Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath)

Aerobic stopped-flow experiments have confirmed that component C is the methane monooxygenase component responsible for interaction with NADH. Reduction of component C by NADH is not the rate-limiting step for component C in the methane monooxygenase reaction. Removal and reconstitution of the redox centres of component C suggest a correlation between the presence of the FAD and Fe2S2 redox centres and NADH: acceptor reductase activity and methane monooxygenase activity respectively, consistent with the order of electron flow: NADH----FAD----Fe2S2----component A. This order suggests that component C functions as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for transfer to component A via the one-electron-carrying Fe2S2 centre. Electron transfer has been demonstrated between the reductase component, component C and the oxygenase component, component A, of the methane monooxygenase complex from Methylococcus capsulatus (Bath) by three separate methods. This intermolecular electron transfer step is not rate-determining for the methane monooxygenase reaction. Intermolecular electron transfer was independent of component B, the third component of the methane monooxygenase. Component B is required to switch the oxidase activity of component A to methane mono-oxygenase activity, suggesting that the role of component B is to couple substrate oxidation to electron transfer, via the methane monooxygenase components.     

Blockade of voltage-dependent 42K efflux from rat brain synaptosome by minaprine and tetrahydroaminoacridine

The effect of minaprine (3-(2-morpholinoethylamino)-4-methyl-6-phenylpyridazine) on the K+ channels was studied by means of 42K efflux from rat brain synaptosomes, comparing the effects of 4-aminopyridine and 9-amino-1,2,3,4-tetrahydroacridine (THA). 42K efflux from rat brain synaptosomes was classified into five components: a resting component (R), a rapidly inactivating, voltage-dependent component (T), a slowly inactivating, voltage-dependent component (S) and a voltage-dependent, Ca(2+)-dependent component which is divided into a fast phase (CT) and a slower phase (CS). 4-Aminopyridine selectively inhibited 42K efflux of component T. THA blocked both S and T components. The inhibitory effect of THA on the 42K efflux of component S was quite pronounced compared with that of component T. Minaprine inhibited the 42K efflux of components S and T but the inhibitory effect on component S was observed with a lower dose of minaprine than that needed for the effect on component T. Minaprine had no effect on the Ca(2+)-dependent component while THA blocked component CT. 42K efflux of the resting component was not changed by minaprine, THA or 4-aminopyridine. These results suggest that minaprine blocks Ca2+ independent voltage-dependent K+ channel is involved in the pharmacological actions of minaprine.     

Computing the uniform component of shape variation

Any change in shape of a configuration of landmark points in two or three dimensions includes a uniform component, a component that is a wholly linear (affine) transformation. The formulas for estimating this component have been standardized for two-dimensional data but not for three-dimensional data. We suggest estimating the component by way of the complementarity between the uniform component and the space of partial warps. The component can be estimated by regression in either one space or the other: regression on the partial warps, followed by their removal, or regression on a basis for the uniform component itself. Either of the new methods can be used for both two- and three-dimensional landmark data and thus generalize Bookstein's (1996, pages 153-168 in Advances in morphometrics [L. F. Marcus et al., eds.], Plenum, New York) linearized Procrustes formula for estimating the uniform component in two dimensions.     

Rat liver membrane secretory component is larger than free secretory component in bile: evidence for proteolytic conversion of membrane form to free form

Secretory component is a receptor for polymeric immunoglobulins on epithelial cells and hepatocytes that facilitates transport of polymeric immunoglobulins into external secretions. Little is known about the transcellular migration of secretory component-polymeric IgA complexes or the membrane forms of secretory component. We therefore examined rat bile and liver membranes to identify and compare the various molecular species of secretory component. Bile or liver membrane proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Protein profiles on blots were probed with antisecretory component antiserum, and the immunoreactive bands were visualized by indirect immunoperoxidase staining. Bile collected in the presence of proteolytic inhibitors showed an immunoreactive doublet band (Mr = 82,000 and 78,000) in the molecular weight range of free secretory component. By contrast, free secretory component in bile collected in the absence of proteolytic inhibitors and purified by affinity chromatography migrated as a single protein with an Mr = 70,000. Both components of the free secretory component doublet bound dimeric IgA when blots were probed with human dimeric IgA. Crude liver membranes prepared in the presence of proteolytic inhibitors showed two immunoreactive secretory component-containing bands, Mr = 107,000 and 99,000, whereas membranes prepared without proteolytic inhibitors showed two smaller immunoreactive bands; one of these proteolytically severed proteins comigrated with the 82,000-dalton free secretory component in bile. These results indicate that membrane forms of secretory component are present in rat liver. The observations that the membrane secretory component is larger than biliary free secretory component and yields biliary SC-like forms of secretory component upon proteolysis support the hypothesis that free secretory component in bile is a proteolytic product of larger liver membrane-associated secretory component.     

Signals, resistance to change, and conditioned reinforcement in a multiple schedule

The effect of signals on resistance to change was evaluated using pigeons responding on a three-component multiple schedule. Each component contained a variable-interval initial link followed by a fixed-time terminal link. One component was an unsignaled-delay schedule, and two were equivalent signaled-delay schedules. After baseline training, resistance to change was assessed through (a) extinction and (b) adding free food to the intercomponent interval. During these tests, the signal stimulus from one of the signaled-delay components (SIG-T) was replaced with the initial-link stimulus from that component, converting it to an unsignaled-delay schedule. That signal stimulus was added to the delay period of the unsignaled-delay component (UNS), converting it to a signaled-delay schedule. The remaining signaled component remained unchanged (SIG-C). Resistance-to-change tests showed removing the signal had a minimal effect on resistance to change in the SIG-T component compared to the unchanged SIG-C component except for one block during free-food testing. Adding the signal to the UNS component significantly increased response rates suggesting that component had low response strength. Interestingly, the direction of the effect was in the opposite direction from what is typically observed. Results are consistent with the conclusion that the signal functioned as a conditioned reinforcer and inconsistent with a generalization-decrement explanation.     

Contractile activation in the retractor unguis muscle of the cockroach Periplaneta americana

1. The K⁺-induced contracture consists of a phasic and a sustained component. Both were eliminated in Ca²⁺-free saline, but the sustained component recovered on the addition of Ca²⁺ to the muscle.2. Procaine mainly inhibited the phasic component. 3. Unlike the sustained component, the phasic component was inhibited by nifedipine in a concentration dependent manner.4. Divalent cations such as Mn²⁺, Co²⁺ and Ni²⁺ markedly increased the sustained component at low concentrations, but decreased it at high concentrations. The cations also modified the phasic component differentially, but to a lesser extent. High concentration abolished the phasic component.5. Ouabain markedly enhanced the sustained component.6. Caffeine contracture was a phasic type. Its duration and amplitude were augmented by pre-soaking the muscle in Na⁺-reduced salines. Immediate pre-treatment with caffeine eliminated the phasic component of the 160mM K⁺-induced contracture.7. These results suggest that a Na-Ca exchange mechanism may play a role in excitation-contraction coupling in insect muscle. Calcium ions flowing into the cell upon membrane depolarization may specifically activate the phasic component by way of a calcium-induced calcium releasing mechanism.     

Low-level inversion of the L component of pseudorabies virus is not dependent on sequence homology.

Pseudorabies virus has a class 2 genome in which the S component is found in two orientations relative to the L component. The L component is bracketed by sequences that are partially homologous; it is found mainly in one orientation, but a small proportion is inverted (J. M. DeMarchi, Z. Lu, G. Rall, S. Kuperschmidt, and T. Ben-Porat, J. Virol. 64:4968-4977, 1990). We have ascertained the role of the patchy homologous sequences bracketing the L component in its inversion. A viral mutant, vYa, from which the sequences at the right end of the L component were deleted was constructed. Despite the absence of homologous sequences bracketing the L component in vYa, its L component inverted to an extent similar to that of the L component in the wild-type virus. These results show the following. (i) The low-frequency inversion of the L component of PrV is not mediated by homologous sequences bracketing this component. (ii) Cleavage of concatemeric DNA at the internal junction between the S and L components is responsible for the appearance of the minority of genomes with an inverted L component in populations of pseudorabies virus. (iii) The signals present near or at the end of the S component are sufficient to allow low-frequency cleavage of concatemeric DNA; the sequences at the end of the L component are not essential for cleavage, although they enhance it considerably.     

Crotoxin, a phospholipase A2 neurotoxin from the South American rattlesnake Crotalus durissus terrificus: purification of several isoforms and comparison of their molecular structure and of their biological activities

Crotoxin, the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus is a mixture of several isoforms that differ slightly in their molecular structure. The toxin consists of two nonidentical subunits: a basic and weakly toxic phospholipase A2, component B, and an acidic and nontoxic subunit, component A. In the present investigation, we have used fast-performance liquid chromatography (FPLC) on anionic and cationic exchange columns to purify isoforms of both crotoxin subunits. Two component A isoforms and four component B isoforms were obtained in a homogeneous state, and their purity was verified by isoelectric focusing in polyacrylamide gels. The amino acid composition of the purified component A and component B isoforms was in good agreement with the protein sequences determined previously with mixtures of isoforms. The amino acid compositions indicated that for both crotoxin components the isoforms differed only by the replacement of few amino acid residues. Eight crotoxin complexes have been prepared in a homogeneous state by reassociation of pure component A and component B isoforms. The quantitative comparison of enzymatic and pharmacological properties of the reconstituted crotoxins indicated that the two component A isoforms had identical properties, whereas the four component B isoforms fell in two classes: crotoxin complexes formed with component B isoforms of the first class were enzymatically less active and pharmacologically more potent than those obtained with component B isoforms of the second class.     

Electric and hydrodynamic properties of polypeptides in solution. 3. Aggregation of poly(L-glutamic acid) in aqueous methanol solvents studied by electric birefringence

The rise and decay of electric birefringence for poly(L -glutamic acid) (PLGA) in aqueous solvents containing 20 and 10 vol % methanol have been found to be unusual. The decay curves have been analyzed on the assumption that there exist two kinds of particles, namely, one (component I) with a shorter relaxation time exhibiting positive birefringence and the other (component II) with a longer relaxation time exhibiting negative birefringence at low fields. From the field strength dependence of the steadystate birefringence the permanent dipole moment, the anisotropy of electric polarizability, and the saturation value of birefringence have been determined for each component. Furthermore, from the relaxation time the length of component I and the diameter of component II have been computed on the models of cylindrical rod and oblate ellipsoid, respectively. The dipole moment, the anisotropy of electric polarizability, and the relaxation time of component II are much larger than those of component I. Both the anisotropy of electric polarizability and the optical anisotropy factor are positive in sign for component I and negative for component II. It is concluded that component I is the helical PLGA molecule itself and component II is the side-by-side (antiparallel) aggregate composed of many helical PLGA molecules. The optical anisotropy factor of each component has been discussed on the basis of Peterlin-Stuart theory.     

Calcium-dependent aggregation of human serum amyloid P component. Inhibition by the cyclic 4,6-pyruvate acetal of galactose

Serum amyloid P component is a normal plasma glycoprotein which is the precursor of amyloid P component, a minor but universal constituent of amyloid deposits. When isolated human P component is exposed to free ionised Ca2+ it aggregates and precipitates. This phenomenon is completely inhibited by the presence of 10(-4)-10(-2) M methyl 4,6-O-(1-carboxyethylidene)-beta-D-galactopyranoside, a recently synthesised specific ligand for amyloid P component. This observation suggests that the autoaggregation of human amyloid P component involves the Ca2+ dependent specific ligand binding property of P component, but does not distinguish between receptor-site-mediated and allosteric mechanisms.     

Calcium channels and excitation-contraction coupling in cardiac cells. I. Two components of contraction in guinea-pig papillary muscle

Biphasic contractions have been obtained in guinea-pig papillary muscle by inducing partial depolarization in K+-rich solution (17 mM) containing 0.3 microM isoproterenol; whereas in guinea-pig atria, the same conditions led to monophasic contractions corresponding to the first component of contraction in papillary muscle. The relationships between the amplitude of the two components of the biphasic contraction and the resting membrane potential were sigmoidal curves. The first component of contraction was inactivated for membrane potentials less positive than those for the second component. In Na+-low solution (25 mM), biphasic contraction became monophasic subsequent to the loss of the second component, but tetraethylammonium unmasked the second component of contraction. The relationship between the amplitude of the first component of contraction and the logarithm of extracellular Ca2+ concentration was complex, whereas for the second component it was linear. When Ca2+ ions were replaced by Sr2+ ions, only the second component of contraction was observed. It is suggested that the first component of contraction may be triggered by a Ca2+ release from sarcoplasmic reticulum, induced by the fast inward Ca2+ current and (or) by the depolarization. The second component of contraction may be due to a direct activation of contractile proteins by Ca2+ entering the cell along with the slow inward Ca2+ current and diffusing through the sarcoplasm. These results do not exclude the existence of a third "tonic" component, which could possibly be mixed with the second component of contraction.     

Nitrogenase V The effect of Mo,W and V on the synthesis of nitrogenase components in Azotobacter vinelandii

When Azotobacter vinelandii OP is derepressed for nitrogenase synthesis in a medium containing no added Mo, component II but not component I is detected. Derepression with W in the medium allows the cells to produce inactive component I that can be activated by addition of molybdate to the medium. This activation does not require protein synthesis. Addition of V to Mo-free media does not increase the synthesis of component I protein. It is possible that Mo is an inducer of component I synthesis and that W also is capable of inducing component I, but that this component I is inactive because it lacks Mo.     

Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase

The Rhodospirillum rubrum pyridine dinucleotide transhydrogenase system is comprised of a membrane-bound component and an easily dissociable soluble factor. Active transhydrogenase complex was solubilized by extraction of chromatophores with lysolecithin. The membrane component was also extracted from membranes depleted of soluble factor. The solubilized membrane component reconstituted transhydrogenase activity upon addition of soluble factor. Various other ionic and non-ionic detergents, including Triton X-100, Lubrol WX, deoxycholate, and digitonin, were ineffectual for solubilization and/or inhibited the enzyme at higher concentrations. The solubilized membrane component was significantly less thermal stable than the membrane-bound component. None of the pyridine dinucleotide substrate affected the thermostability of the solubilized membrane-bound component, whereas NADP⁺ and NADPH afforded protection to membrane-bound component. NADPH stimulated trypsin inactivation of membrane-bound component to a greater extent than NADP⁺, but inactivation of solubilized membrane component was stimulated to the same extent by both pyridine dinucleotides. The solubilized membrane component appears to have a slightly higher affinity for soluble factor than does the membrane-bound component.Abbreviations AcPyAD⁺ oxidized 3-acetylpyridine adenine dinucleotide - BChl bacteriochlorophyll - C_T-particles chromatophores depleted of soluble transhydrogenase factor and devoid of transhydrogenase activity This work was supported by Grant GM 22070 from the National Institutes of Health, United States Public Health Service. Paper I of this series is R. R. Fisher et al. (1975)     

Simplified method for purification of colostrum to obtain secretory component of immunoglobulin A, using secretory component as a reference protein in tracheal aspirate fluid

Many studies employ bronchoalveolar lavage fluid for assessment of biologically active substances secreted from the lung. However, investigators continue to search for a useful reference standard to correct for the inevitable but variable degree of dilution of this fluid. The glycoprotein, soluble secretory component of IgA, may serve as a valid reference protein. We report a simplified method for the purification of secretory component from colostrum. Soluble secretory component was isolated from human colostrum using serial centrifugation, size-exclusion fractionation and ion-exchange chromatography. Secretory component rich fractions were assayed by enzyme immunoassay. They were also evaluated for total amino acid content and distribution and sequence determination with satisfactory agreement with published results. We then demonstrated that soluble secretory component concentration in tracheal aspirate fluid did not correlate with either albumin or with total protein measured in the same samples. Therefore, we conclude that the secretory component of IgA serves as a useful reference marker because its use may avoid errors resulting from leakage of plasma proteins into epithelial lining fluid. Advantages of this method for establishing a standard for secretory component include ready availability of soluble secretory component, simplicity of the method and relative rapidity of the techniques.     

Effect of pH buffer molecules on the light-induced currents from oriented purple membrane

The effect of pH buffers on the microsecond photocurrent component, B2, of oriented purple membranes has been studied. We found that under low salt conditions (less than 10 mM monovalent cationic salt) pH buffers can dramatically alter the waveform of the B2 component. The effect is induced by the protonation process of the buffer molecules by protons expelled from the membrane. These effects can be classified according to the charge transition upon protonation of the buffer. Buffers that carry two positive charges in their protonated form add a negative current component (N component) to B2. Almost all of the other buffers add a positive current component (P component) to B2, which is essentially a mirror image of the N component. Buffers with a pK less than 5.5 have only a small positive buffer component. The pH dependence of the buffer effect is closely related to the pK of the buffer; it requires that the buffer be in its unprotonated form. The rise time of the buffer component increases with the concentration of the buffer molecules. All the buffer effects can be inhibited by the addition of 5 mM of a divalent cation such as Ca2+. Reducing the surface potential slows down the N component but accelerates the P component without affecting the amplitude of the buffer effect significantly. Many of the buffer effects can be explained if we assume that upon protonation of the buffer by a proton expelled from the membrane by light, the buffer molecules move toward the membrane. This backward movement of buffer molecules forms a counter current very similar to that due to cations discussed in Liu, S. Y., R. Govindjee, and T. G. Ebrey. (1990. Biophys. J. 57:951-963).     

Preparation of protein components exhibiting myosin light chain kinase activities from bovine aorta: discrepancies between its enzyme activity and actomyosin activating effect

1) Two protein components, 155 and 130 kDa in their electrophoretic molecular weights, respectively, were isolated in a homogeneous state from bovine aorta; they showed both the superprecipitation-inducing effect on desensitized natural actomyosin and the myosin light chain kinase (MLCK) action on gizzard myosin. 2) The superprecipitating activity of the 155 kDa component was 5 time higher than that of the 130 kDa component on the basis of equivalent MLCK activity. 3) The same procedure was applied to bovine stomach, giving rise to a 155 kDa component in a homogeneous state as in the case of aorta, but the 130 kDa component thus prepared was contaminated by higher molecular weight components. 4) If compared on the basis of equivalent MLCK activity, bovine stomach 155 kDa component showed more than 10 times higher superprecipitating activity than the fraction that contained the 130 kDa component as the main constituent. 5) The discrepancy between the superprecipitating activity and MLCK activity mentioned above was discussed in relation to the Ca2+ regulation mechanism in smooth muscle contraction. The possibility that the 130 kDa component might be a proteolytic product of the 155 kDa component was also discussed.     

黑龙江省五大连池枝状地衣多样性

报道了五大连池地区枝状地衣4属32种,其中圆鳞石蕊(Cladonia trassii Ahti)为中国新记录种,裸柄石蕊(Cladonia gymnopoda Vain.)为中国大陆新记录种,7种为黑龙江省新记录种。对32种地衣地理成分进行了分析,分属于6个地理成分:环北极成分(50%),广布成分(25%),东亚成分(9%),欧亚成分(6%),泛热带成分(6%),中国特有成分(3%);并对32种地衣的基物及分布特点进行了简要分析。     

Simulated calcium current can both cause calcium loading in and trigger calcium release from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell

Skinned canine cardiac Purkinje cells were stimulated by regularly repeated microinjection-aspiration sequences that were programmed to simulate the fast initial component of the transsarcolemmal Ca2+ current and the subsequent slow component corresponding to noninactivating Ca2+ channels. The simulated fast component triggered a tension transient through Ca2+-induced release of Ca2+ from the sarcoplasmic reticulum (SR). The simulated slow component did not affect the tension transient during which it was first introduced but it potentiated the subsequent transients. The potentiation was not observed when the SR function had been destroyed by detergent. The potentiation decreased progressively when the slow component was separated by an increasing time interval from the fast component. The potentiation was progressive over several beats under conditions that decreased the rate of Ca2+ accumulation into the SR (deletion of calmodulin from the solutions; a decrease of the temperature from 22 to 12 degrees C). In the presence of a slow component, an increase of frequency caused a positive staircase, and the introduction of an extrasystole caused a postextrasystolic potentiation. There was a negative staircase and no postextrasystolic potentiation in the absence of a slow component. These results can be explained by a time- and Ca2+-dependent functional separation of the release and accumulation processes of the SR, rather than by Ca2+ circulation between anatomically distinct loading and release compartments. The fast initial component of transsarcolemmal Ca2+ current would trigger Ca2+ release, whereas the slow component would load the SR with an amount of Ca2+ available for release during the subsequent tension transients.     

A Component of Wheat Flour Globulin Polymerized at Alkaline Sides and Depolymerized by Reduction Reversibly

1. The SDS-disc electrophoretical analysis of wheat flour globulin revealed that upon incubation with “Kansui,” a mixture of alkali carbonates, one (No. 9) of the components disappeared and another component (No. 11) increased significantly whose molecular weight was estimated to be more than one million. Incubation of flour globulin with “Kansui” resulted in decrease of SH groups of the globulin.

2. However, a mild reduction of the globulin incubated with “Kansui” caused reappearance of component No. 9 and a decrease of component No. 11.

3. Previous treatment of flour globulin with PCMB or AN completely inhibited such effects of “Kansui” as described above.

4. A fraction containing component No. 9 of flour globulin which was the main component responsible to polymerization by “Kansui,” was isolated from “Kansui”-treated globulin by chromatography on columns of Sephadex G200 and Sepharose 4B.

5. The component protein thus isolated showed a typical phenomenon of reversibility in depolymerization to the original component corresponding to No. 9 component by chemical reduction and polymerization to a component corresponding to No. 9 at alkaline sides.