Analysis of protein sequences and protein complexes by matrix-assisted laser desorption/ionization mass spectrometry.

In the context of proteome analysis, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) can fulfil the two tasks of primary structure verification and protein identification. As an illustration of the first of these tasks, the sequence of Eschericha coli isoleucyl-tRNA synthetase, a protein with 15 reported sequence conflicts, has been established by means of MALDI mass mapping. The identification of mitochondrial proteins participating in a yeast supramolecular complex exhibiting NADH dehydrogenase activity highlights the performances of MALDI-MS for the second task. The spectral suppression phenomenon occurring for complex peptide mixtures analysed by MALDI is discussed, as well as the role of post-source decay analysis for confident protein identification.

Yeast mitochondrial dehydrogenases are associated in a supramolecular complex.

Separation of yeast mitochondrial complexes by colorless native polyacrylamide gel electrophoresis led to the identification of a supramolecular structure exhibiting NADH-dehydrogenase activity. Components of this complex were identified by N-terminal Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. The complex was found to contain the five known intermembrane space-facing dehydrogenases, namely two external NADH-dehydrogenases Nde1p and Nde2p, glycerol-3-phosphate dehydrogenase Gut2p, D- and L-lactate-dehydrogenases Dld1p and Cyb2p, the matrix-facing NADH-dehydrogenase Ndi1p, two probable flavoproteins YOR356Wp and YPR004Cp, four tricarboxylic acids cycle enzymes (malate dehydrogenase Mdh1p, citrate synthase Cit1p, succinate dehydrogenase Sdh1p, and fumarate hydratase Fum1p), and the acetaldehyde dehydrogenase Ald4p. The association of these proteins is discussed in terms of NADH-channeling.

Evidence of a subunit 4 (subunit b) dimer in favor of the proximity of ATP synthase complexes in yeast inner mitochondrial membrane.

Yeast mitochondria having either the D54C or E55C mutations in subunit 4 (subunit b), which is a component of the ATP synthase stator, displayed a spontaneous disulfide bridge between two subunits 4. This dimer was not soluble upon Triton X-100 extraction either at concentrations which extract the yeast ATP synthase or at higher concentrations. Increasing detergent concentrations led to a lack of the oligomycin-sensitive ATPase activity, thus showing an uncoupling between the two sectors of the mutated enzymes due to the dissociation of the subunit 4 dimer from the mutant enzyme. There is only one subunit 4 (subunit b) per eukaryotic ATP synthase. As a consequence, the results are interpreted as the proximity of ATP synthase complexes within the inner mitochondrial membrane.

Effect of the lack of light impulses on the hypothalamic PACAP and C-fos immunoreactivities in rats.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the secretin family. It is widely distributed in the central and peripheral nervous systems. The highest concentration of PACAP was found in the hypothalamus. In the present work it has been studied whether PACAP is involved in the mediation of photic stimuli to the anterior pituitary gland. We have examined the effect of the lack of light impulses on the hypothalamic PACAP and C-fos immunoreactivities. In adult rats 10 days after the removal of the eyes (surgical enucleation) and in those received monosodium glutamate treatment neonatally (chemical enucleation). The PACAP immunostaining enhanced in the hypothalamic magnocellular nuclei and in the extemal zone of the median eminence. C-fos immunoreactivity also enhanced in a few hypothalamic nuclei 2 hours after the surgical enucleation indicating that the lack of light impulses activated hypothalamic neurons which, in turn, might stimulate the release of PACAP into the portal circulation. It has been concluded that PACAP may be involved in photoendocrine regulations.

Separation by blue native and colorless native polyacrylamide gel electrophoresis of the oxidative phosphorylation complexes of yeast mitochondria solubilized by different detergents: specific staining of the different complexes.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) or colorless native polyacrylamide gel electrophoresis (CN-PAGE) allowed separation of the oxidative phosphorylation complexes of yeast mitochondria. These complexes were characterized by specific staining related to their enzymatic activity. Solubilization of mitochondria by different nonionic detergents such as Triton X-100, dodecyl maltoside, Nonidet P-40, Lubrol, octyl glucoside, or Hecameg led to the separation of F1-FO ATPase complexes exhibiting distinct apparent molecular masses related to different contaminating proteins and lipids. All these different forms were active in ATP hydrolysis as revealed directly on the gel. Analysis of the subunit composition of these complexes was carried out by a two-dimensional Tricine-SDS-PAGE and showed that the purest F1-FO ATPase complex was obtained with Lubrol, whereas with Hecameg and octyl glucoside, only the F1 part of ATPase was solubilized.

Antibodies against subunits of F0 sector of ATP synthase from Saccharomyces cerevisiae. Stimulation of ATP synthase by subunit-8-reactive antibodies and inhibition by subunit-9-reactive antibodies.

Polyclonal antibodies against the three purified proteolipids of the F0 sector [subunit 6 (Su6), subunit 8 (Su8), subunit 9 (Su9)] and against the beta subunit (F1) of ATP synthase were raised in rabbits. All antisera showed ELISA reactivities with F0F1-ATPase. Antisera used to immunoblot partially purified ATP synthase labeled a single band migrating with the same molecular mass as that of the purified protein. Mitochondria were incubated with IgG of each antiserum and oxidative phosphorylation was measured. Anti-Su6 IgG, as anti-Su beta IgG, was without effect whereas anti-Su9 IgG decrease both respiration and ATP-synthesis rates, resulting in a decrease of ATP/O. In contrast, anti-Su8 IgG enhanced respiratory control and stimulated the ATP-synthesis rate, resulting in an increase of ATP/O. In the same manner, anti-Su9 IgG inhibited ATP hydrolysis whereas anti-Su8 IgG stimulated this activity. Antimycin titration of phosphorylation and respiration rates demonstrated that anti-Su9 IgG decreased the H+/ATP ratio and promoted a H+ leak, whereas anti-Su8 IgG increased H+/ATP without modification of the proton permeability. Anti-Su9 IgG decreased proton-motive force whereas anti-Su8 IgG did not. It is proposed that both antibodies promoted opposite mechanistic changes of the H+/ATP stoichiometry of the ATP synthase, and that in vivo Su8 could have a negative regulatory role in the oxidative phosphorylation process.

Detection of purine cytosine permease of S. cerevisiae: use of antibodies against a synthetic peptide corresponding to a predicted sequence in the N-terminal domain of the protein.

A synthetic peptide, selected in the predicted N-terminal amino-acid sequence of the purine cytosine permease (gene FCY2), linked to albumins proved a remarkably good immunogen in rabbits. In ELISA, sera reacted with the synthetic peptide and with specific proteins of plasma-membrane-enriched fractions of mutant Saccharomyces cerevisiae pAB strains (amplified FCY2 gene) with high titers and high avidity. Western blots of plasma membrane proteins of pAB strain probed with antisera showed two bands: a major (45 kDa) and minor band (50 kDa). On the contrary, plasma-membrane-enriched fractions of mutant S. cerevisiae pJDB strain (deficient in FCY2 gene) gave no signal when probed in the same conditions. These results demonstrate the specificity of the antisera and also suggest that the 45 kDa and 50 kDa proteins are both products of the FCY2 gene.

Photoaffinity labelling of the purine-cytosine permease of Saccharomyces cerevisiae.

8-Azidoadenine was used as a photoaffinity reagent to characterize the purine-cytosine permease of Saccharomyces cerevisiae. It is a potent competitive inhibitor of cytosine uptake and irradiation of the cells incubated with the label induced the irreversible inactivation of cytosine uptake. Addition of excess cytosine prevented this labelling which was restricted to the outer face of the plasma membrane since it was not accumulated by the cells. In the strain with the amplified purine-cytosine permease gene the maximum cytosine uptake rate was increased 4-5-fold relative to wild type without a modification of the Michaelis constant of uptake (Kt); no uptake could be measured in the deleted strain. The relative amounts of specific labelling determined for the cells and for membrane preparations were 0, 1 and 4 for the null, the wild-type and the amplified strains, respectively. One major band specifically labelled by [3H]azidoadenine, corresponding to a polypeptide with an apparent molecular mass of 45 kDa, was observed in the wild type, amplified in the strain carrying the multicopy plasmid and not detected in the deleted strain. Therefore this polypeptide corresponds to the purine-cytosine permease.

The use of auto-anti-idiotypes for the visualization of acetylcholine receptors in an invertebrate skeletal muscle.

Polyclonal auto-anti-idiotypic antibodies (AAIs), detected in antisera of rabbits immunized with acetylcholine conjugate, have already been characterized. In this paper, we report on a cytochemical application of these AAIs on skeletal muscle motor endplates of an invertebrate. We have shown that there is a non-uniformly distributed reaction in the synaptic cleft. The deposits seem to be associated with the muscular plasma membrane. The specificity of the cytochemical method is discussed. On this kind of muscle, the AAIs are able to detect, at the ultrastructural level, acetylcholine binding sites which most likely are acetylcholine receptors.

Methodology for purification of large hydrophobic peptides by high-performance liquid chromatography.

To aid in structural studies of pig cardiac myosin light chains (L27 and L28), a procedure of ion-exchange chromatography (IEC) on Trisacryl M (noted for its high capacity) in combination with reversed-phase high-performance liquid chromatography (RP-HPLC) and volatile buffers has been developed. In contrast with other IEC methods (resins or HPIEC), the use of Trisacryl M facilitates subsequent peptide purifications by RP-HPLC. The advantage of the present combination of techniques is also that it enables the isolation of hydrophobic peptides in high yield, e.g., the N-terminal chymotryptic peptide from L27 was thus purified and, after sub-digestion with trypsin, its sequence has been established.

Phylogenetic studies of cardiac myosins from amphibia to mammals.

Comparison between pig atrial and ventricular myosins was performed on the light chains (using SDS-PAGE) and on the heavy chains (using Ca2+-ATPase measurements and NTCBA peptide mapping). Light chain composition of pig cardiac myosins was compared to three other species ones (frog, chicken and human). Up to birds, atrial and ventricular myosin light chain composition was identical whereas in mammals atrial and ventricular myosin light chain composition was different; likewise the heavy chains. Six cardiac myosin isoenzymes have been thus characterized. No correlation can be established between cardiac myosin light chain pattern and species evolution.

Perineal rhabdomyosarcoma in a newborn child: pathological and biochemical studies with emphasis on contractile proteins.

Histological and ultrastructural studies have been undertaken on a perineal rhabdomyosarcoma from a newborn child. The spontaneous tumour has the typical feature of mesenchymoma. The recurrent tumour, however, displays some rhabdopoietic characteristics. The myosin of the recurrent tumour has been extracted and compared with human fetal myosin. These two myosins are identical in their synthetic filaments and their light-chain composition. Nevertheless, whereas the ATPase activity of fetal myosin can be stimulated considerably by increasing the ca2+ concentration, that of tumoral myosin remains very low. These results show that there are isoenzymes of myosins and there must be differences in the myosin heavy chains, particularly in the active sites. These findings are identical with those seen in experimental rhabdomyosarcoma.

Pig cardiac myosin isoenzymes.

Pig heart myosins isolated from the free wall of the right ventricle and the free wall of the left ventricle were compared with respect to structural and enzymatic properties. The following parameters were studied (1) activation of myosin ATase by Ca2+ and K+j(2) molecular weight of the heavy and light chains of myosins as determined by electrophoretic migration in polyacrylamide sodium dodecyl sulfate (SDS) gels; (3) ability of the heavy chains to form aggregates at low ionic strength as revealed by electron microscopy; (4) sensitivity to the action of chymotrypsin. Differences were observed between left and right ventricular myosins (L-myosin and R-myosin) for all these parameters except for the molecular weight of heavy and light chains. The existence of large amounts of short synthetic filaments for R-myosin compared with L-myosin as revealed by the length repartition of the filaments, and the production of smaller quantities of HMM-S by chymotryptic digestion for R-myosin, strongly suggest the presence of different cardiac myosin heavy chain species.

Tumoral myosins of Ni3S2-induced rhabdomyosarcomas in rat and rabbit: comparative studies with adult and fetal myosins of skeletal muscle.

Tumoral myosins were isolated from rat and rabbit rhabdomyosarcomas and compared with normal adult and fetal skeletal myosins. The synthetic filaments, the light-chain composition and the Ca2+ ATP-ase activity were studied. In the presence of Mg2+, normal myosins precipitated as bipolar filaments (0.5 micrometer), fetal and tumoral myosins, however, precipitated as long fusiform filaments (1 to 10 micron). SDS-PAGE revealed that tumoral myosins contain the same light-chains as fetal myosin (25000 and 18000 daltons, L25-L18). The third light-chain of the normal muscle myosin (16000 daltons, L16) was absent. In addition, Urea-PAGE revealed the absence of the phosphorylated form of the L18 in fetal and tumoral myosins. Ca2+ ATPase activity measurements performed in function of the Ca2+ concentration showed similarities between fetal and adult muscle myosins. The Ca2+-ATPase activity of tumoral myosins, however, was very low and slightly activated by increasing the Ca2+ concentration (0.01 to 10 mM). The investigation has shown that fetal and tumoral myosins are identical concerning the ultrastructure of their synthetic filaments and their light-chain composition. This was not so in regard to the Ca2+ ATPase activity. This is probably the result of the expression of a new myosin- or of one of its polypeptides-, which has a different Ca2+-ATPase activity.

[Isolation of myosin light chain from porcine heart by preparative electrophoresis and study of the polypeptides obtained by cyanogen bromide action]. / Isolement des chaînes légères de la myosine cardiaque de porc par électrophorèse préparative et étude des polypeptides obtenus après action du bromure de cyanogène.

A simple, rapid and efficient procedure is developed to isolate proteins with identical or different isoelectric points such as pig cardiac myosin light chains. Preparative electrophoresis on discontinuous polyacrylamide slab gels in the presence of urea allows a very good separation of each light chain (L27 and L18) and heavy chain from highly purified myosin. An original elution procedure of the proteins fixed and localized by amido schwartz allows the isolation of the L27 and L18 light chains in quantities sufficient to carry out structural studies. Homogeneity of light chains thus isolated is checked by the analysis of cyanogen bromide peptides. Structural similarities can be demonstrated between myosin light chains of beef and pig heart.