonal antibodies to human fibroblast procollagenase. Inhibition of enzymatic activity, affinity purification of the enzyme, and evidence for clustering of epitopes in the NH2-terminal end of the activated enzyme

This study describes 11 monoclonal antibodies (Mabs) against human fibroblast collagenase that (i) inhibit the specific catalytic activity of the enzyme and/or (ii) react with one or more forms of the enzyme on Western blots. Each of the Mabs specifically immunoprecipitated the Mr 57,000/52,000 procollagenase from [35S]methionine-labeled culture medium. Five Mabs, designated VI-3, VI-4, 2C5, 4A2, and 7C2, inhibited the activity of fibroblast-type collagenase against soluble monomeric collagen and against reconstituted collagen fibrils but did not inhibit the genetically distinct human PMN leukocyte collagenase. The interstitial collagenase produced by human mucosal keratinocytes (SCC-25) was also inhibited, whereas the corresponding enzyme from rat was not. Assignment of epitopes to structural domains within the molecule based on immunoperoxidase staining of Western blots of collagenase and its autocatalytic fragments revealed that 9 of 11 epitopes, including those recognized by 4 inhibitory Mabs, were clustered in a 169-residue domain, which constitutes the NH2-terminal part of the Mr 46,000/42,000 active enzyme. One Mab (X-2a) specifically recognized the Mr 57,000/52,000 zymogen species and failed to react with the active Mr 46,000/42,000 form. The inhibitory Mab VI-3 was used for immunoaffinity purification of procollagenase from culture media with a recovery better than 80% and a yield of approximately 1.4 mg of enzyme/L of medium.     

Purification and characterization of a streptomycete collagenase

A soil streptomycete designated as Streptomyces sp. A8 produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase' as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-a rginine into two tripeptides. The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G-150 gel filtration. The purified enzyme had an apparent molecular weight of about 75,000 by SDS-polyacrylamide gel electrophoresis. Treatment with lithium chloride did not dissociate it into subunits. A strong inhibition was observed with chelating agents such as alpha-alpha-dipyridyl and 8-hydroxyquinoline. Ethylene diamine tetraacetate completely inhibited the enzyme activity. Among the cations tested only Ca2+ and Mg2+ enhanced the collagenase activity. Heavy metal ions like Pb2+, Ag+, Cu2+ and Zn2+ strongly inhibited the enzyme. The EDTA inhibition could be reversed with Ca2+. Cysteine and reduced glutathione caused significant reduction in enzyme activity. Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase. Amino acid analysis revealed the absence of cysteine and tyrosine. Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus.     

Purification and characterization of cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Effects of divalent cations on activity

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase purified greater than 13,000-fold to apparent homogeneity from calf liver exhibited a single protein band (Mr approximately 102,000) on polyacrylamide gel electrophoresis under denaturing conditions. Enzyme activity comigrated with the single protein peak on analytical polyacrylamide gel electrophoresis, sucrose density gradient centrifugation, and gel filtration. From the sedimentation coefficient of 6.9 S and Stokes radius of 67 A, an Mr of 201,000 and frictional ratio (f/fo) of 1.7 were calculated, suggesting that the native enzyme is a nonspherical dimer of similar, if not identical, peptides. The effectiveness of Mg2+, Mn2+, and Co2+ in supporting catalytic activity depended on the concentration of cGMP and cAMP present as substrate or effector. Over a wide range of substrate concentrations, optimal concentrations for Mg2+, Mn2+, and Co2+ were about 10, 1, and 0.2 mM, respectively. At concentrations higher than optimal, Mg2+ inhibited activity somewhat; inhibition by Co2+ (and in some instances by Mn2+) was virtually complete. At low substrate concentrations, activity with optimal Mn2+ was equal to or greater than that with Co2+ and always greater than that with Mg2+. With greater than or equal to 0.5 microM cGMP or 20 to 300 microM cAMP and for cAMP-stimulated cGMP or cGMP-stimulated cAMP hydrolysis, activity with Mg2+ greater than Mn2+ greater than Co2+. In the presence of Mg2+, the purified enzyme hydrolyzed cGMP and cAMP with kinetics suggestive of positive cooperativity. Apparent Km values were 15 and 33 microM, and maximal velocities were 200 and 170 mumol/min/mg of protein, respectively. Substitution of Mn2+ for Mg2+ increased apparent Km and reduced Vmax for cGMP with little effect on Km or Vmax for cAMP. Co2+ increased Km and reduced Vmax for both. cGMP stimulated cAMP hydrolysis approximately 32-fold in the presence of Mg2+, much less with Mn2+ or Co2+. In the presence of Mg2+, Mn2+ and Co2+ at concentrations that increased activity when present singly inhibited cGMP-stimulated cAMP hydrolysis. It appears that divalent cations as well as cyclic nucleotides affect cooperative interactions of this enzyme. Whereas Co2+ effects were observed in the presence of either cyclic nucleotide, Mn2+ effects were especially prominent when cGMP was present (either as substrate or effector).     

Synthesis of collagenase and collagenase inhibitors by osteoblast-like cells in culture

A rat osteosarcoma cell clone (ROS 17/2), and osteoblast-enriched populations from rat calvaria cultured in the presence of concanavalin A, have been shown to produce latent collagenase and collagenase inhibitors. The enzymes and inhibitor activities from the ROS 17/2 cells were concentrated by ammonium sulphate precipitation and separated by gel filtration on AcA 54 resin. The size of the latent collagenase (Mr approximately equal to 58000) was reduced on conversion to active enzyme (Mr approximately equal to 48000) by p-aminophenylmercuric acetate. Latent and active forms of gelatinase activity, similar in size to the corresponding forms of collagenase, were also resolved. The collagenase inhibitor activity, which was sensitive to organomercurials, was recovered in two peaks (Mr approximately equal to 68000 and 30000). The active collagenase cleaved interstitial collagens (type I = III greater than II) producing typical 3/4 and 1/4 fragments. This activity was inhibited by the metal ion chelators ethylenediaminetetraacetic acid and o-phenanthroline. Additional specific cleavages of native collagen were also observed which, from the susceptibility of this activity to phenylmethylsulphonyl fluoride, leupeptin and antipain, suggested the presence of a second collagenolytic enzyme. This synthesis of collagenolytic enzymes by these osteoblast-like cells suggests that individual osteoblasts, like fibroblasts, are capable of both synthesizing and degrading their respective organic matrices in vivo.     

Interactions of tervalent lanthanide ions with bacterial collagenase (clostridiopeptidase A)

Tervalent cations of the lanthanide (rare-earth) elements reversibly inhibit bacterial collagenase (clostridiopeptidase A; EC 3.4.24.3). Sm(3+), whose ionic radius is closest to that of Ca(2+), is the most effective inhibitor, completely suppressing clostridiopeptidase activity at a concentration of 100mum in the presence of 5mm-Ca(2+). Er(3+) and Lu(3+), which both have ionic radii smaller than either Ca(2+) or Sm(3+), inhibit less efficiently, and La(3+), which is slightly larger than Ca(2+) or Sm(3+), inhibits only weakly. These findings indicate a closely fitting, stereospecific, Ca(2+)-binding pocket in clostridiopeptidase, which excludes ions that are only slightly larger than Ca(2+) [ionic radius 0.099nm (0.99 A)]. By contrast, trypsin, an enzyme whose activity does not depend on Ca(2+), requires lanthanide concentrations 50-100-fold greater for inhibition. Furthermore, the relative efficiency of inhibition of trypsin by lanthanides increases as the lanthanide ions become smaller and the charge/volume ratio increases. At a concentration of 50mum, Sm(3+) lowers the apparent K(m) for the hydrolysis of Pz-peptide by clostridiopeptidase from 5.4mm to 0.37mm and the apparent V(max.) from 0.29 Wünsch-Heidrich unit to 0.018 unit. Thus Sm(3+) enhances the affinity of this enzyme for its substrate; inhibition of hydrolysis of Pz-peptide may result from the excessive stability of the enzyme-Sm(3+)-substrate complex. Inhibition by Sm(3+) is competitive with regard to Ca(2+). The apparent dissociation constant, K(d), of Ca(2+) is 0.27mm, where the K(i) for Sm(3+) is 12mum. Clostridiopeptidase is more thermolabile in the absence of Ca(2+). With Sm(3+), thermoinactivation of the enzyme at 53 degrees C or 60 degrees C is initially accelerated, but then becomes retarded as heating continues. Lanthanide ions bind to gelatin and collagen. In so doing, they appear to protect these substrates from lysis by clostridiopeptidase through mechanisms additional to supplanting Ca(2+) at its binding site on the enzyme. Collagen and gelatin sequester sufficient lanthanide ions to gain partial protection from clostridiopeptidase in the absence of an extraneous source of these inhibitors.     

Regulation of activity of purified guanylate cyclase from liver that is unresponsive to nitric oxide.

Guanylate cyclase was purified from rat liver supernatant. Electrophoresis under denaturing conditions revealed one major peptide of Mr approx. 69 000. On the basis of the Stokes radius (4.7 nm) and S20,w (6.4S), the calculated Mr value of the native enzyme was 133 000, i.e. it is apparently a homodimer. Kinetics of inactivation by diamide (which was reversible with dithiothreitol) suggested that oxidation of a single class of thiol sites was involved. In the absence of other additions, cyclase activity assayed with Mn2+ was over 7 times that assayed with Mg2+; maximal effects were observed with approx. 5 mM of each (with 1 mM-GTP). The purified enzyme was markedly activated by nitrosylhaemoglobin. Relative activation was much greater in assays with Mg2+ than with Mn2+, although maximal activities were similar. When assayed with Mg2+, the enzyme exhibited a single Km (0.35 mM) for GTP; with Mn2+, plots of 1/v versus 1/[S] were non-linear. Activator or nitrosylhaemoglobin increased Vmax, but did not alter Km in the presence of either Mg2+ or Mn2+. The enzyme was inhibited by Na3VO4, Na2WO4 and Na2B4O7. Reduction from VV to VIV abolished the inhibitory effect of vanadate. Na2B4O7 (2 mM) inhibited activity with Mn2+, but not with Mg2+. In assays with Mg2+, but not with Mn2+, FMN, NAD+ and NADH (each 0.5 mM) inhibited activation by protoporphyrin IX and nitrosylhaemoglobin. Rotenone (0.6 mM) inhibited activity with protoporphyrin IX to a greater extent than with nitrosylhaemoglobin. Methylene Blue (1 mM) inhibited activation by nitrosylhaemoglobin, protoporphyrin IX and activator. It appears that this enzyme purified from rat liver lacks haem (and perhaps other components) required for activation by NO, and it should be particularly useful for elucidating the mechanism of action of NO, protoporphyrin IX and other activators.     

The events relating to lanthanide ions enhanced permeability of human erythrocyte membrane: binding, conformational change, phase transition, perforation and ion transport.

The binding and uptake of Gd3+ ions by human erythrocytes in vitro were studied by determining the Gd contents in membrane and in cytosol by means of particle-induced X-ray emission (PIXE) spectrometry. Results obtained from varied incubation time revealed that the Gd3+ ions bind to the membrane proteins and lipids at first. Gd3+ binding to the membrane lipids and proteins lasts 0 approximately 20 and 20 approximately 100 ms respectively, as shown by the stopped-flow studies. Then a fraction of Gd3+ ions diffuses through the membrane. The kinetics of Gd3+ binding indicates that the binding to phospholipids is prior to that to the membrane proteins, but a portion of the lipid-bound Gd3+ redistributed later to the proteins. PIXE studies showed that the entry of Gd3+ increased the influx of Ca2+ and Cl-. By monitoring the changes in fluorescence of proteins and that of the Ln3+, the uptake of La3+, Eu3+, Gd3+ and Tb3+ was shown to be a process comprising a series of events. Binding to the membrane molecules induces the phase transition of lipid bilayer and conformational changes and aggregation of membrane proteins. Conformational changes of the proteins were characterized by Fourier transform IR spectroscopy (FT-IR) deconvolved spectra, i.e. alpha-helix content decreases while beta-sheet increases. ESR spectra of MSL-labeled proteins reflect the aggregation state related with the conformational change. [31P]NMR spectra of membrane lipid bilayer revealed the Ln3+ ions induced hexagonal (H(II)) phase formation. Phase transition and aggregation of membrane proteins cause the formation of domain structure and perforation in the membrane. These alterations in membrane structure are responsible for the Ln3+ enhanced membrane permeability. Thus the previous Ln3+ binding will facilitate the across-membrane transport of other Ln3+ ions through the membrane.     

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.     

Inositol phosphate metabolism in bradykinin-stimulated human A431 carcinoma cells. Relationship to calcium signalling.

A high-Mr neutral endopeptidase-24.5 (NE) that cleaved bradykinin at the Phe5-Ser6 bond was purified to apparent homogeneity from human lung by (NH4)2SO4 fractionation, ion-exchange chromatography and gel filtration. The final enzyme preparation produced a single enzymically active protein band after electrophoresis on a 5% polyacrylamide gel. Human lung NE had an Mr of 650,000 under non-denaturing conditions, but after denaturation and electrophoresis on an SDS/polyacrylamide gel NE dissociated into several lower-Mr components (Mr 21,000-32,000) and into two minor components (Mr approx. 66,000). The enzyme activity was routinely assayed with the artificial substrate Z-Gly-Gly-Leu-Nan (where Z- and -Nan represent benzyloxycarbonyl- and p-nitroanilide respectively). NE activity was enhanced slightly by reducing agents, greatly diminished by thiol-group inhibitors and unchanged by serine-proteinase inhibitors. Human lung NE was inhibited by the univalent cations Na+ and K+. No metal ions were essential for activity, but the heavy-metal ions Cu2+, Hg2+ and Zn2+ were potent inhibitors. With the substrate Z-Gly-Gly-Leu-Nan a broad pH optimum from pH 7.0 to pH 7.6 was observed, and a Michaelis constant value of 1.0 mM was obtained. When Z-Gly-Gly-Leu-Nap (where -Nap represents 2-naphthylamide) was substituted for the above substrate, no NE-catalysed hydrolysis occurred, but Z-Leu-Leu-Glu-Nap was readily hydrolysed by NE. In addition, NE hydrolysed Z-Gly-Gly-Arg-Nap rapidly, but at pH 9.8 rather than in the neutral range. Although human lung NE was stimulated by SDS, the extent of stimulation was not appreciable as compared with the extent of SDS stimulation of NE from other sources.     

Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.