Proton magnetic resonance studies of histidines in human, rhesus monkey, and bovine carbonic anhydrases.

Histidine C-2 proton resonances in rhesus monkey carbonic anhydrase B (carbonate hydro-lyase, EC 4.2.1.1) and bovine carbonic anhydrase were investigated using 270-MHz proton magnetic resonance. The results suggest that there are extensive three-dimensional homologies between the human B and rhesus B enzymes and between the human C and bovine enzymes. Resonances from solvent exchangeable protons have been observed in the 11-16 ppm range in the NMR spectra of human carbonic anhydrases B and C and bovine carbonic anhydrase. Up to five of these are sensitive to changes of pH and the presence of inhibitors. Three of these resonances are assigned to NH protons of the metal coordinated imidazole groups. These results are discussed in relation to various models for the catalytic mechanism of carbonic anhydrase.     

Paramagnetic probes attached to a light chain on the myosin head are highly disordered in active muscle fibers.

We have measured the orientation of a region of the myosin head, close to the junction with the rod, during active force generation. Paramagnetic probes were attached specifically to a reactive cysteine (Cys 125) of purified myosin light chain 2 (LC2) and exchanged into myosin heads in glycerinated rabbit psoas muscle. Electron paramagnetic resonance spectroscopy was used to monitor the orientation of the probes. Previous work has shown that the LC2 bound spin probes are significantly ordered in rigor and muscle in the presence of adenosine diphosphate (ADP). In contrast, there is a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, all of the LC2 bound spin probes (98 +/- 1.6%) show an angular distribution similar to that of relaxed muscle. These findings contrast with results obtained from probes attached to Cys 707 on the cross-bridge, located close to the actin binding site, where, during active force generation, a proportion of the spin probes were ordered as in rigor, whereas the remaining probes were disordered as in relaxation. To test the hypothesis that this ordered component is due to modification of Cys 707, we measured the spectra obtained from probes attached to LC2 in fibers modified at Cys 707. The modification of Cys 707 did not produce an ordered component in these spectra. The absence of an ordered component at the LC2 site limits the populations of some states in active fibers. An actin/myosin/ADP state is thought to be the major force-producing state. Our present results show that the populations of states with ordered probes on LC2 are < 2% in active fibers; thus, the major force-producing state is different from the one obtained by addition of ADP to rigor fibers.     

Expression of membrane-bound carbonic anhydrases IV, IX, and XIV in the mouse heart.

Expression of membrane-bound carbonic anhydrases (CAs) of CA IV, CA IX, CA XII, and CA XIV has been investigated in the mouse heart. Western blots using microsomal membranes of wild-type hearts demonstrate a 39-, 43-, and 54-kDa band representing CA IV, CA IX, and CA XIV, respectively, but CA XII could not be detected. Expression of CA IX in the CA IV/CA XIV knockout animals was further confirmed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Cardiac cells were immunostained using anti-CA/FITC and anti-alpha-actinin/TRITC, as well as anti-CA/FITC and anti-SERCA2/TRITC. Subcellular CA localization was investigated by confocal laser scanning microscopy. CA localization in the sarcolemmal (SL) membrane was examined by double immunostaining using anti-CA/FITC and anti-MCT-1/TRITC. CAs showed a distinct distribution pattern in the sarcoplasmic reticulum (SR) membrane. CA XIV is predominantly localized in the longitudinal SR, whereas CA IX is mainly expressed in the terminal SR/t-tubular region. CA IV is present in both SR regions, whereas CA XII is not found in the SR. In the SL membrane, only CA IV and CA XIV are present. We conclude that CA IV and CA XIV are associated with the SR as well as with the SL membrane, CA IX is located in the terminal SR/t-tubular region, and CA XII is not present in the mouse heart. Therefore, the unique subcellular localization of CA IX and CA XIV in cardiac myocytes suggests different functions of both enzymes in excitation-contraction coupling.     

Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase. A resonance energy transfer study

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R-NH2 group, presumably the epsilon-NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535-1558 (1962)] provides clear evidence that a pH 8 eight R-NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non-essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s). One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate. In the doubly labeled protein non-radiative single-singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (+/- 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8-1.1, 1.9-2.1, 2.9-3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8-5.1 nm from the donor site and third site being 6.4 nm apart from the donor. Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein-bound dansyl chloride and eosin isothiocyanate was observed. Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate-binding site with one lysyl residue crucial for activity.     

Histidine-200 alters inhibitor binding in human carbonic anhydrase B. A carbon-13 nuclear magnetic resonance identification.

We have previously prepared 13C-enriched NT-carboxymethylhistidine-200 human carbonic anhydrase B (CmHCAB) by reacting the native enzyme with 90% [1-13C]bromoacetate (Strader, D.J., and Khalifah, R.G. (1976), J. Am. Chem. Soc. 98, 5043). The 13C nuclear magnetic resonance signal of the enriched carboxylate of CmHCAB proved sensitive to active-site events, permitting, among other things, the determination of the microscopic pKa of the modified histidine. This report extends the study to the complexes of CmHCAB with the inhibitors iodide and azide. It is found that the pKa of histidine-200 is significantly increased when these inhibitors bind. A quantiative comparison of the iodide-induced pKa shift with literature data (Whitney, P. L., and Brandt, H. (1976), J. Biol, Chem. 251, 3862) showing that the binding of iodide is influenced by the ionization of an active-site group of pKa 6.1 allowed the clear identification of histidine-200 as the perturbing group. Other important implications of the magnetic resonance results are also discussed.     

Parameters involved in binding of porcine pancreatic alpha-amylase with black bean inhibitor: role of sulfhydryl groups, chloride, calcium, solvent composition and temperature

The amylase inhibitor of black (kidney) beans (Phaseolus vulgaris; MW 53,000) forms a 1:1 stoichiometric complex with porcine pancreatic alpha-amylase (MW 52,000) at pH 5.40. The single sulfhydryl group of the inhibitor and the two sulfhydryl groups of alpha-amylase are not involved in recognition and binding. Chloride ions, required for activity of alpha-amylase at both pH 5.40 and 6.90, are important for inhibitor--enzyme binding at pH 6.90 but not at pH 5.40. Calcium-free alpha-amylase binds with the inhibitor. An increase in the ionic strength of the solvent increases the rate of binding of the inhibitor with alpha-amylase; a decrease in the dielectric constant decreases the rate of binding; and decreasing the temperature increases the dissociation constant, Kd, of the complex. These data support the hypothesis that hydrophobic interaction is of primary importance in complex formation. The activation energy, Ea, for complex formation was found to be 12.4 kcal/mol at pH 5.40 and 24.2 kcal/mol at pH 6.90. In the presence of the poor substrate, p-nitrophenyl-alpha-D-maltoside, the Ea for complex formation was 4.1 kcal/mol at pH 6.90.     

The role of sulfhydryl groups in human neutrophil adhesion, movement and particle ingestion

Recent studies have suggested that ectosulfhydryl groups may play a role in cell contact phenomena. We have studied the possible role of ecto- and endosulfhydryl groups in the morphology, adhesiveness, random and directed (chemotaxis) motility and phagocytosis of human polymorphonuclear neutrophils. The rapidly penetrating sulfhydryl binding reagents HgCl₂ and NEM inhibited adhesiveness, motility and phagocytosis when studied at > 0.1 mM in plasma or > 0.01 mM in buffer. The difference in inhibitory concentration was shown to be due to the difference in albumin content of the two media. D-cysteine prevented the effect of HgCl₂ and NEM on cell morphology, adhesiveness, motility and phagocytosis indicating that their effects were on cell sulfhydryl groups. PCMBS, a very slowly penetrating organic mercurial, had no effect on neutrophil morphology, adhesiveness, motility or phagocytosis. However, PCMBS inhibited platelet aggregation, assuring its potency. These studies indicate that ectosulfhydryl groups are either not present or not participants in the maintainence of structure and functions of human neutrophilic granulocytes.     

Intermediates in the refolding of ribonuclease at subzero temperatures. 2. Monitoring by inhibitor binding and catalytic activity

The kinetics of refolding of ribonuclease A were monitored by the return of catalytic activity and inhibitor binding at -15 degrees C in 35% methanol cryosolvent at pH* 3.0 and 6.0. Catalytic activity was measured with cytidine 2',3'-cyclic monophosphate as substrate; inhibitor binding was determined with the competitive inhibitor cytidine 2'-monophosphate. Biphasic kinetics were observed at pH* 3.0 for both return of catalytic activity and inhibitor binding. At pH* 6.0 the rate of return of catalytic activity was monophasic, whereas that of inhibitor binding was biphasic. For both inhibitor binding and catalytic activity one of the observed rates was pH-dependent. Full return of catalytic activity was obtained at the completion of the refolding process. The observations are interpreted in terms of two parallel pathways of refolding for slow-refolding ribonuclease, with several native-like, partially folded intermediate states on the minor slow-refolding pathway. Of particular note is the presence of at least one such species that has inhibitor-binding capacity but not catalytic activity. This may be rationalized in terms of the known native structure. In addition, an intermediate is postulated which has the incorrect Pro-93 conformation and only partial catalytic activity (42% of the native). The slowest observed transient is attributed to the isomerization of this proline residue and return of full catalytic activity.     

Application of fluorescent probes to study structural changes in Aspergillus fumigatus exposed to amphotericin B, itraconazole, and voriconazole

The broad objective of this study was to document patterns of structural changes following antifungal treatment, and to determine any relationship with minimum inhibitory concentration (MIC) of an antifungal. Three clinical isolates of Aspergillus fumigatus, with high, intermediate, and low amphotericin B (AB), itraconazole (IZ), and voriconazole (VZ) MICs were studied in 24-well plates with cover slips. The fluorescent probes used were Calcofluor White (cell wall), propidium iodide (nucleus), and MitoTracker Green FM (mitochondria). Fluorescent microscopy as early as 3-h after exposure revealed that AB treated hyphae had intact cell wall with deformed mitochondria and nuclei while IZ and VZ treated hyphae revealed no intact cell wall, and deformation of mitochondria and nuclei. At 48 h, AB treated cells revealed rupture of hyphae and disintegration of mitochondria, and nuclei, IZ treated hyphae were swollen with disintegration of mitochondria, and nuclei while VZ treated hyphae showed rupture and disintegration of mitochondria and nuclei. The structural changes for the three strains studied were similar in fluorescent microscopy as long as the incubation time and their respective MICs were used. Thus, AB, IZ, and VZ induced gross organelle defects in A. fumigatus nuclei, mitochondria, and cell wall, which were consistent with respective MICs of antifungals used.     

Application of nile blue and nile red, two fluorescent probes, for detection of lipid droplets in human skeletal muscle

Using frozen sections from human muscle biopsies, we assessed the value of Nile blue and Nile red, two fluorescent probes, as stains for lipid droplets in normal and pathological skeletal muscle fibers. In normal muscle, lipid storage disorders, and mitochondrial myopathies, Nile blue stained the lipid droplets as yellow-gold fluorescent structures. The lipid droplets were also seen as yellow-gold fluorescent structures in Nile red-stained sections, but the outstanding feature in these preparations was the staining of the membrane network of the muscle fibers and membrane proliferations in pathological muscle as red-orange fluorescent structures. These results suggest that both Nile blue and Nile red stains are useful for visualization of lipid droplets and membrane proliferations in pathological muscle biopsies.     

Function of sulfhydryl groups in ribosome-elongation factor G reactions. Assignment of guanine nucleotide binding site to elongation factor G.

Titration of elongation factor G (EF-G) with the thiol reagents 5,5'-dithiobis(2-nitrobenzoate) (DNTB), p-hydroxymercuribenzoate (HMB), and N-ethylmaleimide and analysis of cysteic acid after performic acid oxidation revealed a total of four sulfhydryl groups per EF-G molecule. One of these is exposed in the native state and could be used to distinguish between two different conformations of EF-G in our preparations according to its rate of reaction with DTNB and HMB. No evidence for disulfide bridges was obtained. Among the different nucleotides tested, GTP, GDP, and GMP were able to protect the native sulfhydryl group against reaction with DTNB in the absence of ribosomes. Their Kd values with the faster reacting EF-G were 3.4 x 10(-4) M, 0.3 X 10(-4)M, and 2.0 x 10(-4) M, respectively. Because of the specificity of protection by guanine nucleotides and the correspondence of the Kd values with Ki values for GDP and GMP in the ribosome-EF-G GTPase reaction, their binding site on EF-G should be closely related to the active center for ribosome-dependent GTP hydrolysis. Blockage of the native sulfhydryl group of EF-G with a variety of irreversible thiol reagents reduced its activity from one to two-thirds in ribosome-dependent complex formation, GTP hydrolysis, and poly(U)-directed poly(phenylalanine) synthesis. A test of the N-ethylmaleimide-treated EF-G showed both the Km and Vmax of the GTPase reaction to be affected. Thus, the native sulfhydryl group, although important, appears not to be located in the GTPase active center. Denaturation of EF-G with guanidine-HCl and random blockage of any of the three masked sulfhydryl groups caused inactivation, likely due to steric interference with proper chain folding upon renaturation. Treatment of ribosomes or ribosomal subunits with six different thiol reagents at a concentration of 0.27 mM had little or no effect on the ribosome-EF-G GTPase, except for the case with HMB which inactivated the 30 S subunit. An interaction of EF-G with the 30 S subunit in addition to that known to occur with the 50 S subunit is suggested by a rapid and preferential exchange of HMB from the native sulfhydryl group of EF-G to the 30 S subunit of 70 S ribosomes.     

Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: a pentacoordinated binding of the SCN- ion to the zinc at high pH

The binding of four inhibitors--mercuric ion, 3-acetoxymercuri-4-aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion--to human carbonic anhydrase II (HCA II) has been studied with X-ray crystallography. The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 A resolution. Mercuric ions are observed at both nitrogens in the His-64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys-206. The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 A, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closest to the zinc ion is 3.1 A away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc. The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 A resolution using the least-squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN- ion is 1.9 A from the zinc ion but shifted 1.3 A with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the O gamma l atom of Thr-199. This is due to the inability of the O gamma l atom of Thr-199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN- molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so-called "deep" water molecule of the native enzyme. The zinc-bound water molecule is 2.2 A from the zinc ion and 2.4 A from the SCN- nitrogen. In addition, this water is hydrogen bonded to the O gamma l atom of Thr-199 and to another water molecule. We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3- ion are ligated to the zinc ion.     

The reaction of PGA1 with sulfhydryl groups; a component in the binding of A-type prostaglandins to proteins.

Prostaglandins of the A-type (PGAs) were found to react with cysteine or reduced glutathione to yield water-soluble adducts, an effect due to a reaction of the sulfhydryl group of cysteine with the unsaturated carbonyl function of these prostaglandins. The binding of tritiated PGA1 to the supernatant fraction of rabbit papilla homogenates reported by Attallah and Lee (4) appears to be related to this phenomenon since ethacrynic acid, a compound also highly reactive with the thiol group of cysteine, effectively competes with PGAs for the binding site in this soluble kidney preparation. Evidence is also presented to show that this binding of PGAs to the "acceptor' of the rabbit kidney is related to an interaction with a thiol group of 15-hydroxy prostaglandin dehydrogenase, the enzyme chiefly involved in the metabolism of prostaglandins.     

Region- and cell-specific differences in the distribution of carbonic anhydrases II, III, XII, and XIV in the adult rat epididymis.

We employed RT-PCR followed by light microscope immunocytochemistry on St. Marie's- and Bouin's-fixed tissues to define the distribution of carbonic anhydrase (CA) isoforms in the male reproductive tract. The data revealed that CA II, III, IV, XII, and XIV were expressed in rat epididymis. Whereas CA III was found in principal cells of all epididymal regions, CA II was localized in narrow cells of the initial segment and principal cells of all regions. CA XII expression was most intense in the corpus and proximal cauda regions, where it appeared over the basolateral plasma membranes of principal cells. Narrow cells of the initial segment also revealed intense reactions, as did basal cells of the corpus and proximal cauda regions. Principal cells of the initial segment and proximal caput regions showed diffuse apical cytosolic reactions and occasional basolateral staining for CA XIV, whereas principal cells of distal regions showed more diffuse cytosolic reactions highlighting both apical and basal regions of the cell, with basal cells also being reactive. These data suggest subtle differences in cell type and subcellular- and region-specific distributions for CAs in their role of fine-tuning pH in the lumen, cell cytosol, and intervening intercellular spaces of the epididymis.     

Evaluation of substrate and inhibitor binding to yeast and human isoprenylcysteine carboxyl methyltransferases (Icmts) using biotinylated benzophenone-containing photoaffinity probes

The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coli cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme.     

Protein adsorption orientation in the light of fluorescent probes: mapping of the interaction between site-directly labeled human carbonic anhydrase II and silica nanoparticles

Little is known about the direction and specificity of protein adsorption to solid surfaces, a knowledge that is of great importance in many biotechnological applications. To resolve the direction in which a protein with known structure and surface potentials binds to negatively charged silica nanoparticles, fluorescent probes were attached to different areas on the surface of the protein human carbonic anhydrase II. By this approach it was clearly demonstrated that the adsorption of the native protein is specific to limited regions at the surface of the N-terminal domain of the protein. Furthermore, the adsorption direction is strongly pH-dependent. At pH 6.3, a histidine-rich area around position 10 is the dominating adsorption region. At higher pH values, when the histidines in this area are deprotonated, the protein is also adsorbed by a region close to position 37, which contains several lysines and arginines. Clearly the adsorption is directed by positively charged areas on the protein surface toward the negatively charged silica surface at conditions when specific binding occurs.