An Internal View of the Spherical Body of Treponema macrodentium as Revealed by Scanning Electron Microscopy

The osmium-dimethyl sulfoxide-osmium method for clear visualization of intracellular structure was used to observe the detailed inner structure of the spherical bodies produced in vitro by a human oral treponeme. Scanning electron microscopy of the cracked spherical body revealed no morphological differences between the outer and inner surfaces of the spherical body membrane, and that multiple folded or somewhat linear main bodies adhere closely to the inner surface. In addition, axial flagella partially free from the main bodies spread widely within the body to make a network, and a number of blebs ranging from approximately 1 μm to 0.2 μm in diameter were located near the terminal or subterminal areas of the main bodies. The origin of the blebs and the mechanism of spherical body formation are discussed.     

Humans but Not Chimpanzees Vary Face-Scanning Patterns Depending on Contexts during Action Observation

Human and nonhuman primates comprehend the actions of other individuals by detecting social cues, including others’ goal-directed motor actions and faces. However, little is known about how this information is integrated with action understanding. Here, we present the ontogenetic and evolutionary foundations of this capacity by comparing face-scanning patterns of chimpanzees and humans as they viewed goal-directed human actions within contexts that differ in whether or not the predicted goal is achieved. Human adults and children attend to the actor’s face during action sequences, and this tendency is particularly pronounced in adults when observing that the predicted goal is not achieved. Chimpanzees rarely attend to the actor’s face during the goal-directed action, regardless of whether the predicted action goal is achieved or not. These results suggest that in humans, but not chimpanzees, attention to actor’s faces conveying referential information toward the target object indicates the process of observers making inferences about the intentionality of an action. Furthermore, this remarkable predisposition to observe others’ actions by integrating the prediction of action goals and the actor’s intention is developmentally acquired.     

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.     

Mechanism of function of dicyclohexylcarbodiimide-sensitive Na+/H+-antiporter in Halobacterium halobium: pH effect

The regulatory roles of medium pH, a transmembrane pH gradient (delta pH), and an electrical potential (delta phi) on the activation of the N,N'-dicyclohexylcarbodiimide-sensitive Na+/H+-antiporter were studied in the membrane vesicle of Halobacterium halobium in the dark. Neither delta pH nor delta phi independently activated the antiporter but a combination could. The initial rate of Na+ extrusion did not proportionally relate to the size of delta microH+ imposed. The delta microH+-coupled Na+ efflux in the presence of delta phi (-140 mV) increased as external pH decreased, regardless of the size of delta pH, suggesting the existence of one external H+-binding site (apparent pKa 4.6) whose protonation determines primarily the Na+/H+-exchange activity. On the other hand, the dependence of the Na+ efflux on cytoplasmic pH varied with the size of delta pH imposed and the apparent pKa for the cytoplasmic H+ increased with elevating delta pH. The resulting pKa difference across the membrane seems to be the key mechanism for the facilitation of Na+-coupled H+ influx. In other words, delta pH modulates Na+/H+-exchange activity through manipulating the H+ affinity on the cytoplasmic regulatory site. The Na+ extrusion was gated by the threshold delta phi of -100 mV regardless of the size of existing delta pH. delta phi acts on the protonated antiporter and converts it into an active state which becomes delta pH reactive.     

The membrane-associated ATPase from Sulfolobus acidocaldarius is distantly related to F1-ATPase as assessed from the primary structure of its alpha-subunit

Isolation of novel membrane-associated ATPases, presumably soluble parts of the H+-ATPases, from archaebacteria has been recently reported, and their properties were found to be significantly different from the usual F1-ATPase. In order to assess the relationship of the archaebacterial ATPases to the F1-ATPases and other known ATPases, the amino acid sequence of the alpha subunit of the ATPase from Sulfolobus acidocaldarius, an acidothermophilic archaebacterium, was compared with the sequences of other ATPases. The gene encoding its alpha subunit was cloned from the genomic library of S. acidocaldarius, and the nucleotide sequence was determined. The 591-amino acid sequence deduced from the nucleotide sequence contains a small number of short stretches that shows sequence similarity to the alpha and beta subunits of F1-ATPase. However, the overall similarity is too weak to consider it to be a typical member of the F1-ATPase family when the highly conserved sequences of the F1-ATPase subunits among various organisms are taken into account. Moreover, most of these stretches overlap the consensus sequences that are commonly found in some nucleotide-binding proteins. There is no significant sequence similarity to the ion-translocating ATPases, which form phosphorylated intermediates, such as animal Na+,K+-ATPases. Thus, the S. acidocaldarius ATPase and probably other archaebacterial ATPases also appear to belong to a new group of ion-translocating ATPases that has only a distant relationship to F1-ATPase.     

Amino acid sequence of the calcium-binding light chain of myosin from the lower eukaryote, Physarum polycephalum

We have established a new method for preparing Physarum myosin whose actin-activated ATPase activity is inhibited by micromolar levels of Ca2+. This Ca2+-inhibition is mediated by the Ca2+ binding to the myosin rather than by the Ca2+-dependent modification of the phosphorylated state of the myosin (Kohama, K., and Kendrick-Jones, J. (1986) J. Biochem. (Tokyo) 99, 1433-1446). Ca2+-binding light chain (CaLC) has been suggested to be primary importance in this Ca2+ inhibition (Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, T., and Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). The amino acid sequence of CaLC was determined; it was composed of 147 amino acid residues and the N terminus was acetylated. The molecular weight was calculated to be 16,131. The homology of CaLC in the amino acid sequence with 5,5'-dithiobis-(2-nitrobenzoic acid) light chain and alkali light chain of skeletal muscle myosin were rather low, i.e., 25% and 30%, respectively. Interestingly, however, the CaLC sequence was 40% homologous with brain calmodulin. This amino acid sequence was confirmed by sequencing the cloned phage DNA accommodating cDNA coding CaLC. Northern and Southern blot analysis indicated that 0.8-kilobase pair mRNA was transcribed from a single CaLC gene. This is the first report on the amino acid sequence of myosin light chain of lower eukaryotes and nucleotide sequence of its mRNA.     

Primary structure of chain I of the heterodimeric hemoglobin from the blood clamBarbatia virescens

The blood clamBarbatia virescens has a heterodimeric hemoglobin in erythrocytes. Interestingly, the congeneric clamsB. reeveana andB. lima contain quite different hemoglobins: tetramer and polymeric hemoglobin consisting of unusual didomain chain. The complete amino acid sequence of chain I ofB. virescens has been determined. The sequence was mainly determined from CNBr peptides and their subpeptides, and the alignment of the peptides was confirmed by sequencing of PCR-amplified cDNA forB. virescens chain I. The cDNA-derived amino acid sequence matched completely with the sequence proposed from protein sequencing.B. virescens chain I is composed of 156 amino acid residues, and the molecular mass was calculated to be 18,387 D, including a heme group. The sequence ofB. virescens chain I showed 35–42% sequence identity with those of the related clamAnadara trapezia and the congeneric clamB. reeveana. An evolutionary tree forAnadara andBarbatia chains clearly indicates that all of the chains are evolved from one ancestral globin gene, and that the divergence of chains has occurred in each clam after the speciation. The evolutionary rate for clam hemoglobins was estimated to be about four times faster than that of vertebrate hemoglobin. We suggest that blood clam hemoglobin is a physiologically less important molecule when compared with vertebrate hemoglobins, and so it evolved rapidly and resulted in a remarkable diversity in quaternary and subunit structure within a relatively short period.     

Effects of Na+ and K+ on Artemia salina (Na,K)-ATPase solubilized with a zwitterionic detergent, CHAPS

The membrane bound (Na,K)-ATPase prepared from Artemia salina nauplii was solubilized with a zwitterionic detergent, 3[3(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and then purified on a Bio-Gel A-1.5 m column in the presence of the detergent. 1) Upon solubilization, both NaCl and KCl protected the enzyme against loss of activity, KCl being more effective than NaCl. 2) Gel filtration of the solubilized enzyme on a Bio-Gel A-1.5 m column in the presence of 5 mM CHAPS resulted in loss of the enzyme activity even when one of the cations was added. Most of the phospholipids in the solubilized enzyme preparation were removed during the gel filtration (delipidation) and 10-25 phospholipids were left on a protomer (alpha beta) of the enzyme irrespective of the cation present during the gel filtration. With the addition of exogenous phospholipids, the activity was restored. The activity of the enzyme delipidated in the presence of KCl was restored to 3-4 times higher than in the case of that delipidated in the presence of NaCl. 3) Relipidation experiments with a fluorescent phospholipid, dansyl phosphatidylethanolamine (Dans-PE), suggested that the enzyme delipidated in the presence of KCl reassociated with phospholipids more firmly than the enzyme delipidated in the presence of NaCl. From these results we concluded that K+ stabilized the (Na,K)-ATPase more effectively than Na+, even when the enzyme was delipidated.     

Membrane potential in liposomes measured by the transmembrane distribution of 86Rb+, tetraphenylphosphonium or triphenylmethylphosphonium: effect of cholesterol in the lipid bilayer

Valinomycin-induced potassium diffusion potential (delta psi, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb+, tetraphenylphosphonium (TPP+) or triphenylmethylphosphonium (TPMP+). In any liposome, the values of membrane potential obtained by 86Rb+ uptake (delta psi Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both delta psi TPP and delta psi TPMP showed smaller values than delta psi Rb when the cholesterol content in liposomes increased. delta psi TPMP at a stationary state was much smaller than delta psi TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP+ or TPMP+ into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.     

Binding of immunoglobulin G from patients with autoimmune thyroid diseases to solubilized guinea pig fat cell membranes crosslinked with 125I-TSH

Binding of immunoglobulin G (IgG) to Triton-solubilized fat cell membranes crosslinked with 125I-TSH was studied by an indirect immunoprecipitation method. Guinea pig fat cell membranes (FCM) containing TSH receptors with an association constant of 1.92 X 10(9) M-1 were first reacted with 125I-TSH, then treated with a crosslinker, dissuccinimidyl suberate. The dissociation of 125I-TSH from the crosslinked 125I-TSH-FCM complexes due to the addition of 100 mU/ml unlabeled TSH was 9.0%, while it was 33% without the treatment. To the Triton-solubilized FCM crosslinked with 125I-TSH, 50 micrograms each of IgG from 20 normal controls, 20 patients with Graves' disease and 20 with Hashimoto's disease was added and precipitation was effected by adding anti-human IgG. In patients with Graves' disease, 125I-TSH-FCM complexes immunoprecipitated ranged from 1.10 to 4.18% with an average of 2.4 +/- 0.99 (S. D.) % which was significantly higher than those in normal controls (1.6 +/- 0.29%). The values in the patients with Hashimoto's disease averaged 1.7 +/- 0.53 (S. D.) which did not differ significantly from those of controls. The value did not correlate with either TSH-binding inhibiting activities or titers of anti-microsomal antibodies. These data suggest the presence of TSH-receptor antibodies which react with antigens other than TSH-binding sites in the patients with Graves' disease.