Long-QT syndrome-associated caveolin-3 mutations differentially regulate the hyperpolarization-activated cyclic nucleotide gated channel 4.

Background Caveolin-3 (cav-3) mutations are linked to the long-QT syndrome (LQTS) causing distinct clinical symptoms. Hyperpolarization-activated cyclic nucleotide channel 4 (HCN4) underlies the pacemaker current If. It associates with cav-3 and both form a macromolecular complex. Methods To examine the effects of human LQTS-associated cav-3 mutations on HCN4-channel function, HEK293-cells were cotransfected with HCN4 and wild-type (WT) cav-3 or a LQTS-associated cav-3 mutant (T78M, A85T, S141R, or F97C). HCN4 currents were recorded using the whole-cell patch-clamp technique. Results WT cav-3 significantly decreased HCN4 current density and shifted midpoint of activation into negative direction. HCN4 current properties were differentially modulated by LQTS-associated cav-3 mutations. When compared with WT cav-3, A85T, F97C, and T78M did not alter the specific effect of cav-3, but S141R significantly increased HCN4 current density. Compared with WT cav-3, no significant modifications of voltage dependence of steady-state activation curves were observed. However, while WT cav-3 alone had no significant effect on HCN4 current activation, all LQTS-associated cav-3 mutations significantly accelerated HCN4 activation kinetics. Conclusions Our results indicate that HCN4 channel function is modulated by cav-3. LQTS-associated mutations of cav-3 differentially influence pacemaker current properties indicating a pathophysiological role in clinical manifestations.

Emerging therapies for Parkinson's disease: from bench to bedside.

The prevalence of Parkinson's disease (PD) increases with age and is projected to increase in parallel to the rising average age of the population. The disease can have significant health-related, social, and financial implications not only for the patient and the caregiver, but for the health care system as well. While the neuropathology of this neurodegenerative disorder is fairly well understood, its etiology remains a mystery, making it difficult to target therapy. The currently available drugs for treatment provide only symptomatic relief and do not control or prevent disease progression, and as a result patient compliance and satisfaction are low. Several emerging pharmacotherapies for PD are in different stages of clinical development. These therapies include adenosine A2A receptor antagonists, glutamate receptor antagonists, monoamine oxidase inhibitors, anti-apoptotic agents, and antioxidants such as coenzyme Q10, N-acetyl cysteine, and edaravone. Other emerging non-pharmacotherapies include viral vector gene therapy, microRNAs, transglutaminases, RTP801, stem cells and glial derived neurotrophic factor (GDNF). In addition, surgical procedures including deep brain stimulation, pallidotomy, thalamotomy and gamma knife surgery have emerged as alternative interventions for advanced PD patients who have completely utilized standard treatments and still suffer from persistent motor fluctuations. While several of these therapies hold much promise in delaying the onset of the disease and slowing its progression, more pharmacotherapies and surgical interventions need to be investigated in different stages of PD. It is hoped that these emerging therapies and surgical procedures will strengthen our clinical armamentarium for improved treatment of PD.

Inactivation of enolase from carp (Cyprinus carpio) muscle by 2,3-butanedione.

Enolase from white muscle of carp (Cyprinus carpio) is inactivated by 2,3-butanedione in borate buffer. Magnesium ions as well as substrate--2-phosphoglycerate markedly altered the rate and extent of inactivation. The partially inactivated enzyme shows unaltered Km but decreased Vmax after 10 min incubation with butanedione, however after 60 min incubation the Km value increased 2.5 fold.

The effect of temperature on catalytic function of glyceraldehyde-3-phosphate dehydrogenase from muscle of pig and carp Cyprinus carpio.

1. The temperature dependence of the kinetics of glyceraldehyde-3-phosphate dehydrogenase from white muscle of carp and skeletal muscle of pig was examined. 2. The Km values of carp muscle enzyme were stable over the temperature range 5-35 degrees C, but increased for pig muscle enzyme with increasing temperature. 3. The Arrhenius plot for pig muscle enzyme is linear but non-linear for carp muscle enzyme. 4. The differences indicate that glyceraldehyde-3-phosphate dehydrogenase from white carp muscle may contribute to the adaptive mechanism of carp to varied temperature conditions.

Derivative spectrophotometry of dimer and monomer of enolase.

1. SDS causes significant polar exposure of aromatic amino acids of enolase. The alpha-helix content remains unchanged. The enzyme lost all its activity. 2. The presence of 1 M K Br in enzyme solution results in a smaller increase of polarity of aromatic amino acids residues environment. The amount of alpha-helix does not decrease in comparison to native enzyme. Enzyme lost nearly 80% of its initial activity. 3. The extreme pH values and the presence of 6 M Gnd.HCl influence the whole structure of enolase. It is accompanied by a large polar shift of aromatic amino acids and significant decrease of alpha-helix content of the protein.

Purification and some properties of glyceraldehyde-3-phosphate dehydrogenase from carp muscle.

Glyceraldehyde-3-phosphate dehydrogenase was purified from carp white muscle. On CM-Sephadex chromatography two well separated active peaks were obtained. Both of them show a single protein band on gel electrophoresis and have the same molecular and kinetic properties; they differ only by the amount of bound NAD, the enzyme in the second peak being coenzyme-free. Significant differences were observed between the properties of carp and pig muscle enzymes. Glyceraldehyde-3-phosphate dehydrogenase from carp is more resistant to heat and proteolytic inactivation. Moreover NAD does not protect it against inactivation. Only one sulphydryl group per subunit is able to react with 5,5'-dithiobis(2-nitrobenzoate), irrespective of the kind of the buffer. The structure of glyceraldehyde-3-phosphate dehydrogenase from white muscle of carp seems to be more compact and therefore more inaccessible to some agents than that of the enzyme from pig muscle.

Chemical modification of histidine, tyrosine, tryptophan and cysteine residues in carp (Cyprinus carpio) muscle enolase.

Enolase from carp (Cyprinus Carpio) muscle was modified by diethylpyrocarbonate, tetranitromethane, N-bromosuccinimide and 5,5'-dithiobis(2-nitrobenzoic acid). The extent and rate of modification and its effect on the enzyme activity were determined. Modification of histidine, tyrosine and tryptophan residues caused complete inactivation of the enzyme; Mg2+ as well as 2-phosphoglycerate markedly altered the rates of modification and inactivation. The above-mentioned amino acid residues seem to be essential for the functioning of muscle enolases. Modification of cysteine residues had no effect on the enolase activity.

Immobilization of pig muscle enolase. Studies on the activity of subunits.

The native dimeric form of enolase from pig muscle was immobilized on Sepharose 4B activated with cyanogen bromide. The amount of matrix-bound enolase, its specific activity and kinetic properties depend on the extent of gel activation with CNBr. Only on the Sepharose activated with small quantities of CNBr the amount of protein which remained after treatment with Gdn.HCl was about 50% of the initially bound enolase, indicating that the enzyme was bound covalently to the matrix through a single subunit. The matrix-bound monomers obtained in this way were inactive and were unable to reassociate to dimers on addition of free subunits. The matrix-bound monomers obtained after KBr treatment were inactive but retained the ability to reassociate into active dimers after addition of free subunits. The results indicate that single matrix-bound subunits of pig muscle enolase are enzymatically inactive and dimeric structure is essential for catalytic activity.

Localization of arginyl residues modified with butanedione in glyceraldehyde-3-phosphate dehydrogenase from pig muscle.

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from pig muscle was inactivated by incubation with butanedione in triethanolamine buffer, pH 8.3. The inactivation was reversible after short treatment with butanedione; it became irreversible after 12-15 h, with a concomitant loss of two arginyl residues per subunit. The modified enzyme was digested with TPCK-trypsin and the peptides were purified by chromatography and electrochromatography. Two new peptides were obtained as the result of modification. From their partially determined sequence the modified arginyl residues were identified as Arg-13 and Arg-231 in the primary structure of pig muscle enzyme.

Human muscle glyceraldehyde-3-phosphate dehydrogenase: reactivity of sulphydryl groups.

1. The kinetics of the reaction of thiol groups of glyceraldehyde-3-phosphate dehydrogenase from human muscle with 5,5'-dithiobis-2-nitrobenzoate (DTNB) was studied spectrophotometrically using the conventional and stopped-flow methods. 2. Each of the three thiol groups present in the enzyme subunit reacts with a different velocity. 3. The reaction with DTNB of the four sulphydryl groups of Cys-149, essential for the enzymatic activity, is biphasic, depends on the amount of NAD bound and is strongly slowed down when one mole of coenzyme is bound to the tetramer. NAD bound is only partially released from the holoenzyme treated with DTNB. 4. In the presence of borate, thiol groups of Cys-153 and Cys-244 do not react with DTNB.

Glyceraldehyde-3-phosphate dehydrogenase from human muscle: cleavage of tryptophanyl peptides with o-iodosobenzoic acid.

The new procedure of protein fragmentation with o-iodosobenzoic acid (Mahoney & Hermodson, 1979; Biochemistry, 18, 3810-3814) was used to split three tryptophanyl peptide bonds in glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) from human skeletal muscle. The mixture obtained was separated into four homogeneous peptides by a one-step chromatographic procedure. High specificity of the cleavage reaction was proved by the amino acid analysis and determination of the N-terminal sequence of the peptide products. Both specificity ahd high yield obtained by this procedure confirmed its utility in the establishment of the protein primary structure.

Glyceraldehyde-3-phosphate dehydrogenase from human muscle: primary structure.

The complete sequence of 334 amino acids of glyceraldehyde-3-phosphate dehydrogenase (GPDH) from human muscle was established through the analysis of tryptic digests of the enzyme treated alternatively with bromocyanate, o-iodosobenzoic acid and maleic acid. Comparison of the primary structure of the enzyme from human muscle with that of the other known GPDH showed high homology; the closest similarity of the amino acid sequence was found between the enzymes from human and pig muscle.

The structure/activity relationship in glyceraldehyde-3-phosphate dehydrogenase of pig muscle.

The macromolecular digestion product obtained on limited proteolysis of glyceraldehyde-3-phosphate dehydrogenase (GPDH, EC 1.2.1.12) was fractionated into seven polypeptides. Their position in the known sequence of GPDH was established. The sites of chymotrypsin action at Phe-34, Tyr-91, Val-116, Leu-154, Leu-221 and Leu-246 were identified on the basis of N-terminal sequence and molecular weight of the peptides isolated. Cleavage of particular peptide bonds was related to the enzymatic activity.

The sites of chymotrypsin action on the pig muscle glyceraldehyde 3-phosphate dehydrogenase during limited proteolysis.

1. The sites of chymotrypsin action on glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde 3-phosphate) : DNA+ oxidoreductase (phosphorylating), EC 1.2.1.12) was established; limited proteolysis by chymotrypsin results in lowering of the phosphorolytic activity of the enzyme without affecting its oxidative activity. 2. The low-molecular fraction of the chymotrypsin digest separated by Sephadex G-100 chromatography, was fractionated on Bio-gels. Determination of the amino acid composition of the nine peptides isolated, and of their amino acid sequence, permitted to relate cleavage of Leu-64, Trp-84, Leu-109, Leu-141, Phe-165, Lys-212, Val-239, Leu-242, Leu-271 (or Phe-315) bonds in the enzyme to the decrease of the phosphorolytic activity.

Effect of borate on the catalytic activities of muscle glyceraldehyde-3-phosphate dehydrogenase.

The effect of borate on glyceraldehyde-3-phosphate dehydrogenase from human, pig and rabbit muscle was studied. At lower concentration of borate only the dehydrogenase activity is inhibited, reversibly and competitively against NAD. At concentration of borate above 6 mM the plots of 1/v versus borate concentration become nonlinear and the inhibition is extended to the esterase and acetylphosphatase activities. In certain conditions a time-dependent inactivation and reactivation was observed. The direct interaction between borate (if present at concentration of at least 6 mM) and glyceraldehyde-3-phosphate dehydrogenase is postulated, the possible site of the reaction being the histidine residue(s). The esterase activity of the human muscle enzyme and the effect of borate on it are different from the other mammalian enzymes.

Kinetic studies of the reactivity of the sulfhydryl groups of glyceraldehyde-3-phosphate dehydrogenase.

The reaction of sulfhydryl groups of glyceraldehyde-3-phosphate dehydrogenase from rabbit and pig muscles with a large molar excess of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) shows three-phasic pseudo-first-order kinetics. Since the fastest reaction between active cysteine-149 and Nbs2 is apparently biphasic, half-of-the-sites reactivity towards Nbs2 is suggested. Further sulfhydryl groups become reactive as an effect of conformational changes in the protein molecule after formation of a mixed disulfide on cysteine-149. In the presence of 40 mM borate the reaction is biphasic only, and two sulfhydryl groups per subunit react very quickly. The bound NAD+ is only partially released even after a long reaction with Nbs2. It was demonstrated that the two NAD+ binding sites with the highest dissociation constants have no significant effect on the reaction between cysteine-149 and Nbs2.

Amino acid sequence of human muscle glyceraldehyde-3-phosphate dehydrogenase. Isolation and amino acid sequences of tryptic peptides.

1. The amino acid compostion, N- and C-terminal amino acid sequences, and the subunit molecular weight of glyceraldehyde phosphate dehydrogenase from human muscle, were determined. The obtained results and the maps of tryptic peptides suggest that the enzyme is composed of four identical or very similar polypeptide chains. 2. From the tryptic digest of performic acid-oxidized enzyme, 32 peptides were isolated. The amino acid sequence analysis showed a high degree of homology with the corresponding tryptic peptides of the dehydrogenase from pig muscle, with 9 replacements and probably two additional amino acids in the examined sequences of the human muscle enzyme.