Molecular cloning and immunological characterization of the gamma polypeptide, a small protein associated with the Na,K-ATPase

The gamma subunit of the Na,K-ATPase is a small membrane protein that copurifies with the alpha and beta subunits of the enzyme. Strong evidence that the gamma subunit is a component of the Na,K-ATPase comes from studies indicating that the subunit is involved in forming the site for cardiac glycoside binding. We have isolated and characterized the cDNAs coding the gamma subunit from several species. The gamma subunit is a highly conserved protein consisting of 58 amino acids with a molecular weight of 6500. Hydropathy analysis reveals the presence of a single hydrophobic domain that is sufficient to cross the membrane. There are no sites for N-linked glycosylation. Northern blot analysis revealed that the gamma subunit mRNA is expressed in a tissue-specific fashion and is present in all tissues characterized. gamma-specific antibodies have been used to verify that the sequenced protein is the same protein labeled by [3H]nitroazidobenzoyl-ouabain (NAB-ouabain), and that this protein, the gamma subunit of the Na,K-ATPase, has a distribution pattern along nephron segments that is identical with the alpha subunit. In addition, coimmunoprecipitation of the alpha, beta and gamma subunits demonstrate specific association of the subunits. These results are consistent with the notion that the gamma subunit is specifically associated with and may be an important component of the Na,K-ATPase.     

Statistical approach for subwavelength measurements with a conventional light microscope

A method is developed theoretically that will permit subwavelength measurements of objects that differ from the surroundings by any contrast enhancing parameter, such as fluorescence, second harmonic generation, reflection et cetera, using a statistical analysis of a picture obtained with a conventional light microscope through a set of subwavelength apertures or by repeated scanning of a laser beam over a defined area. It is demonstrated that with this methodology information can be obtained on microdomains that are thirty times less than the diameter of the aperture. For example, for apertures that are 0.3 μm in diameter it is possible to measure the dimension of objects that are ~10 nm. A technology is described by which it is possible to produce masks with the appropriate apertures. Instrumentation is described that would allow for the realization of these statistical methodologies with either apertures or scanning laser beams.     

Sau3AI, a monomeric type II restriction endonuclease that dimerizes on the DNA and thereby induces DNA loops

Here, we report that Sau3AI, an unusually large type II restriction enzyme with sequence homology to the mismatch repair protein MutH, is a monomeric enzyme as shown by gel filtration and ultracentrifugation. Structural similarities in the N- and C-terminal halves of the protein suggest that Sau3AI is a pseudo-dimer, i.e. a polypeptide with two similar domains. Since Sau3AI displays a nonlinear dependence of cleavage activity on enzyme concentration and a strong preference for substrates with two recognition sites over those with only one, it is likely that the functionally active form of Sau3AI is a dimer of a pseudo-dimer. Indeed, electron microscopy studies demonstrate that two distant recognition sites are brought together through DNA looping induced by the simultaneous binding of two Sau3AI molecules to the DNA. We suggest that the dimeric form of Sau3AI supplies two DNA-binding sites, one that is associated with the catalytic center and one that serves as an effector site.     

Carnitine-acyltransferase activity of mitochondria from mung-bean hypocotyls

Carnitine-acyltransferase activity assayed with acetyl-CoA, octanoyl-CoA, or palmitoyl-CoA is associated with the mitochondrial but not with the peroxisomes of mung-bean hypocotyls. Using mitochondria as an enzyme source, a half-maximal reaction rate is obtained with a palmitoyl-CoA concentration approximately twice that required with acetyl-CoA. In the presence of a saturating acetyl-CoA concentration the carnitine-acyltransferase activity is not enhanced by palmitoyl-CoA as additional substrate. However, palmitoylcarnitine is formed in addition to acetylcarnitine, and the formation of acetylcarnitine is competitively inhibited by palmitoyl-CoA. It is concluded that the mitochondria of mung-bean hypocotyls possess a carnitine acyltransferase of broad substrate specificity with respect to the chainlength of the acyl-CoA and that the demonstration of a carnitine-palmitoyltransferase activity in plant mitochondria does not indicate the presence of a specific carnitine long-chain acyltransferase.     

Serine acetyltransferase from Escherichia coli is a dimer of trimers

Equilibrium sedimentation studies show that the serine acetyltransferase (SAT) of Escherichia coli is a hexamer. The results of velocity sedimentation and quasi-elastic light scattering experiments suggest that the identical subunits are loosely packed and/or arranged in an ellipsoidal fashion. Chemical cross-linking studies indicate that the fundamental unit of quaternary structure is a trimer. The likelihood, therefore, is that in solution SAT exists as an open arrangement of paired trimers. Crystals of SAT have 32 symmetry, consistent with such an arrangement, and the cell density function is that expected for a hexamer. Electron microscopy with negative staining provides further evidence that SAT has an ellipsoidal subunit organization, the dimensions of the particles consistent with the proposed paired trimeric subunit arrangement. A bead model analysis supports the view that SAT has a low packing density and, furthermore, indicates that the monomers may have an ellipsoidal shape. Such a view is in keeping with the ellipsoidal subunit shape of trimeric LpxA, an acyltransferase with which SAT shares contiguous repeats of a hexapeptide motif.     

Fluctuation and linkage relations in macromolecular solution

It is shown in the appendix that the derivatives of the excess free energy of a macromolecule in solution, with respect to the activities of other solution components, lead to fluctuation and linkage relations among these other components. Solution fluctuation theory is used, but it is specialized to the fluctuations and correlations associated with the presence of a macromolecule, and is developed with a modified ensemble. The relations of the appendix are used to analyze the interaction of two solution components, A and B, with the macromolecule and with one another. Three cases are considered: (1) A and B are ligands that bind stoichiometrically to the macromolecule. This case reduces to Wyman's binding polynomial analysis. (2) A and B are two substances at high concentration that interact selectively with the macromolecule. (3) A is a species that binds stoichiometrically to the macromolecule, while B is a component at high concentration that interacts weakly with the macromolecule.     

Interactions of ellipticine with model or natural membranes. A spectrophotometric study

Ellipticine is the type of a group of substances used against human cancer. It was shown that ellipticine and 9-methoxyellipticine strongly interact with monolayers of negatively charged phospholipids. In the present paper it is shown by spectrophotometric methods that ellipticine interacts with suspensions of a model membrane with a 1/1 charge neutralization, and with a natural membrane, both negatively charged. At a physiological pH, ellipticine undergoes protonation in the presence of these membranes. Its apparent pK is increased by 1, and its spectral behaviour, when the pH is changed, indicates that the drug is in a less polar environment, suggesting that hydrophobic bonds link the drug and the lipids. The existence of such bonds is suggested by the fact that the drug is not significantly released from anionic membranes when the pH is increased to 10, at which value ellipticine is not ionized. The consequences of these ionic and hydrophobic interactions are briefly discussed.     

Is glycine a surrogate for a d‐amino acid in the collagen triple helix?

Collagen is the most abundant protein in animals. Every third residue in a collagen strand is a glycine with phi, psi = -70 degrees, 175 degrees. A recent computational study suggested that replacing these glycine residues with D-alanine or D-serine would stabilize the collagen triple helix. This hypothesis is of substantial importance, as the glycine residues in collagen constitute nearly 10% of the amino acid residues in humans. To test this hypothesis, we synthesized a series of collagen mimic peptides that contain one or more D-alanine or D-serine residues replacing the canonical glycine residues. Circular dichroism spectroscopy and thermal denaturation experiments indicated clearly that the substitution of glycine with D-alanine or D-serine greatly disfavors the formation of a triple helix. Host-guest studies also revealed that replacing a single glycine residue with D-alanine is more destabilizing than is its replacement with L-alanine, a substitution that results from a common mutation in patients with collagen-related diseases. These data indicate that the glycine residues in collagen are not a surrogate for a D-amino acid and support the notion that the main-chain torsion angles of a glycine residue in the native structure (especially, phi > 0 degrees ) are critical determinants for its beneficial substitution with a D-amino acid in a protein.     

Activation mechanism of HSP16.5 from Methanococcus jannaschii

The small heat shock proteins are the ubiquitous proteins found in a wide range of organisms and function as molecular chaperones by binding to the folding intermediates of their substrates. Although the crystal structure of HSP16.5, a small heat shock protein from Methanococcus jannaschii, revealed that it is a hollow sphere composed of 24 identical subunits, its activation mechanism remains unclear. We found out that HSP16.5 is active only at high temperatures and forms a stable complex with substrate in a stoichiometric manner. We also observed that the conformational change of HSP16.5 is correlated with the increasing hydrophobic site and its activation as a molecular chaperone. However, it is revealed that the conformational change is not accompanied with the change of the secondary structure of a subunit, but correlated with the increasing diameter of HSP16.5. Therefore, it is proposed that the activation mechanism of HSP16.5 involves temperature induced conformational change with size increment of the complex resulting in the exposure of hydrophobic substrate-binding site.     

The genus Theissenia: T. pyrenocrata, T. cinerea sp. nov., and T. eurima sp. nov

The diagnosis of Theissenia is emended to include taxa that lack a definite central perithecial columella and taxa that feature ascospores with a germ slit. Theissenia cinerea is described as a new species that lacks a perithecial columella; T. eurima is described as a new species with ascospores having a germ slit. The type species, T. pyrenocrata, is redescribed and compared with the new species. The taxonomic position of Theissenia is discussed.     

Prenatally diagnosed foetal malformations and termination of pregnancy: the case of Lebanon

Termination of pregnancy (TOP) is offered in many countries, for foetuses prenatally diagnosed with congenital malformations that are deemed incompatible with life or that are associated with a high morbidity. In Lebanon, a middle income country where religion plays a focal role, the law prohibits any form of TOP unless it is the only means to save the mother's life. It is the contention of the authors of this article that even if the foetus is a person, if it were medically revealed that there is a substantial risk that the newborn will suffer severe physical abnormalities that will cause it to be seriously handicapped; it is morally acceptable to terminate the pregnancy. Hence, TOP carried out for these indications is justified in the interest of the foetus and the child. Whatever the status of the foetus is, once born, it will become a full-fledged sentient being with all that this entails. When given the option of starting an existence, this person-to-be has the right to a minimum that allows him/her to enjoy a relatively good quality of life. Today, Lebanese obstetricians are confronted with the burden placed on them under the law to refuse TOP, or, when performing them, to forge records or deny having done them. This is why we strongly believe that the Lebanese policy on abortion should be amended.     

The aboral pore of hydra: evidence that the digestive tract of hydra is a tube not a sac

The digestive tract of Bilateria is a tube with a mouth at one end and an anus at the other end. Radiata, that include the phylum Cnidaria, have a blind-sac form of digestive tract with only one opening. It has therefore been commonly believed that the evolution of the body plan from Radiata to Bilateria included the change of the digestive tract from a blind sac to a tube. In this study, we report that there is a very narrow opening at the aboral end of hydra termed the aboral pore. This confirms a classical finding by Kanajew (Zool Anz, 76:37-44, 1928), but we confirmed it in both asexually reproduced and sexually reproduced polyps, demonstrating that the aboral pore represents innate morphology. We also find that the opening coincides with the site where synthesis of an extracellular matrix-degrading enzyme, hydra matrix metalloprotease, is elevated suggesting that the pore is maintained by extracellular matrix degradation. Finally, we find that there is material transfer through the opening in both inward and outward directions. From these observations, we conclude that the digestive tract and the body plan of hydra is not a blind sac as formerly believed but is a tube with a tapered end.     

Activity of pyruvate dehydrogenase A (PDHA) in hamster spermatozoa correlates positively with hyperactivation and is associated with sperm capacitation

Unravelling the molecular basis of capacitation is crucial to our understanding the basis of acquisition of fertilization competence by spermatozoa. In two recent studies, we have demonstrated that dihydrolipoamide dehydrogenase, which is a post-pyruvate metabolic enzyme and one of the components of pyruvate dehydrogenase complex, undergoes capacitation-dependent tyrosine phosphorylation, and that the activity of the enzyme correlates with capacitation events in the hamster spermatozoa. However, it is not clear as to whether other components of the pyruvate dehydrogenase complex are also crucial for sperm capacitation. In this report, we have identified pyruvate dehydrogenase A2 (PDHA2), a constituent of pyruvate dehydrogenase A (PDHA), which is a component of pyruvate dehydrogenase complex that exhibits tyrosine phosphorylation during hamster spermatozoal capacitation. This is the first report showing that hamster sperm PDHA2 is a testis-specific phosphotyrosine that is associated with the fibrous sheath of hamster spermatozoa. The localization of PDHA2 in spermatozoa was investigated using antibodies to PDHA, which is the active tetrameric protein that consists of a homodimer of PDHA2 and PDHB. Both immunofluorescence and confocal studies indicated a unique non-canonical, extramitochondrial localization for PDHA in the principal piece of hamster spermatozoa. It was also observed that PDHA colocalized with AKAP4 in the fibrous sheath of the spermatozoon. The enzymatic activity of PDHA was positively correlated with hyperactivation but not with the acrosome reaction. Given the localization of PDHA and the evidence that its activity correlates positively with hyperactivation and that its PDHA2 subunit exhibits capacitation-associated protein tyrosine phosphorylation, it appears that PDHA2 is associated with the process of capacitation.     

Equivalent Circuit of Frog Atrial Tissue as Determined by Voltage Clamp-Unclamp Experiments

The equivalent circuit that has been used in the analysis of nerve voltage-clamp data is that of the membrane capacity in parallel with the membrane resistance. Voltage-clamp experiments on frog atrial tissue indicate that this circuit will not suffice for this cardiac tissue. The change in membrane current associated with a step change in membrane potential does not show a rapid spike of capacitive current as would be expected for the simple parallel resistance-capacitance network. Rather, there is a step change in current followed by an exponential decay in current with a time constant of about 1 msec. This relatively slow capacitive charging current suggests that there is a resistance in series with the membrane capacity. A possible equivalent circuit is that of a series resistance external to the parallel resistance-capacitance network of the cell membranes. Another possible circuit assumes that the series resistance is an integral part of the cell membrane. The data presented in this paper demonstrate that the equivalent circuit of a bundle of frog atrial muscle is that of an external resistance in series with the cell membranes.     

Redox regulation of yeast flavin-containing monooxygenase

The flavin-dependent monooxygenase from yeast (yFMO) oxidizes biological thiols such as cysteine, cysteamine, and glutathione. The enzyme makes a major contribution to the pools of oxidized thiols that, together with reduced glutathione from glutathione reductase, create the optimum cellular redox environment. We show that the activity of yFMO, as a soluble enzyme or in association with the ER membrane of microsomal fractions, is correlated with the redox potential. The enzyme is active under conditions normally found in the cytoplasm, but is inhibited as GSSG accumulates to give a redox potential similar to that found in the lumen of the ER. Site-directed mutations show that Cys 353 and Cys 339 participate in the redox regulation. Cys 353 is the principal residue in the redox-sensitive switch. We hypothesize that it may initiate formation of a mixed disulfide that is partially inhibitory to yFMO. The mixed disulfide may exchange with Cys 339 to form an intramolecular disulfide bond that is fully inhibitory.     

Specific in vivo protein-protein interactions between Escherichia coli SOS mutagenesis proteins.

One of the components of the RecA-LexA-controlled SOS response in Escherichia coli cells is an inducible error-prone DNA replication pathway that results in a substantial increase in the mutation rate. It is believed that error-prone DNA synthesis is performed by a multiprotein complex that is formed by UmuC, UmuD', RecA, and probably DNA polymerase III holoenzyme. It is postulated that the formation of such a complex requires specific interactions between these proteins. We have analyzed the specific protein-protein interactions between UmuC, UmuD, and UmuD' fusion proteins, using a Saccharomyces cerevisiae two-hybrid system. In agreement with previous in vitro data, we have shown that UmuD and UmuD' are able to form both homodimers (UmuD-UmuD and UmuD'-UmuD') and a heterodimer (UmuD-UmuD'). Our data show that UmuC fusion protein is capable of interacting exclusively with UmuD' and not with UmuD. Thus, posttranslational processing of UmuD into UmuD' is a critical step in SOS mutagenesis, enabling only the latter protein to interact with UmuC. Our data seem to indicate that the integrity of the entire UmuC sequence is essential for UmuC-UmuD' heterotypic interaction. Finally, in our studies, we used three different UmuC mutant proteins: UmuC25, UmuC36, and UmuC104. We have found that UmuC25 and UmuC36 are not capable of associating with UmuD'. In contrast, UmuC104 protein interacts with UmuD' protein with an efficiency identical to that of the wild-type protein. We postulate that UmuC104 protein might be defective in interaction with another, unknown protein essential for the SOS mutagenesis pathway.     

Gating of Two Mechanoelectrical Transducer Channels Associated with a Single Tip Link

Although gating of mechanoelectrical transducer (MET) channels has been successfully described by assuming that one channel is associated with a tip link in the hair bundle, recent reports indicate that a single tip link is associated with more than one channel. To address the consistency of the model with the observations, gating of MET channels is described here by assuming that each tip link is associated with two identical MET channels, which are connected either in series or in parallel. We found that series connection does not lead to a single minimum of stiffness with respect to hair bundle displacement unless the minimum is above a certain positive value. Thus, negative stiffness must appear in pairs in the displacement axis. In contrast, parallel connection of the two channels predicts gating compliance similar to that predicted by the one-channel-per-tip-link model of channel gating, within the physiological range of parameters. Parallel connection of MET channels is, therefore, a reasonable assumption to explain most experimental observations. However, the compatibility with series connection cannot be ruled out for experimental data on turtle hair cells.     

A calpain-like proteolytic activity produces the limited cleavage at the N-terminal regulatory domain of rabbit skeletal muscle AMP deaminase: evidence of a protective molecular mechanism

On storage at 4 degrees C, rabbit skeletal muscle AMP deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.     

The B-chromosome system of myrmeleotettix maculatus (thunb.)

It is established that in populations of Myrmeleotettix maculatus with B-chromosomes these chromosomes occur at stable frequencies and are present to the same extent in both the males and the females of the same population. It is also established that the B-chromosome content of a population is positively correlated with its chiasma frequency and that, within a population, individuals with single B-chromosomes tend to have higher chiasma frequencies than individuals lacking B-chromosomes. Since this effect is not increased by the addition of further supernumeraries it is argued that selection operates in favour of individuals with single B-chromosomes. Finally it is shown that the level of B-chromosomes in a population is related to temperature and especially to rainfall so that B-chromosomes are absent from populations in climatically stringent environments.