Accumulation pattern of amino acid substitutions in protein evolution

Summary A simple method for the evolutionary analysis of amino acid sequence data is presented and used to examine whether the number of variable sites (NVS) of a protein is constant during its evolution. The NVSs for hemoglobin and for mitochondrial cytochrome c are each found to be almost constant, and the ratio between the NVSs is close to the ratio between the unit evolutionary periods. This indicates that the substitution rate per variable site is almost uniform for these proteins, as the neutral theory claims. An advantage of the present analysis is that it can be done without knowledge of paleontological divergence times and can be extended to bacterial proteins such as bacterial c-type cytochromes. It is suggested that the NVS of cytochrome c has been almost constant even over the long period (ca. 3.0 billion years) of bacterial evolution but that at least two different substitution rates are necessary to describe the accumulated changes in the sequence. This two clock interpretation is consistent with fossil evidence for the appearance times of photosynthetic bacteria and eukaryotes.     

Purification of cardiac (Na+,K+)-activated adenosine triphosphatase from rat

A procedure is described for preparation of highly active (Na+,K+)-ATPase from rat heart which has a specific activity of 200-600 mumol Pi/mg/h. The procedure is simple and can be applied to small amounts of heart muscle (approximately 1 g). The ATPase activity was more than 90% sensitive to ouabain (at concentrations up to 1 mM). The ouabain sensitivity is biphasic with about 20% of the ATPase activity being inhibited at approximately 3 X 10(-7) M ouabain.     

Selective potentiation of 5-methyl-2-(1-methylcyclohexyl)-4-oxazoleacetic acid (AD-4610) on glucose-induced insulin secretion

In the perfused pancreas from normal SD rats, AD-4610 (0.01-0.1 mM) potentiated biphasic insulin secretion induced by 7.5 mM of glucose. The concentration-response curve of insulin secretion to glucose was shifted leftwards with AD-4610 (0.1 mM) without altering either the threshold concentration of glucose to induce insulin secretion or the maximal insulin response to glucose, indicating increased sensitivity of the pancreatic B-cells to glucose. On the other hand, AD-4610 was 10-fold less effective in altering insulin secretion induced by arginine and glyceraldehyde. The effect of AD-4610 on insulin secretion and glucose metabolism was compared with that of tolbutamide in vivo. AD-4610 (100 mg/kg) potentiated insulin secretion induced by an intravenous glucose load, and also accelerated glucose metabolism without altering basal insulin secretion in normal rats. On the other hand, tolbutamide (20 mg/kg) increased basal insulin secretion, but slightly decreased glucose-induced insulin secretion. In yellow KK mice with hyperglycemia, AD-4610 (10-100 mg/kg) had a dose-dependent hypoglycemic action, but tolbutamide did not. Thus, AD-4610 stimulated insulin secretion in a glucose-dependent fashion and enhanced glucose metabolism in vivo. These results suggest that AD-4610 selectively potentiates glucose-induced insulin secretion by increasing the sensitivity of pancreatic B-cells to glucose and may be useful for treating human NIDDM through a different mechanism than that of tolbutamide.     

Fluctuation in the microtubule sliding movement driven by kinesin in vitro.

We studied the fluctuation in the translational sliding movement of microtubules driven by kinesin in a motility assay in vitro. By calculating the mean-square displacement deviation from the average as a function of time, we obtained motional diffusion coefficients for microtubules and analyzed the dependence of the coefficients on microtubule length. Our analyses suggest that 1) the motional diffusion coefficient consists of the sum of two terms, one that is proportional to the inverse of the microtubule length (as the longitudinal diffusion coefficient of a filament in Brownian movement is) and another that is independent of the length, and 2) the length-dependent term decreases with increasing kinesin concentration. This latter term almost vanishes within the length range we studied at high kinesin concentrations. From the length-dependence relationship, we evaluated the friction coefficient for sliding microtubules. This value is much larger than the solvent friction and thus consistent with protein friction. The length independence of the motional diffusion coefficient observed at sufficiently high kinesin concentrations indicates the presence of correlation in the sliding movement fluctuation. This places significant constraint on the possible mechanisms of the sliding movement generation by kinesin motors in vitro.     

Divergence pattern and selective mode in protein evolution: The example of vertebrate myoglobins and hemoglobin chains

Summary The evolutionary relation of vertebrate myoglobin and the hemoglobin chains including the agnathan hemoglobin chain is investigated on the basis of a new view of amino acid changes that is developed by canonical discriminant analysis of amino acid residues at individual sites. In contrast to the clear discrimination of amino acid residues between myoglobin, hemoglobin a chain, and hemoglobin chain in warm-blood vertebrates, the three types of globins in the lower class of vertebrates show so much variation that they are not well discriminated. This is seen particularly at the sites that are ascertained in mammals to carry the amino acid residues participating in stabilizing the monomeric structure in myoglobin and the residues forming the subunit contacts in hemoglobin. At these sites, agnathan hemoglobin chains are evaluated to be intermediate between the myoglobin and hemoglobin chains of gnathostomes. The variation in the phylogenetically lower class of globins is also seen in the internal region; there the amino acid residues of myoglobin and hemoglobin chains in the phylogenetically higher class exhibit an example of parallel evolution at the molecular level. New quantities, the distance of sequence property between discriminated groups and the variation within each group, are derived from the values of discriminant functions along the peptide chain, and this set of quantities simply describes an overall feature of globins such that the distinction between the three types of globins has been clearer as the vertebrates have evolved to become jawed, landed, and warm-blooded. This result strongly suggests that the functional constraint on the amino acid sequence of a protein is changed by living conditions and that severe conditions constitute a driving force that creates a distinctive protein from a less-constrained protein.Offprint requests to: J. Otsuka     

ALEXANDRIUM SATOANUM SP. NOV. (DINOPHYCEAE) FROM MATOYA BAY,CENTRAL JAPAN1

The gonyaulacoid dinofiagellate Alexandrium satoanum Yuki et Fukuyo sp. nov. is described from Matoya Bay, Pacific coast of central Japan. The species is distinctive in its conical epitheca with almost straight sides and dorsal concavity of the hypotheca. The plate formula is Po, pc, 4′, 6″, 6c, 10s, 5″″, and 2″″, including two accessory plates inside the sulcus. The apical pore plate is triangular and possesses an anterior attachment pore at the right margin. The first apical plate does not make contact with the apical pore plate and lacks a ventral pore. A posterior attachment pore lies at the center of the posterior sulcal plate. In Matoya Bay, vegetative cells occur as solitary cells or sometimes in pairs during late spring and early summer in low concentrations. In connection with this study, the following new combination is proposed: Alexandrium pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov.     

Identification of the Sites for Suppressor Mutations on the Hemagglutinin Molecule to Temperature-Sensitive Phenotype of the Influenza Virus

A temperature-sensitive (ts) mutant of the influenza virus A/WSN/ 33 strain, ts-134, possessed a defect in intracellular transport at the nonpermissive temperature and marked thermolability of hemagglutinin (HA) activity at 51 C. These were caused by a change at amino acid residue 157 from tyrosine to histidine in the HA protein. We isolated 37 spontaneous revertant clones from ts-134 at the nonpermissive temperature and determined their HA sequences. The deduced amino acid sequences demonstrated that one was a true revertant and the others were revertants with suppressor mutations, each of which had an additional amino acid change besides those of ts-134. The changed amino acids were located at 14 positions on the HA molecule, and eight of them were found in multiple revertants. These were located in five to six distinct regions on the three-dimensional structure of the HA molecule. However, the heat stability of HAs in the revertants was recovered differently depending on the sites of the changed amino acids. The kinetics of transport of the HA protein in the revertants were slightly delayed compared to the wild-type both at permissive and nonpermissive temperatures.     

Isolation of restriction enzyme EcoVIII, an isoschizomer of HindIII, produced by Escherichia coli E1585-68

A restriction endonuclease designated EcoVIII, an isoschizomer of HindIII, was isolated from Escherichia coli E1585-68 and purified by dextran-polyethylene glycol (DPG) phase partition, ammonium sulfate precipitation, phospho- and DEAE-cellulose chromatography, and hydroxylapatite chromatography. The purified EcoVIII was stable during the purification procedure and its high specific activity required 10 mM Mg²⁺. Unlike HindIII, Eco VIII exhibited a high specific activity even at low pH (pH 6.3) and showed the highest activity at 48° C. Transformation of purified plasmid DNA from E. coli E1585-68 into K-12 indicated that the EcoVIII gene was carried on a multicopy 4.4-kb miniplasmid. EcoVIII seems to be preferable to HindIII for its production and use because of easier handling of producer cells and a wider range of activity.     

Non-polymerizable tropomyosin and control of the superprecipitation of actomyosin.

Non-polymerizable tropomyosin was prepared by the digestion of several C-terminal residues of tropomyosin with carboxypeptidase A [EC 3.4.12.2]. The intrinsic viscosity and molecular weight of the non-polymerizable tropomyosin were almost the same as those of untreated tropomyosin. Like untreated tropomyosin, the non-polymerizable tropomyosin in combination with troponin repressed the superprecipitation of actomyosin in the absence of calcium, while this repression was released by addition of calcium. However, the curve representing the superprecipitation rate as a function of pCa was less steep than that found with actomyosin containing untreated tropomyosin: in the former case, the rate increased to a plateau over about 2 pCa units, while in the latter case, it did so over about 1 pCa unit. These experimental results provide evidence that the "co-operation" in the regulation mechanism of skeletal muscle contraction, which is indicated by the steep curve of the contraction versus pCa relation, is mediated by tropomyosin-tropomyosin interaction along the thin filament.