A low-barrier hydrogen bond between histidine of secreted phospholipase A2 and a transition state analog inhibitor

This work describes in-depth NMR characterization of a unique low-barrier hydrogen bond (LBHB) between an active site residue from the enzyme and a bound inhibitor: the complex between secreted phospholipase A(2) (sPLA(2), from bee venom and bovine pancreas) and a transition-state analog inhibitor HK32. A downfield proton NMR resonance, at 17-18 ppm, was observed in the complex but not in the free enzyme. On the basis of site-specific mutagenesis and specific 15N-decoupling, this downfield resonance was assigned to the active site H48, which is part of the catalytic dyad D99-H48. These results led to a hypothesis that the downfield resonance represents the proton (H(epsilon 2) of H48) involved in the H-bonding between D99 and H48, in analogy with serine proteases. However, this was shown not to be the case by use of the bovine enzyme labeled with specific [15N(epsilon 2)]His. Instead, the downfield resonance arises from H(delta1) of H48, which forms a hydrogen bond with a non-bridging phosphonate oxygen of the inhibitor. Further studies showed that this proton displays a fractionation factor of 0.62(+/-0.06), and an exchange rate protection factor of >100 at 285 K and >40 at 298 K, which are characteristic of a LBHB. The pK(a) of the imidazole ring of H48 was shown to be shifted from 5.7 for the free enzyme to an apparent value of 9.0 in the presence of the inhibitor. These properties are very similar to those of the Asp em leader His LBHBs in serine proteases. Possible structural bases and functional consequences for the different locations of the LBHB between these two types of enzymes are discussed. The results also underscore the importance of using specific isotope labeling, rather than extrapolation of NMR results from other enzyme systems, to assign the downfield proton resonance to a specific hydrogen bond. Although our studies did not permit the strength of the LBHB to be accurately measured, the data do not provide support for an unusually strong hydrogen bond strength (i.e. >10 kcal/mol).     

Measurement of extracellular pH,K(+), and lactate in ischemic heart

Simultaneous and continuous measurements of extracellular pH, potassium (K(+)), and lactate (L(-)) in ischemic rabbit papillary muscle are presented for the first time. Potentiometric pH and K(+) sensors and an amperometric lactate biosensor were used. These miniature electrodes were previously developed and individually tested for this purpose. The pH sensor was based on an iridium oxide layer electrodeposited on a planar platinum electrode fabricated on a flexible substrate. The potentiometric K(+) sensor was based on a polymeric membrane and valinomycin ionophore. The L(-) biosensor was based on lactate oxidase and an organic conducting salt polarized at 0.15V vs Ag/AgCl reference electrode. The utility of this novel analytical system to cardiovascular research was demonstrated by using the system to study the interrelationship of cellular K(+) and lactate loss in ischemic myocardium, and the role of extracellular pH and buffer capacity on this relationship. The results indicated: (i) sequential brief episodes of ischemia produced reproducible trends of L(-), pH, and K(+) changes during the first three episodes, (ii) extracellular L(-) increased with increasing buffer capacity of extracellular compartment, (iii) the patterns of extracellular L(-) and K(+) changes were not related directly, and (iv) L(-) transport and lactic acid diffusion were not the primary cause of extracellular acidosis during ischemia.     

Social interactions and cortisol treatment increase the production of aggressive electrocommunication signals in male electric fish,Apteronotus leptorhynchus

Brown ghost knife fish, Apteronotus leptorhynchus, continually emit a weakly electric discharge that serves as a communication signal and is sensitive to sex steroids. Males modulate this signal during bouts of aggression by briefly (approximately 15 ms) increasing the discharge frequency in signals termed "chirps." The present study examined the effects of short-term (1-7 days) and long-term (6-35 days) male-male interaction on the continuous electric organ discharge (EOD), chirping behavior, and plasma levels of cortisol and two androgens, 11-ketotestosterone (11KT) and testosterone. Males housed in isolation or in pairs were tested for short-term and long-term changes in their EOD frequency and chirping rate to standardized sinusoidal electrical stimuli. Within 1 week, chirp rate was significantly higher in paired fish than in isolated fish, but EOD frequency was equivalent in these two groups of fish. Plasma cortisol levels were significantly higher in paired fish than in isolated fish, but there was no difference between groups in plasma 11KT levels. Among paired fish, cortisol levels correlated positively with chirp rate. To determine whether elevated cortisol can cause changes in chirping behavior, isolated fish were implanted with cortisol-filled or empty Silastic tubes and tested for short-term and long-term changes in electrocommunication signals and steroid levels. After 2 weeks, fish that received cortisol implants showed higher chirp rates than blank-implanted fish; there were no difference between groups in EOD frequency. Cortisol implants significantly elevated plasma cortisol levels compared to blank implants but had no effect on plasma 11KT levels. These results suggest that male-male interaction increases chirp rate by elevating levels of plasma cortisol, which, in turn, acts to modify neural activity though an 11KT-independent mechanism.     

Fluorescence resonance energy transfer detection of synaptophysin I and vesicle-associated membrane protein 2 interactions during exocytosis from single live synapses

To investigate the molecular interactions of synaptophysin I and vesicle-associated membrane protein 2 (VAMP2)/synaptobrevin II during exocytosis, we have used time-lapse videomicroscopy to measure fluorescence resonance energy transfer in live neurons. For this purpose, fluorescent protein variants fused to synaptophysin I or VAMP2 were expressed in rat hippocampal neurons. We show that synaptophysin I and VAMP2 form both homo- and hetero-oligomers on the synaptic vesicle membrane. When exocytosis is stimulated with alpha-latrotoxin, VAMP2 dissociates from synaptophysin I even in the absence of appreciable exocytosis, whereas synaptophysin I oligomers disassemble only upon incorporation of the vesicle with the plasma membrane. We propose that synaptophysin I has multiple roles in neurotransmitter release, regulating VAMP2 availability for the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and possibly participating in the late steps of exocytosis.     

Aposymbiotic culture of the sepiolid squid Euprymna scolopes: role of the symbiotic bacterium Vibrio fischeri in host animal growth, development, and light organ morphogenesis

The sepiolid squid Euprymna scolopes forms a bioluminescent mutualism with the luminous bacterium Vibrio fischeri, harboring V. fischeri cells in a complex ventral light organ and using the bacterial light in predator avoidance. To characterize the contribution of V. fischeri to the growth and development of E. scolopes and to define the long-term effects of bacterial colonization on light organ morphogenesis, we developed a mariculture system for the culture of E. scolopes from hatching to adulthood, employing artificial seawater, lighting that mimicked that of the natural environment, and provision of prey sized to match the developmental stage of E. scolopes. Animals colonized by V. fischeri and animals cultured in the absence of V. fischeri (aposymbiotic) grew and survived equally well, developed similarly, and reached sexual maturity at a similar age. Development of the light organ accessory tissues (lens, reflectors, and ink sac) was similar in colonized and aposymbiotic animals with no obvious morphometric or histological differences. Colonization by V. fischeri influenced regression of the ciliated epithelial appendages (CEAs), the long-term growth of the light organ epithelial tubules, and the appearance of the cells composing the ciliated ducts, which exhibit characteristics of secretory tissue. In certain cases, aposymbiotic animals retained the CEAs in a partially regressed state and remained competent to initiate symbiosis with V. fischeri into adulthood. In other cases, the CEAs regressed fully in aposymbiotic animals, and these animals were not colonizable. The results demonstrate that V. fischeri is not required for normal growth and development of the animal or for development of the accessory light organ tissues and that morphogenesis of only those tissues coming in contact with the bacteria (CEAs, ciliated ducts, and light organ epithelium) is altered by bacterial colonization of the light organ. Therefore, V. fischeri apparently makes no major metabolic contribution to E. scolopes beyond light production, and post-embryonic development of the light organ is essentially symbiont independent. J. Exp. Zool. 286:280-296, 2000.     

Selective coupling of G protein beta gamma complexes to inhibition of Ca2+ channels

Several mechanisms couple heterotrimeric guanine nucleotide-binding proteins (G proteins) to cellular effectors. Although alpha subunits of G proteins (Galpha) were the first recognized mediators of receptor-effector coupling, Gbetagamma regulation of effectors is now well known. Five Gbeta and 12 Ggamma subunit genes have been identified, suggesting through their diversity that specific subunits couple selectively to effectors. The molecular determinants of Gbetagamma-effector coupling, however, are not well understood, and most studies of G protein-effector coupling do not support selectivity of Gbetagamma action. To explore this issue further, we have introduced recombinant Gbetagamma complexes into avian sensory neurons and measured the inhibition of Ca(2+) currents mediated by an endogenous phospholipase Cbeta- (PLCbeta) and protein kinase C-dependent pathway. Activities of Gbetagamma in the native cells were compared with enzyme assays performed in vitro. We report a surprising selective activation of the PLCbeta pathway by Gbetagamma complexes containing beta(1) subunits, whereas beta(2)-containing complexes produced no activation. In contrast, when assayed in vitro, PLCbeta and type II adenylyl cyclase did not discriminate among these same Gbetagamma complexes, suggesting the possibility that additional cellular determinants confer specificity in vivo.     

Catalytic cysteine of thymidylate synthase is activated upon substrate binding

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.