Low temperature optical absorption spectroscopy: an approach to the study of stereodynamic properties of hemeproteins

In this short review we show how suitable analysis of the temperature dependence of the optical absorption spectra of metalloproteins can give insight into their stereodynamic properties in the region of the chromophore. To this end, the theory of coupling between an intense allowed electronic transition of a chromophore and Franck-Condon active vibrations of the nearby atoms is applied to the Soret band of hemeproteins to obtain an analytical expression suitable for fitting the spectral profile at various temperatures. The reported approach enables one to separate the various contributions to the overall bandwidth together with the parameters that characterize the vibrational coupling. The thermal behavior of these quantities gives information on the dynamic properties of the active site and on their dependence upon protein structure and ligation state. The Soret band of hemeproteins appears to be coupled to high frequency vibrational modes of the heme group (as already shown by resonance Raman spectroscopy) and to a bath of low frequency modes most likely deriving from the bulk of the protein. For the deoxy derivatives inhomogeneous broadening arising from conformational heterogeneity appears to contribute substantially to the linewidth. The data indicate the onset; at temperatures near 180 K, of large scale anharmonic motions that can be attributed to jumping among different conformational substates of the protein.Abbreviations MbCO Carbonmonoxy-myoglobin - Mb Deoxymyoglobin - Mb³⁺ Aquomet-myoglobin - SWMbCO Spermwhale carbonmonoxy-myoglobin - SWMb Spermwhale deoxy-myoglobin Correspondence to: A. Cupane     

Stereodynamic properties of the cooperative homodimeric Scapharca inaequivalvis hemoglobin studied through optical absorption spectroscopy and ligand rebinding kinetics.

The study of the thermal evolution of the Soret band in heme proteins has proved to be a useful tool to understand their stereodynamic properties; moreover, it enables one to relate protein matrix fluctuations and functional behavior when carried out in combination with kinetic experiments on carbon monoxide rebinding after flash photolysis. In this work, we report the thermal evolution of the Soret band of deoxy, carbonmonoxy, and nitric oxide derivatives of the cooperative homodimeric Scapharca inaequivalvis hemoglobin in the temperature range 10-300 K and the carbon monoxide rebinding kinetics after flash photolysis in the temperature range 60-200 K. The two sets of results indicate that Scapharca hemoglobin has a very rigid protein structure compared with other hemeproteins. This feature is brought out i) by the absence of nonharmonic contributions to the soft modes coupled to the Soret band in the liganded derivatives, and ii) by the almost "in plane" position of the iron atom in the photoproduct obtained approximately 10(-8) s after dissociating the bound carbon monoxide molecule at 15 K.     

Reducing enzyme conformational flexibility by multi-point covalent immobilisation

Summary A thermostable esterase was immobilised to glyoxyl-agarose under conditions designed to generate limited-linkage and multi-point covalent derivatives. The multi-point derivative was 830-fold more thermostable than the limited-linkage derivative and retained more activity in the presence of sodium chloride and organic solvents. Medium chain (C8) aliphatic p-nitrophenyl ester substrates, which inactivate the soluble enzyme, were shown to be more readily hydrolysed. Together these data support the contention that multi-point covalent immobilisation results in a more rigid, less conformationally flexible protein structure.     

Optical absorption spectra of azurin and stellacyanin in glycerol/water and ethylene glycol/water solutions in the temperature range 290-20 K

We have measured the optical absorption spectra of azurin and stellacyanin in the wavelength range 1100-350 nm and in the temperature interval 290-20 K. Samples are protein aqueous solutions containing 65% (v/v) glycerol or ethylene glycol as cryoprotectants and remain homogeneous and transparent throughout the whole temperature range investigated. Spectra are deconvoluted into Gaussian components and the temperature dependence of the zeroth, first and second moments of the various bands is analyzed, within the harmonic Franck-Condon approximation, to obtain information on the stereodynamic properties of the active sites of these proteins. Sizable differences of the integrated intensities of all the bands with temperature are observed and are attributed to variations of the metal-ligand relative positions (i.e., deformations of the active site) that occur as the temperature is lowered. The mean effective frequency of the nuclear vibrations coupled to all the observed bands is about 150 cm(-1) for both proteins in both solvents used; this indicates that the electronic transitions from which the optical spectrum originates are substantially coupled with low-frequency vibrational modes, likely ligand-metal-ligand deformations. The relevance of the stereodynamic properties of azurin and stellacyanin, investigated in this work, to their functional behavior is also suggested.     

β-structures of polypeptides with L- and D-residues

Summary A series of five alternating poly(leucyl-lysyl) samples with varying amounts of L- and D-residues randomly distributed along the chain, but evenly shared out amongst leucyl and lysyl residues were synthesized by condensation of a mixture of the four diastereoisomeric dipeptidep-nitro-phenylesters. Their behavior in aqueous solution at various ionic strengths was studied by infrared spectroscopy which allowed measurement of the total amount of-structures, and by circular dichroism which gives the excess of L-residues over D-residues in the same structures. Comparison with the properties of the all L-poly(Lys-Leu-Lys-Leu) shows that incorporation of a few D-residues in a L-chain seems to reduce the width of the-sheets obtained in presence of salt. Higher proportions of D-isomers prevent the coil transition from occurring when the ionic strength is increased except for segments containing at least 6 to 7 adjacent residues of the same configuration.     

Low temperature optical spectroscopy of cobalt-substituted hemocyanin from Carcinus maenas

In this work we report the optical absorption spectra of three cobalt-substituted derivatives of hemocyanin (He) from Carcinus maenas, in the temperature range 300–20 K. The derivatives studied are the mononuclear (Co²⁺)-He with a single cobalt ion in the Cu_A site, the binuclear (Co²⁺)₂-He and the binuclear mixed metal (Co²⁺-Cu¹⁺)-He. At low temperature three main bands are clearly resolved; the temperature dependence of their zeroth, first and second moments sheds light on the stereodynamic properties in the surroundings of the chromophore. Within the limits of the reported analysis, in the binuclear derivatives the motions coupled to the chromophore appear to be essentially harmonic in the whole temperature range investigated; moreover the data are consistent with the presence of an exogenous ligand strongly bound to the two metal ions. For the mononuclear derivative an essentially harmonic behavior is evident only up to 200 K where the data are consistent with the presence of an exogenous ligand much less strongly bound, while at higher temperatures the behavior of the spectra indicates the onset of very large anharmonic contributions to motions, that plausibly involve the above exogenous ligand and, quite likely, the entire active site.Abbreviations He Hemocyanin - M₀ zeroth moment - M₁ first moment - M₂ second moment - (Co^2–)₂-He binuclear bicobalt hemocyanin derivative - (Co²⁺)-He mononuclear monocobalt hemocyanin derivative - (Co²⁺-Cu¹⁺)-He binuclear mixed metals hemocyanin derivative - LFT ligand field theory - CT charge transfer - EPR electronic paramagnetic resonance - XANES X-ray absorption near edge structure Correspondence to: L. Cordone     

Task Relevance Enhances Early Transient and Late Slow-Wave Activity of Distributed Cortical Sources

The primary purpose of these studies was to link together concepts related to attention/working memory and feedforward/feedback activity using MEG response profiles obtained in humans. Similar to recent studies of attention in monkeys, we show early spike-like activity (<200 ms poststimulus), most likely reflecting an early transient excitatory response mixed with feedback influences, followed by slow-wave activity (>200 ms poststimulus) in MEG cortical response profiles evoked by a visual working memory task. We experimentally dissociated the early transient activity from the later sustained activity (predominately feedback) by conducting an auditory size classification task. Words, representing common objects, evoked activity in occipital cortex (presumably due to imagery) even though visual stimuli were not present in this task. The initial spike was absent from the response profile obtained from occipital cortex, leaving only slow-wave activity, thereby allowing us to characterize or profile feedback activity in this situation. Attention or task relevance enhanced the initial spike and slow-wave activity in visually responsive areas. Prefrontal activity, along the superior frontal sulcus, evoked by the working memory task, was active later in time than initial activity in visual cortex and later than the earliest effect of attention modulation in visual cortex.     

Peasants in primitive accumulation: Western Europe and Venezuela compared

Conclusion: The internal market and capitalist development The basic point of this comparison has already been implied. I noted above that primitive accumulation consists of two processes: (1) capital accumulation, and (2) proletarianization. These are, in turn, part of a more general process: the creation of an internal market for capitalism, i. e., a market for means of production and a market for labor power. Yet what is internal and what is external depends on our levels of analysis. When we refer to the development of capitalism in a particular country, e.g. Venezuela or England or Russia, we must examine internal and external markets with reference to the economic formation. But when we are referring to a market for goods, the importance of the distinction between internal and external markets (i.e., intra-state or national market vs. foreign trade) is reduced. When, however, we refer to the development of a market for labor power, the problem takes on special significance. Whereas goods as commodities may flow easily across state boundaries, labor power as a commodity cannot be as free [59]. We must direct our analysis primarily to the market for capital and labor power.It has been argued that a capitalist mode of production developed in England and other countries of Western Europe because an internal market for capitalism emerged within particular economic formations. While these formations depended on foreign markets for goods, they developed an internal market for capital and labor power. Venezuela was part of the internal market of the capitalist system, but its position in that system prohibited the development of an internal market for capital and labor power within the economic formation of Venezuela. This difference can be explained with reference to the respective positions of the formations within the inter-state capitalist system: Western Europe at the center; Venezuela at the periphery.But this study implies another, cognate conclusion which I cannot pursue here, namely, that capitalism cannot be rationalized either as the relatively progressive fate of under-developed peoples, or as an inevitable forerunner of socialism in whatever place the struggle for socialism develops [60].     

DNA polymorphism in cytokine genes based on length variation in simple-sequence tandem repeats

The possibility of the involvement of cytokines in the genetic predisposition to various diseases has been suggested by a large variety of studies. However, the study of potential disease linkage of cytokine genes has been hampered by a lack of sufficiently polymorphic markers at the restriction fragment length polymorphism (RFLP) level. We have investigated the distribution, the length polymorphism, the informativeness, and the efficiency of analysis, of simple-sequence tandem repeats in the mouse cytokine genes. Highly polymorphic sequences have been identified in the IL-1, IL-1ra, IL-2, IL-4, IL-6, IL-7, and IFN- genes. The utility and the value of these sequences as gene markers is exemplified by mapping the IL-7 gene to mouse chromosome 3 close to pgk-1ps3 and Car-2 loci and the IFN- gene to chrrmosome 10 near the pg locus. Advantages of short tandemly repeated sequences as genetic markers are discussed in comparison with RFLPs.     

The sieve tube wall and its relation to translocation

Summary The sieve tube wall possesses a broad inner layer often with pronounced radial striations. The plasmalemma of the sieve tube appears to penetrate this wall in the form of a brush border of irregular microvilli, greatly increasing its surface area. It is suggested that this is the site of active transport of potassium, which circulates electroosmotically through the sieve plate pores and back through the thick wall. The function of the companion cells is the care and maintenance of the active brush border sites; in conjunction with their activity in supplying high-energy intermediates movement in the column acts regeneratively and fully polarises the plates. Many of the lamellar stacks and curvilinear membrane aggregates hitherto regarded as endoplasmic reticulum are, it is suggested, plasmalemma displaced from the wall. These findings have important consequences for the electroosmotic theory.     

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting regeneration of transgenic plants

We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587–593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by -glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.Abbreviations BAP benzylaminopurine - CTAB hexadecyltrimethylammoniumbromide - Na₂EDTA ethylenediamine-tetra-acetate ferric-sodium salt - FeNaEDTA ethylenediamine-tetra-acetate ferric-sodium salt - GA₃ gibberellic acid 3 - GusA -glucuronidase - gusA -glucuronidase gene of Escherichia coli - IAA indole acetic acid - IBA indole butyric acid - 2iP N6-2-isopentenyl adenine - NAA naphthalene acetic acid - nptII neomycinphosphotransferase II gene - bar phosphinothricin acetyl transferase gene - PCR polymerase chain reaction - PPT phosphinothricin - STS silver thiosulphate - T-DNA transferred DNA - TDZ thidiazuron - X-Gluc 5-bromo-4-chloro-3-indolyl -D-glucuronide - Zea trans-Zeatin     

Arsenic‐induced NFκβ transactivation through Erks‐ and JNKs‐dependent pathways in mouse epidermal JB6 cells

Carnosine, a alanylLhistidine dipeptide with antioxidant properties is present at high concentrations in skeletal muscle tissue. In this study, we report on the antioxidant activity of carnosine on muscle lipid and protein stability from both in vitro and in vivo experiments. Carnosine inhibited lipid peroxidation and oxidative modification of protein in muscle tissue prepared from rat hind limb homogenates exposed to in vitro Fenton reactant (Fe²⁺, H₂O₂)generated free radicals. The minimum effective concentrations of carnosine for lipid and protein oxidation were 2.5 and 1 mM, respectively. Histidine and alanine, active components of carnosine, showed no individual effect towards inhibiting either lipid or protein oxidation. Skeletal muscle of rats fed a histidine supplemented diet for 13 days exhibited a marked increase in carnosine content with a concomitant reduction in muscle lipid peroxidation and protein carbonyl content in skeletal muscle caused by subjecting rats to a Fenitrilotriacetate administration treatment. This significant in vitro result confirms the in vivo antioxidant activity of carnosine for both lipid and protein constituents of muscle under physiological conditions.     

Structure of tanacetan,a pectic polysaccharide from tansy Tanacetum vulgare L

Tanacetan TVF was found to have a branched structure with a backbone of linear -1,4-D-galacturonan. The ramified regions consist of linear -1,2-L-rhamno--1,4-D-galacturonan as the core. The side chains appear to attach to the 4-position of the L-rhamnopyranose residues. They are present as single -galactopyranose residues or a branching -1,4-galactopyranan bearing 4,6-substituted -D-galactopyranose residues as branched points. In addition, the ramified regions contain side chains of a branched -1,5-arabinofuranan possessing 2,5- and 3,5-substituted -L-arabinofuranose residues as branching points. Some side chains of rhamnogalacturonan appear to be arabinogalactan which contains branched sugar chains of -1,5-arabinofuranan attached to the linear chains of -1,4-galactopyranan by 1,3- and 1,6-linkages. The residues of -L-arabinofuranose seem to occupy the terminal positions of the arabinogalactan side chains.     

Molecular species of membrane phospholipids containing arachidonic acid and linoleic acid contribute to the interindividual variability of red blood cell Na+-Li+ countertransport: In vivo and in vitro evidence

Summary Previous studies indicate a particular sensitivity of red blood cell Na⁺-Li⁺ countertransport activity to small variations in the fatty acid composition of membrane phospholipids. To assess whether the interindividual variability of Na⁺-Li⁺ countertransport is related to differences in the species pattern of erythrocyte phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vivo, the molecular species composition of PC and PE as well as the kinetics of Na⁺-Li⁺ countertransport were analyzed in parallel in normo- and hyperlipidemic donors. Both in diacyl PC and in diacyl-PE the species 160/204 and 160/182 were, respectively, positively and negatively related to the apparent maximal velocity of Na⁺-Li⁺ countertransport. The sum of all species with 204 at sn₂ of diacyl-PE exhibited a strong positive (r = 0.82, 2p < 0.001), and those containing 182 a negative correlation (r = –0.63, 2p < 0.01) to the transport activity. Essentially similar connections were observed between these species and the apparent affinity of the transport system for intracellular Na⁺. To evaluate whether the associations between molecular species of membrane phospholipids and Na⁺-Li⁺ countertransport activity were indicative of a causal relationship, the species 160/204-PC and 160/182-PC were selectively introduced into the erythrocyte membrane by means of the PC-specific transfer protein. Replacement of 11% of native PC by 160/182-PC inhibited the transport rate by about 25%. Exchange of 6 and 9% of PC with 160/204-PC, in contrast, accelerated the transport rate by 30 and 60%, respectively. The accordance between the in vivo relations and the results of the in vitro modification strongly suggests that elevations and reductions in the arachidonic acid and linoleic acid content of membrane PC and PE contribute to the interindividual variability of red blood cell Na⁺-Li⁺ counter-transport activity and its acceleration in hyperlipidemias.The authors wish to thank Dr. W.O. Richter (II. Medizinische Klinik, Klinikum Großhadern, Universität München) for selection of the patients and Dr. T. Brosche (Universität ErlangenNürnberg) for gaschromatographic analyses. This study was supported in part by a grant of the Wilhelm-Sander-Stiftung to B.E.     

Hyperpolarizing and depolarizing mechanoreceptor potentials inStylonychia

Summary The surface ofStylonychia was mechanically stimulated with a piezo-crystal driven microneedle of 0.5-2 m distal diameter and maximal amplitudes of 13 m. Stimulation of the anterior surface of the cell produced a membrane depolarization, while stimulation of the posterior surface elicited a hyperpolarizing response. The analysis of electric responses to mechanical stimuli, driven by pulses varied in duration, amplitude, rate and acceleration, revealed that the hyperpolarizing receptor potential (hRP) rose in parallel with the stimulus velocity. Stimulus amplitudes beyond 12 m and at rates larger than 4 mm/s did not increase the amplitude of the membrane response. Sustained stimuli slowed down the repolarization to the resting level. Adaptation of the receptor response was seen with small and sustained velocities of the stimulating probe. The depolarizing receptor response (dRP) triggered an action potential consisting of two regenerative components, one graded, the other all-or-none. Positive conditioning current pulses reversed the polarity of the dRP which was primarily Ca-dependent (22.4 mV/log [Ca]₀).The dRP was isolated from the action potential by negative membrane conditioning. The reversal potential of the hyperpolarizing receptor response was negative of the resting potential and completely K-dependent (58.5 mV/log [K]_o).Submaximal hyperpolarizing and subthreshold depolarizing receptor potentials showed summation. No refractoriness of the hRP was detected. Summation of depolarizing responses beyond the threshold activated a regenerative membrane depolarization.Abbreviations hRP Hyperpolarizing receptor potential - dRP Depolarizing receptor potential Dedicated to Professor J. Schwartzkopff on the occasion of his sixtieth birthdaySupported by the Deutsche Forschungsgemeinschaft (SFB 114, TP A5)     

A molecular piston mechanism of pumping protons by bacteriorhodopsin

Summary In this review the proton-pumping mechanism proposed recently for bacteriorhodopsin [Chou, K. C. (1993) Journal of Protein Chemistry, 12: 337–350] is illustrated in terms of a phenomenological model. According to the model, the-ionone of the retinal chromophore in bacteriorhodopsin can be phenomenologically imagined as a molecular piston. The photon capture by bacteriorhodopsin would pull it up while the spontaneous decrease in potential energy would push it down so that it would be up and down alternately during the photocycle process. When it is pulled up, the gate of pore is open and the water channel for the proton translocation is through; when it is pushed down, the gate of pore is closed and the water channel is shut up. Such a model not only is quite consistent with experimental observations, but also provides useful insights and a different view to elucidate the protonpumping mechanism of bacteriorhodopsin. The essence of the model might be useful in investigating the mechanism of ion-channels of other membrane proteins.Abbreviations bR bacteriorhodopsin - All-trans bR bacteriorhodopsin with all-trans retinal chromophore - 13-cis bR bacteriorhodopsin with 13-cis retinal chromophore - All-trans bundle the 7-helix bundle in the all-trans bR - 13-cis bundle the 7-helix bundle in the 13-cis bR - rms root-mean-square     

Redox-linked proton translocation by direct-coupled ligand conduction

The term direct-coupled is considered in the context of redox-linked proton translocation mechanisms, and the origins of this concept, its philosophical implications, applications, and contributions to the development of bioenergetics, are discussed.     

A temporal study at the ultrastructural level of the developing pro-oocyte of Drosophila melanogaster

The first pro-oocyte in developing pupal germaria of females grown at 25 ° has been followed at 6 h intervals from its formation at 129 h post-ovipostion until Stage 1, to provide an unambiguous temporal order. EM autoradiographs were made of sectioned germaria, scanned at lower magnification for location of the pro-oocyte(s) within the most posterior 16-cell cyst and photographed at higher magnification to show the presence of label indicating DNA replication and synaptonemal complex indicating synapsis in the same pro-oocyte nucleus. Label, detected at 132 h and at all subsequent intervals up to and including 162 h, delimited an S-phase of 30 h and identified this period as premeiotic interphase. Extensive SCs (av. length 50 m/genome) measured in serial sections at 132 h provide irrefutable evidence that synapsis in Drosophila begins close to both pro-oocyte formation and initiation of premeiotic interphase. Measurements of SCs at 6 h intervals during interphase reveal a sharp increase between 132 and 138 h, a peak length (75 m/genome) at 144 h, a decrease and subsequent plateau ( 60 m/genome) from 150–162 h and a further drop (R~50 m/genome) at Stage 1. Maximal extension of SC at 144 h coincides with maximal genome response to heat (Grell and Day, 1974) and with midpremeiotic-S. Spherical nodules, detected at 1/genome between 138 and 150 h would, on the questionable assumption that they are the sites of recombination, provide proof of recombination during early interphase, as genetic evidence strongly implies. Evidence contrary to interpretations of fibrillar material within the nucleus as either precursor of the central region of the SC or sagittal sections of the central element of the SC, is presented. No structure corresponding to the polytene chromocenter was observed.     

Pteroylpolyglutamates

Summary Reduced derivatives of the vitamin pteroylglutamic acid (folic acid) are essential coenzymes for the biosynthesis of purine nucleotides, methionine, thymidylate and for many other enzyme catalyzed reactions involving the transfer, oxidation and reduction of single carbon units. Pteroylglutamic acid is found in tissues in the form of poly--glutamyl derivatives of varying chain length. The present review covers the detection, distribution, synthesis, degradation, coenzyme function and inhibitory activities of pteroyl--glutamates. The biosynthesis and inhibitory activities of poly--glutamyl derivatives of methotrexate, an analog of pteroylglutamic acid having antitumor activity, are also considered. An hypothesis on the coenzymatic role of pteroylpoly--glutamates in the coordination of sequential enzymatic steps in the metabolism of single carbon units is presented.     

Immunocytochemical evidence for the translocation of α-granule membrane glycoprotein IIb/IIIa (integrin αIIbβ3) of human platelets to the surface membrane during the release reaction

Summary The localization of glycoprotein (GP) IIb/IIIa (integrin _IIb3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the pre-embedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0±2.7 gold particles/m of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of -granules, and the density of gold particles was 19.7±3.6 particles/m. At 5 min, the -granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0±3.7 particles/m; p<0.01). In immunostained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the -granule membranes of resting platelets. At 30 s after thrombin stimulation the -granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that -granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS. Also the translocation of -granule membrane GPIIb/IIIa gives rise to an actual increase in GPIIb/IIIa on the surface membrane during the release reaction induced by thrombin.