Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A metalloprotease from Bothrops jararaca venom (J protease) was purified by DEAE-Sephacel, CM-cellulose, Sephacryl S-200 and Sephadex G-75 chromatograph. The proteolytic activity was inactivated by EDTA, o-phenanthroline and DTNB. Phosphoramidon and cysteine protease inhibitors (leupeptin, E64 and its derivatives) were inactive on this enzyme. J protease was activated by calcium and the metal content analysis showed the presence of one mole each of tightly bond zinc and calcium per mole of this J protease. The amino acid composition, N-terminal amino acid sequence (29 residues) and the cleavage sites on the oxidized insulin B chain and angiotensin I were determined.
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PMID:Purification and some characteristics of a zinc metalloprotease from the venom of Bothrops jararaca (jararaca). 278 74

9-(Dicyanovinyl)julolidine (DCVJ) is a fluorescent dye whose intramolecular rotational relaxation is solvent dependent. Since its quantum yield increases with decreasing free volume, this molecule has been very useful in monitoring synthetic polymer reactions and measuring local microviscosity changes in phospholipid bilayers [Loutfy, R. O. (1986) Pure Appl. Chem. 58, 1239-1248; Kung, C. E., & Reed, J. K. (1986) Biochemistry 25, 6114-6121]. We have used DCVJ to follow the polymerization of tubulin, a protein that can assemble into a variety of polymorphic microstructures. DCVJ binding to free tubulin is accompanied by an increase in quantum yield, indicating that DCVJ has become partially immobilized. At 4 degrees C, DCVJ binds to a single population of high-affinity hydrophobic sites (Kd = 1.12 +/- 0.26 microM) with a stoichiometry that is protein concentration dependent. n, the number of moles of DCVJ bound per mole of alpha beta dimer, approaches 1 at concentrations less than or equal to 0.5 mg/mL but decreases to a lower limit of approximately 0.3 at concentrations greater than or equal to 2.0 mg/mL. The quantum yield also increases with increasing protein concentration. This trend is unaltered by the presence of microtubule-associated proteins. These results are analyzed in terms of a concentration-dependent oligomerization of tubulin at 4 degrees C. When tubulin is polymerized at 37 degrees C to microtubules or to sheets in the presence of Zn2+, the fluorescence intensity of DCVJ increases although the magnitude of this increase differs significantly. We are able to use the distinct fluorescent and binding characteristics of the bound dye to distinguish between these two polymorphs on a molecular level.
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PMID:Fluorescent molecular rotors: a new class of probes for tubulin structure and assembly. 279 23

A recently introduced hematofluorometer, the "Protofluor-Z" (Helena Laboratories, Beaumont, TX), has several novel features, most notably reporting units expressed as the molar ratio of zinc protoporphyrin (ZPP) to heme, i.e., micromoles of ZPP per mole of heme. We analyzed human blood specimens on the Protofluor-Z and by an ethyl acetate/acetic acid extraction procedure. Data from three laboratories were pooled and used to provide a comparison of the two methods. Results indicate that, with the Protofluor-Z, a value of 70 mumol of ZPP per mole of heme is approximately equivalent to the recommended screening cutoff of 35 micrograms of erythrocyte protoporphyrin per 100 mL of whole blood used in programs for pediatric lead-poisoning prevention. This empirically determined value is slightly lower than either that recommended by the manufacturer or a theoretical cutoff value that was determined mathematically.
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PMID:Empirically determined lead-poisoning screening cutoff for the Protofluor-Z hematofluorometer. 232 55

Lysyl oxidase was purified to about 3,000- to 5,000-fold from epiphyseal cartilages of chick embryos. Purification was accomplished by sequential column chromatographies of collagen-sepharose, DEAE-Cellulose and Sephacryl S-200. The molecular weight of the most purified enzyme from 4 different isomers as determined by gel-filtration technique using Sephacryl S-200 column was 32,000 and the purified enzyme contained one copper atom per mole. When the enzyme (copper content: 1.12 g-atom per mole) was incubated with 1 X 10(-4)M cadmium, followed by gel-filtration, 0.12 g-atom of cadmium was incorporated into the enzyme per mole and consequently 0.23 g-atom of prosthetic copper was released from the enzyme (loss of 21%) with a substantial decrease of the enzyme activity by 22.8%, but 50% loss of copper caused by treatment with 1 X 10(-3)M cadmium did not further decrease the activity. When the enzyme was incubated with 1 X 10(-4)M zinc, 0.38 g-atom of zinc bound to the enzyme and 65% copper was lost, resulting in 34% loss of the activity. Loss of 84% copper (experiment with 1 X 10(-3)M zinc), however, led to 47% inhibition. These findings indicate the existence of metal-metal interaction in the enzyme that can significantly influence the activity, though inhibition of the enzyme was not strictly proportional to either the amount of copper released or the amount of cadmium or zinc bound to the enzyme.
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PMID:Cadmium- or zinc-binding to bone lysyl oxidase and copper replacement. 286 31

Multinuclear 1 and 2 dimensional magnetic resonance methods have been used to investigate the structures and metal binding properties of metallothioneins (MTs) isolated from several different sources. 113Cd NMR studies have unambiguously shown that the 7 g-atoms of Cd2+ bound per mole of the mammalian MT are located in two separate metal clusters, one containing 4 metal ions and the other, 3 metal ions. In the invertebrate (Scylla serrata) MT, similar studies have revealed that the 6 g-atoms of bound Cd2+ are distributed in two distinct 3-metal clusters while in Neurospora MT, the 3 g-atoms of bound Cd2+ are arranged in a pseudo 3-metal cluster. With the exception of one of the Cd2+ sites in this latter cluster, all the Cd2+ ions are tetrahedrally coordinated to four cysteine thiolate ligands with single cysteinyl sulfurs bridging adjacent metals. These conclusions are based on the 113Cd chemical shift data and a detailed analysis of the observed 113Cd-113Cd scalar couplings by both homonuclear decoupling and 2D techniques. In addition, the 113Cd NMR studies have revealed significant differences in the affinity of different metal ions for the two mammalian metal clusters. For the 3-metal cluster, the affinity is found to decrease in the order Cu+ greater than Cd2+ greater than Zn2+ with Cd2+ greater than Zn2+ for the 4 metal cluster and Cd2+ (4-metal cluster) greater than Cd2+ (3-metal cluster). The 113Cd NMR data are currently being integrated with 500 MHz 2D 1H and 1H-113Cd chemical shift correlated multiple quantum data sets to more completely define the structural arrangement of the metal clusters in the tertiary structure of these proteins.
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PMID:NMR analysis of the structure and metal sequestering properties of metallothioneins. 295 2

Zinc is essential to the catalytic activity of angiotensin converting enzyme. The enzyme contains one g-atom of zinc per mole of protein. Chelating agents abolish activity by removing the metal ion to yield the inactive, metal-free apoenzyme. Zinc does not stabilize protein structure since the native and apoenzymes are equally susceptible to heat denaturation. Addition of either Zn2+, Co2+, or Mn2+ to the apoenzyme generates an active metalloenzyme; Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ fail to restore activity. The activities of the metalloenzymes follow the order Zn greater than Co greater than Mn. The protein binds Zn2+ more firmly than it does Co2+ or Mn2+. Hydrolysis of the chromophoric substrate, furanacryloyl-Phe-Gly-Gly, by the active metalloenzymes is subject to chloride activation; the activation constant is not metal dependent. Metal replacement mainly affects Kcat with very little change in Km, indicating that the role of zinc is to catalyze peptide hydrolysis.
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PMID:The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism. 299 78

Efficient delivery of hydrophobic water-insoluble substrates and cofactors to membrane-bound enzymes is a recurring problem which has impeded kinetic analyses. Kinetic analysis of the Escherichia coli sn-1,2-diacylglycerol kinase, an extremely hydrophobic integral membrane protein of 122 residues, was facilitated by the development of a mixed micellar assay. beta-Octyl glucoside micelles quantitatively solubilized diacylglycerol kinase from membranes of strains which overproduced the enzyme up to 250-fold and provided an effective method to disperse and deliver the hydrophobic water-insoluble substrate, sn-1,2-dioleoyglycerol. Diacylglycerol kinase was active in mixed micelles containing octyl glucoside and dioleoyglycerol. Several phospholipids stimulated activity up to 6-fold, suggesting a cofactor function. Activation by phospholipids was not stereospecific and was mimicked partially by fatty acids. Half-maximal activation was observed at 1 mol % cardiolipin, suggesting that a small number of phospholipids are sufficient to activate the enzyme. Activity was dependent on the mole fractions of dioleoylglycerol and phospholipid in the mixed micelles, but independent of micelle number. Several lines of evidence indicated that the transfer of diacylglycerol between micelles was much more rapid than its utilization by the enzyme. Diacylglycerol kinase exhibited Michaelis-Menten kinetics with respect to diacylglycerol and MgATP. A second Mg2+ ion (in addition to MgATP) was required for activity. When Mg2+ was excluded from the assay, Mn2+, Zn2+, Cd2+, and Co2+ supported activity to lesser extents. These data establish a suitable system for in-depth kinetic analysis of the E. coli diacylglycerol kinase and its phospholipid cofactor requirements.
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PMID:sn-1,2-Diacylglycerol kinase of Escherichia coli. Mixed micellar analysis of the phospholipid cofactor requirement and divalent cation dependence. 300 49

When Neurospora crassa is grown in the presence of Cu(II) ions, it accumulates the metal with the concomitant synthesis of a low molecular weight copper-binding protein. The molecule binds 6 g-atom of copper per mole protein (Mr = 2200) and shows a striking sequence homology to the zinc- and cadmium-binding vertebrate metallothioneins. Absorption, circular dichroism, and electron paramagnetic resonance spectroscopy of Neurospora metallothionein indicate the copper to be bound to cysteinyl residues as a Cu(I)-thiolate complex of the polymeric mu-thiolate structure [Cu(I)6RS7]-. This metal-binding mode is also in agreement with the unusual luminescence of the protein. Spectral perturbation studies with HgCl2 and p-(chloromercuri)benzoate suggest that the 6 Cu(I)ions are coordinated to the seven cysteinyl residues in the form of a single metal cluster. Neurospora apometallothionein is also capable of binding in vivo group IIB metal ions [Zn(II), Cd(II), and Hg(II)] as well as paramagnetic Co(II) ions with an overall metal-to-protein stoichiometry of 3. The spectroscopic properties of the fully substituted forms are indicative of a distorted tetrahedral coordination. However, metal titration of the apoprotein shows the third metal ion to be differently coordinated than the other two metal ions. This difference can be explained by the presence of only seven cysteine residues in Neurospora metallothionein as opposed to nine cysteine residues in the three-metal cluster of the mammalian metallothioneins.
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PMID:Primary structure and spectroscopic studies of Neurospora copper metallothionein. 301 91

The reaction of a copper(II) or nickel(II) imidazolate complex (M[CBP-PHEN-4-CHO-Im]) with zinc(II)tetraphenylporphyrin (TPP) in toluene results in the formation of an imidazolate bridged heterobinuclear axial adduct. Conversion of the four-coordinated Zn(TPP) to the five-coordinated species is followed in the visible region between 700 and 500 nm. Isosbestic behavior is exhibited at 523, 556, 588, and 638 nm by solutions of Zn(TPP) to which varying amounts of the metal imidazolate complex are added, indicating the existence of an equilibrium between Zn(TPP) and its axial adduct. The products exhibit maxima beta and alpha bands at 566 and 606 nm, respectively, which are red-shifted from 548 and 588 nm for Zn(TPP) and yield epsilon alpha/epsilon beta ratios of 0.57 and 0.55 for the Ni(II) and Cu(II) adducts, respectively. The binding of the metal imidazolate complexes is thought to closely resemble that of N-methylimidazole, N-CH3Im, rather than imidazolate, owing to the close spectral similarities with the adduct of the former and significant differences from the latter. Formation constants were determined using the 548-nm beta band of Zn(TPP) in the 293-308 K range by the method of Rose and Drago. At 25 degrees C, K = 152,000 M-1 and 110,000 M-1 for the copper and nickel adducts, respectively. Comparison of these values to that of 54,100 M-1 for N-CH3Im indicates that the metal-imidazolate complexes are considerably more reactive. Van't Hoff plots for the two series are very similar with enthalpies of -41.9 and -43.3 kJ/mole respectively. The structural core of these complexes is similar to the imidazolate bridged model of cytochrome c oxidase in that they contain a metal imidazolate axially adducted to a metalloporphyrin.
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PMID:Imidazolate bridged heterobinuclear complexes of zinc(II)tetraphenylporphyrin. Modeling cytochrome c oxidase. 301 87

Metallothioneins isolated from the hepatopancreas of the edible crab (Cancer paqurus) and the plaice (Pleuronectes platessa) after cadmium injection are predominantly cadmium proteins containing only small amounts of zinc and traces of copper. The removal of metal ions from the two metallothioneins by EDTA was studied using proton NMR spectroscopy. The rates of removal of cadmium and zinc were monitored directly from the intensity of the resonances due to the cadmium and zinc-EDTA complexes. Nearly all the zinc present in the protein was extracted by EDTA relatively rapidly, whereas only 10 to 20% of the total cadmium was removed in at least three steps. The total (Cd + Zn) metal removed at equilibrium was 1.2 to 1.8 g-ions/mole protein. Information on conformational changes in the protein were also obtained from studying alterations in the proton resonances of the protein. This was directly correlated with removal of metal from the protein. The coordination environments of the cadmium ions in crab metallothionein were investigated by using 113Cd-NMR, and compared with 113Cd-NMR spectra of rabbit liver MT-II and Scylla serrata MT-I.
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PMID:NMR studies of crab and plaice metallothioneins. 308 76


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