Mammalian neuronal voltage‐gated Ca2+ channels have been implicated as potential mediators of membrane permeability to Zn2+. We tested directly whether voltage‐gated Ca2+ channels can flux Zn2+ in whole‐cell voltage‐clamp recordings from cultured murine cortical neurones.
In the presence of extracellular Zn2+ and no Na+, K+, or other divalent cations, a small, non‐inactivating, voltage‐gated inward current was observed exhibiting a current‐voltage relationship characteristic of high‐voltage activated (HVA) Ca2+ channels. Inward current was detectable at Zn2+ levels as low as 50 μm, and both the amplitude and voltage sensitivity of the current depended upon Zn2+ concentration. This Zn2+ current was sensitive to blockade by Gd3+ and nimodipine and, to a lesser extent, by ω‐conotoxin GVIA.
Zn2+ could permeate Ca2+ channels in the presence of Ca2+ and other physiological cations. Inward currents recorded with 2 mm Ca2+ were attenuated by Zn2+ (IC50= 210 μm), and currents recorded with Zn2+ were unaffected by up to equimolar Ca2+ concentrations. Furthermore, the Zn2+‐selective fluorescent dye Newport Green revealed a depolarisation‐activated, nimodipine‐sensitive Zn2+ influx into cortical neurones that were bathed in a physiological extracellular solution plus 300 μm ZnCl2.
Surprisingly, while lowering extracellular pH suppressed HVA Ca2+ currents, Zn2+ current amplitude was affected oppositely, varying inversely with pH with an apparent pK of 7·4. The acidity‐induced enhancement of Zn2+ current was associated with a positive shift in reversal potential but no change in the kinetics or voltage sensitivity of channel activation.
These results provide evidence that L‐ and N‐type voltage‐gated Ca2+ channels can mediate Zn2+ entry into cortical neurones and that this entry may be enhanced by extracellular acidity.
Limited proteolysis of intact yeast methionine aminopeptidase (MAP1) with trypsin releases a 34 kDa fragment whose NH2-terminal sequence begins at Asp70, immediately following Lys69. These results suggest that yeast MAP may have a two-domain structure consisting of an NH2-terminal zinc finger domain and a C-terminal catalytic domain. To test this, a mutant MAP lacking residues 2-69 was generated, overexpressed, purified and analyzed. Metal ion analyses indicate that 1 mol of wild-type yeast MAP contains 2 mol of zinc ions and at least 1 mol of cobalt ion, whereas 1 mol of the truncated MAP lacking the putative zinc fingers contains only a trace amount of zinc ions but still contains one mole of cobalt ion. These results suggest that the two zinc ions observed in the native yeast MAP are located at the Cys/His rich region and the cobalt ion is located in the catalytic domain. The kcat and Km values of the purified truncated MAP are similar to those of the wild-type MAP when measured with peptide substrates in vitro and it appears to be as active as the wild-type MAP in vivo. However, the truncated MAP is significantly less effective in rescuing the slow growth phenotype of map mutant than the wild-type MAP. These findings suggest that the zinc fingers are essential for normal MAP function in vivo, even though the in vitro enzyme assays indicate that they are not involved in catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
The glyphosate-degrading bacterium, Burkholderia caryophilli PG2982, was observed to utilize glyceryl glyphosate as a sole phosphorus source. The hydrolysis of glyceryl glyphosate to glyphosate by a phosphonate ester hydrolase (PEH) was identified as the first metabolic step in the mineralization pathway. This observation provides the first biological role for a phosphonate ester hydrolase activity. Purified PEH enzyme hydrolyzed several phosphonate esters including p-nitrophenyl phenylphosphonate, beta-naphthyl phenylphosphonate, and 5-bromo-4-chloro-3-indolyl phenylphosphonate. The purified PEH also hydrolyzed some phosphodiesters including p-nitrophenyl 5'-thymidine monophosphate and p-nitrophenyl phosphorylcholine. The most catalytically efficient substrate identified was bis-(p-nitrophenyl) phosphate with a Km of 0.9 mM and a kcat of 6.2 x 10(2) min-1, suggesting that the enzyme may also function in vivo as a phosphodiesterase. The native enzyme was a homotetramer of 58-kDa subunits and exhibited a pI of 4.2. The enzyme activity had a pH activity optimum of 9.0 and was stimulated 14-fold by Mn2+ ions, but a metal cofactor was not essential for activity. N-terminal and tryptic fragment amino acid sequences were obtained from the purified PEH protein and used to clone the B. caryophilli PG2982 gene, designated pehA. The unique substrate specificity of the enzyme and potential use as a novel conditional lethal gene in plants are discussed.
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