Selective replacement of the catalytic zinc of the human stromelysin-1 catalytic domain

 We have selectively replaced the catalytic zinc of the catalytic domain of stromelysin-1 (SCD) with other transition metals. Dialysis of the enzyme against 2 mM 1,10-phenanthroline, 20 mM Hepes, pH 7.5 in the presence of 10 mM CaCl₂ removes the catalytic zinc, leaving the structural zinc site intact. Dialysis with metal-free buffer followed by the new metal ion replaces the catalytic zinc forming a metal hybrid enzyme. Full incorporation of 1 mol Co²⁺, Ni²⁺, or Cd²⁺/mol enzyme is confirmed by atomic absorption spectrometry while the weaker binding Mn²⁺ yields a value of 0.4 mol Mn²⁺/mol enzyme after dialysis against 1 μM Mn²⁺. The activity of the monozinc enzyme is <10% while its activity is restored upon the addition of zinc and other transition metals. The k _cat values for the Co²⁺, Mn²⁺, Cd²⁺, and Ni²⁺ enzymes are respectively 99%, 54%, 19%, and 17% of the value for the native enzyme, while the respective k _cat/K _m values are 36%, 29%, 7%, and 16% toward the fluorescent heptapeptide substrate, DnsPLALRAR. The zinc and metal hybrid SCD cleave DnsPLA↓LRAR, and DnsPLE↓LFAR, exclusively at one bond, while DnsPLA↓L↓WAR and DnsPLA↓L↓FAR are cleaved at two positions. The double cleavage of DnsPLALWAR and DnsPLALFAR catalyzed by SCD is in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis is the different cleavage site specificity of the metal hybrids toward the A-L and L-W bonds of the DnsPLALWAR substrate. Thus the k _cat values of the Co/Zn hybrid for the cleavage of the A-L bond in the DnsPLALRAR and DnsPLAWAR substrates are 5- and 8-fold greater than those for the Cd/Zn hybrid compared to a 140-fold difference for the corresponding k _cat values for the L-W bond cleavage. These results imply that the catalytic metal of SCD is not only involved in catalysis but also influences the substrate specificity of the enzyme. Received: 30 December 1997 / Accepted: 23 February 1998     

Amyloid β-Peptide Inhibits High-Affinity Choline Uptake and Acetylcholine Release in Rat Hippocampal Slices

Abstract: The characteristic pathological features of the postmortem brain of Alzheimer's disease (AD) patients include, among other features, the presence of neuritic plaques composed of amyloid β-peptide (Aβ) and the loss of basal forebrain cholinergic neurons, which innervate the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Aβ accumulation in vivo may initiate and/or contribute to the process of neurodegeneration and thereby the development of AD. However, the mechanisms by which Aβ peptide influences/causes degeneration of the basal forebrain cholinergic neurons and/or the cognitive impairment characteristic of AD remain obscure. Using in vitro slice preparations, we have recently reported that Aβ-related peptides, under acute conditions, potently inhibit K⁺-evoked endogenous acetylcholine (ACh) release from hippocampus and cortex but not from striatum. In the present study, we have further characterized Aβ-mediated inhibition of ACh release and also measured the effects of these peptides on choline acetyltransferase (ChAT) activity and high-affinity choline uptake (HACU) in hippocampal, cortical, and striatal regions of the rat brain. Aβ_1–40 (10^?8M) potently inhibited veratridine-evoked endogenous ACh release from rat hippocampal slices and also decreased the K⁺-evoked release potentiated by the nitric oxide-generating agent, sodium nitroprusside (SNP). It is interesting that the endogenous cyclic GMP level induced by SNP was found to be unaltered in the presence of Aβ_1–40. The activity of the enzyme ChAT was not altered by Aβ peptides in hippocampus, cortex, or striatum. HACU was reduced significantly by various Aβ peptides (10^?14 to 10^?6M) in hippocampal and cortical synaptosomes. However, the uptake of choline by striatal synaptosomes was altered only at high concentration of Aβ (10^?6M). Taken together, these results indicate that Aβ peptides, under acute conditions, can decrease endogenous ACh release and the uptake of choline but exhibit no effect on ChAT activity. In addition, the evidence that Aβ peptides target primarily the hippocampus and cortex provides a potential mechanistic framework suggesting that the preferential vulnerability of basal forebrain cholinergic neurons and their projections in AD could relate, at least in part, to their sensitivity to Aβ peptides.     

Fluorescent nucleotide triphosphate substrates for snake venom phosphodiesterase

The procedure described utilizes a crude cell-free extract from the yeast Saccharomyces cerevisiae as enzymatic source for the synthesis of coproporphyrin III from [¹⁴C]δ-aminolevulinic acid with a high yield of conversion (?60%). Both specific radioactivity and total radioactivity of coproporphyrin III can be adjusted fairly well. This procedure is not time consuming for yeast acellular extracts or porphyrin ester preparations. The acellular extracts can be stored frozen (?30°C) for at least 1 year without loss of enzymatic activity. The same procedure can be used for [¹⁴C]protoporphyrin preparation.     

pH-dependent properties of cobalt(II) carboxypeptidase A-inhibitor complexes.

1H NMR spectroscopy of the isotropically shifted signals in cobalt carboxypeptidase, CoCPD, permits a direct and selective detection of protons belonging to the residues liganded to the metal. The chemical shift of these protons in the free enzyme and enzyme-inhibitor complexes with changing pH monitors the state of ionization of the ligands directly and of other residues in the active center indirectly. The 1H NMR spectrum of CoCPD at pH 6 shows three well-resolved isotropically shifted signals in the downfield region at 62 (a), 52 (c), and 45 (d) ppm which have been assigned to the NH proton of His-69 and to the C-4 H's of His-69 and His-196, respectively. Titration of signal a with pH is characterized by a pKa of 8.8 which is identical to that seen in prior electronic absorption and kinetic studies. The fact that the signal reflecting the NH of His-69 is still observed at pH 10 and no major shifts occur for the signals reflecting the C-4 H's indicates the alkaline pKa in carboxypeptidase A catalysis, pKEH, cannot be ascribed to ionization of the histidyl NH of either His-69 or His-196. Binding of L-Phe shifts this pKa to 7.7 while not greatly perturbing the downfield 1H NMR signals that reflect the ligation shell of the cobalt coordination sphere. These results indicate the pKa of 8.8 in CoCPD and the pKa of 7.7 in the CoCPD.L-Phe adduct reflect ionization of the same group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of energy deprivation and hydrogen peroxide on contraction and myoplasmic free calcium concentrations in isolated myocardial muscle cells

The effect of energy deprivation and H2O2 on the contraction, shape, and intracellular free Ca2+ concentration of myocardial muscle cells was investigated using suspensions of freshly isolated, electrically stimulated rat ventricle heart cells. The mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used to decrease the rate of ATP synthesis. At 0.9 mM extracellular Ca2+, CCCP (0.25 microM) reduced the number of contracting cells by 50% after 5 min, and the number of rod-shaped cells by 40% after 10 min. The effects of CCCP were associated with a substantial decrease in measured cellular ATP concentrations. The deleterious effect of exposure of myocytes to CCCP for periods of up to 5 min was enhanced by an increase in the extracellular Ca2+ concentration, but markedly reduced in the absence of electrical stimulation. Verapamil protected myocytes from the deleterious effects of CCCP during the first 5 min but not at later times. In the presence of 46 mM extracellular K+, CCCP caused a marked increase in the myoplasmic free Ca2+ concentration (measured using quin2). This effect was inhibited by verapamil and was not observed in the absence of K+-induced depolarization. Exposure of myocytes to H2O2 (0.5 mM) caused a substantial decrease both in the number of cells which exhibited normal end-to-end synchronous contraction and in the total number of cells which contracted either partially or fully. The effects of H2O2 were more pronounced at higher concentrations of the peroxide, with longer times of exposure to the agent, and at higher concentrations of extracellular Ca2+, and were partially reversed by dimethyl sulfoxide. The results indicate that both ATP deprivation and H2O2, possibly through the generation of free radicals, cause substantial and rapid damage to cardiac myocytes and induce the movement of additional Ca2+ across the sarcolemma to the myoplasm. In the case of ATP deprivation, this initially occurs through voltage-operated channels.     

Transient apical polarization of Gliotactin and Coracle is required for parallel alignment of wing hairs in Drosophila

In Drosophila, wing hairs are aligned in a distally oriented, parallel array. The frizzled pathway determines proximal-distal cell polarity in the wing; however, in frizzled pathway mutants, wing hairs remain parallel. How wing hairs align has not been determined. We have demonstrated a novel role for the septate junction proteins Gliotactin (Gli) and Coracle (Cora) in this process. Prior to prehair extension, Gli and Cora were restricted to basolateral membranes. During pupal prehair development, Gli and Cora transiently formed apical ribbons oriented from the distal wing tip to the proximal hinge. These ribbons were aligned beneath prehair bases and persisted for several hours. During this time, Gli was lost entirely from the basolateral domain. A Gliotactin mutation altered the apical polarization Gli and Cora and induced defects in hair alignment in pupal and adult stages. Genetic and cell biological assays demonstrated that Gli and Cora function to align hairs independently of frizzled. Taken together, our results indicate that Gli and Cora function as the first-identified members of a long-predicted, frizzled-independent parallel alignment mechanism. We propose a model whereby the apical polarization of Gli and Cora functions to stabilize and align prehairs relative to anterior-posterior cell boundaries during pupal wing development.     

13C NMR studies of carboxylate inhibitor binding to cobalt(II) carboxypeptidase A

Both 13C NMR and electronic absorption spectral studies on cobalt(II) carboxypeptidase A in the presence of acetate and phenylacetate provide evidence for two binding sites for each of these agents. The transverse relaxation rate T2-1 for the 13C-enriched carboxyl groups of the inhibitors is significantly increased when bound to the paramagnetic cobalt carboxypeptidase as compared to the diamagnetic zinc enzyme. The acetate concentration dependence of T2p-1 shows two inflections indicative of sequential binding of two inhibitor molecules. The cobalt-13C distances, calculated by means of the Solomon equation, indicate that the second acetate molecule binds directly to the metal ion while the first acetate molecule binds to a protein group at a distance 0.5-0.8 nm for the metal ion, consistent with it binding to one or more of the arginyl residues (Arg-145, Arg-127, or Arg-71). In the case of phenylacetate, perturbation of the cobalt electronic absorption spectrum shows that binding occurs stepwise. 13C NMR distance measurements indicate that one of the two phenylacetates is bound to the metal in the EI2 complex. These binding sites may correspond to those identified previously by kinetic means (one of which is competitive, the other noncompetitive) with peptide binding. The studies further indicate that it should be possible to map the protein interactions of the carbonyl groups of both substrate and noncompetitive inhibitors during catalysis by means of 13C NMR studies with suitably labeled substrates and inhibitors.     

Do Males Form Social Associations Based on Sexual Attractiveness in a Fission-Fusion Fish Society?

Recent theory predicts that males should choose social environments that maximize their relative attractiveness to females by preferentially associating with less attractive rivals, so as to enhance their mating success. Using the Trinidadian guppy (Poecilia reticulata), a highly social species, we tested for non-random social associations among males in mixed-sex groups based on two phenotypic traits (body length and coloration) that predict relative sexual attractiveness to females and sexual (sperm) competitiveness. Based on a well-replicated laboratory dichotomous-choice test of social group preference, we could not reject the null hypothesis that focal males chose randomly between a mixed-sex group that comprised a female and a rival male that was less sexually attractive than themselves and another mixed-sex group containing a sexually more attractive male. The same conclusion was reached when females were absent from the two groups. As might be expected from these laboratory findings, free-ranging males in the field were not assorted by either body length or colour in mixed-sex shoals. The apparent lack of an evolved and expressed preference in wild male guppies from our study population to form social associations with other males based on their relative sexual attractiveness and competitiveness might be due to the fission-fusion dynamics of guppy shoals in nature. Such social dynamics likely places constraints on the formation of stable phenotype-based social associations among males. This possibility is supported by a simulation model which assumes group departure rules based on relative body size and coloration in males.