Solid state theory of competitive diffusion of associated Na+ and K+ in cells by free cation and vacancy (hole) mechanisms, with application to nerve

If, as recent evidence indicates, most cell potassium is associated with macromolecular fixed charge, then diffusion of potassium ions in cells might occur by (1) diffusion of the small fraction of free potassium in cell water (analogous to electrons in the conduction band of a semiconductor) or by (2) diffusion of vacancies on association sites (analogous to holes in a semiconductor). Derivations of the Fick first law of diffusion predict that partial substitution of sodium for potassium in the cell produces opposite effects on the effective diffusion constant of potassium for those mechanisms. Application of that substitution to nerve data suggests that rubidium ions diffuse by a free cation result when the nerve is clamped at its resting potential, but by a vacancy mechanism when the nerve is clamped at zero voltage.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

Antagonistic enzymes may generate alternate phase transitions leading to ephemeral gels

In some biological processes, two enzymes with antagonistic activities--the one creating a bond, the other destroying it--are involved in a reaction cycle. Several catalysts have the ability to modify the rheological properties of biological media participating in the production of a solid gel phase which later dissolves. Transglutaminase, catalyzing intermolecular protein cross-linking, is considered here as a reverse protease as far as the physical state of a proteic gel is concerned. A kinetic model including diffusion constraints and based on a protease/transglutaminase cycle interconverting insoluble gel and soluble proteolysis fragments showed that alternate sol/gel and gel/sol transitions could occur within such a system, generating transient gel phases. Then, ephemeral gels were obtained in vitro using an experimental system consisting of gelatin, transglutaminase, and thermolysin. Modulating the enzyme activity ratio allows us to "program" the global behavior: polymerization/solubilization cycle of a mixture containing at least one protein and two enzymes without any change in temperature or medium composition.     

Degradation of polyribonucleotides: Biocatalysis and the monitoring of products

Macroporous monolithic materials containing covalently linked ribonuclease A were used to create high-performance flow-through heterogeneous biocatalysts (bioreactors). The kinetic parameters of the degradation of polycytidylic acid were identified, and the properties of the obtained systems were compared. A HPLC method has been developed for monitoring of products of biocatalytic degradation of RNA, and the possibility of using biocatalytic and HPLC columns in RNA degradation processes in a multicomponent mixture of biological molecules was shown.     

A kinetic theory of enzymatic oxidation-reduction reactions based on a postulate of electron conduction in a macromolecular enzyme with an application to active transport of small ions across biological membranes

Experimental evidence of electron conduction within a protein has recently been given by Rosenberg. This paper gives a quantitative kinetic treatment of a hypothetical enzyme reaction that is rate-limited by electron conduction within the enzyme molecule. In particular, a kinetic theory of enzymatic oxidation-reduction has been built considering the enzyme to consist of a large protein molecule catalyzing oxidation-reduction of two different substrates at two different enzymatic sites on the same macromolecule. The electrons on each substrate are assumed in free and rapid equilibrium with the substrate's enzymatic site on the protein molecule. The rate-limiting process is assumed to be electron conduction in the protein molecule between the two sites. The resulting substrate concentrationvs. time curves appear to be zero order in some cases, and appear first order in other cases within narrow substrate concentration limits. Quantitative criteria are given for testing whether experimental data fit this type of kinetics. Oxidation-reduction reactions by this mechanism seem likely to be coupled to countercurrents of small charged ions in the surrounding solution, which suggests that a similar process could produce active transport of small ions across biological membranes. Opinions and conclusions contained in this report are those of the author. They are not to be construed as necessarily reflecting the views or the endorsement of the Navy Department.     

Kinetic criteria of solid state mechanisms in biology

Kinetic criteria for solid state physical mechanisms of electron and ion transport in biological systems are summarized, and the mechanisms are discussed. A reaction which is rate-limited by electron or ion transport across a particle or membrane in accord with Ohm's law will show first order kinetics, with an hyperbolic relationship between rate constant and the sum of substrate plus product. Larger initial substrate concentrations produce smaller rate constants, thus giving the appearance of substrate inhibition. Examples are cytochrome oxidase and peroxidase, and pyruvate carboxylase. Ohmic transport mechanisms may be caused by electron conduction or superconduction through protein, by electron conduction through water, or by conduction of ions through membranes. A reaction which is rate-limited by charge transport across an activation energy barrier at an interface in accord with a logarithmic voltage-current law will show reaction kinetics conforming to the Elovich equation, and will have the appearance of a pair of simultaneous first order processes. Examples include decay of photogenerated free radicals in eye melanin particles and in photosynthetic particles of bacteria, and sodium and potassium ion transport across cell surfaces. The logarithmic voltage-current law may be regarded as an empirical relationship describing behavior of interfaces, justified by extensive experimental data on many types of interfaces, or it may be derived theoretically for individual cases from statistical mechanical and/or solid state physical considerations. Dedicated to Prof. N. Rashevsky and to his enlightened editorial policy, especially to his policy of publishing that which is new, even when he disagrees with it.     

Axillary artery injury combined with delayed brachial plexus palsy due to compressive hematoma in a young patient: a case report

Aim

To prove the possibility of axillary nerve conduction changes following shoulder subluxation due to hemiplegia, in order to investigate the usefulness of screening nerve conduction studies in patients with hemiplegia for finding peripheral neuropathy.

Methods

Forty-four shoulders of twenty-two patients with a first-time stroke having flaccid hemiplegia were tested, 43 ± 12 days after stroke onset. Wasting and weakness of the deltoid were present in the involved side. Motor nerve conduction latency and compound muscle action potential (CMAP) amplitude were measured along the axillary nerve, comparing the paralyzed to the sound shoulder. The stimulation was done at the Erb's point whilst the recording needle electrode was inserted into the deltoid muscle 4 cm directly beneath the lateral border of the acromion. Wilcoxon signed rank test was used to compare the motor conduction between the sound and the paralytic shoulder. Mann-Whitney test was used to compare between plegic and sound shoulder in each side.

Results

Mean motor nerve conduction latency time to the deltoid muscle was 8.49, SD 4.36 ms in the paralyzed shoulder and 5.17, SD 1.35 ms in the sound shoulder (p < 0.001). Mean compound muscle action potential (CMAP) amplitude was 2.83, SD 2.50 mV in the paralyzed shoulder and was 7.44, SD 5.47 mV in the sound shoulder (p < 0.001). Patients with right paralyzed shoulder compared to patients with right sound shoulder (p < 0.001, 1-sided for latency; p = 0.003, 1-sided for amplitude), and patients with left paralyzed shoulder compared to patients with left sound shoulder (p = 0.011, 1-sided for latency, p = 0.001, 1-sided for amplitude), support the same outcomes. The electro-physiological changes in the axillary nerve may appear during the first six weeks after stroke breakout.

Conclusion

Continuous traction of the axillary nerve, as in hypotonic shoulder, may affect the electro-physiological properties of the nerve. It most probably results from subluxation of the head of the humerus, causing demyelinization and even axonopathy. Slowing of the conduction velocities of the axillary nerve in the paralyzed shoulders may be related also to the lowering of the skin temperature and muscular atrophy in the same limb. The usefulness of routine screening nerve conduction studies in the shoulder of hemiplegic patients seems to be advocated.     

Calculation and measurement of semiconduction activation energy and electron mobility in cytochrome oxidase,with evidence that charge carriers are polarons,which may couple oxidation to phosphorylation

It is shown that an electron transport reaction which is rate-limited by electron conduction 4 across a solid biological particle or membrane in accord with Ohm's law should have a first order rate constant approximately proportional to exp (E _a/KT ), whereT is absolute temperature,k is the Boltzmann constant andE _a is the activation energy for semiconduction in the solid particle, where resistance in the semiconductor is proportional to exp (E _a /KT). For two different preparations of cytochrome oxidase, this method yields an average value ofE _a =0.27 ev, which agrees well with direct conductivity measurements on dry solid enzyme, which provide an average value ofE _a =0.26 ev. Electron mobility in dry cytochrome oxidase is estimated to be approximately \gm=10^\t-5 cm² volt^\t-1 sec^\t-1. Elovich decay of current in dry cytochrome oxidase was observed, which parallels the Elovich kinetics of cytochrome oxidase activity in yeast observed previously by M\:uhlig (1966). Finally, the solid state kinetic theory is used to deduce that conduction of polarons may be involved in cytochrome oxidase activity (1 polaron=1 electron + 1 phonon), which provides a link with the solid state phonon phosphorylation theory of Straub.     

The chemotaxonomy ofBeauveria bassiana (Deuteromycotina: Hyphomycetes) isolates from the coffee berry borerHypothenemus hampei (Coleoptera: Scolytidae)

A preliminary chemotaxonomic analysis was carried out on 16 isolates ofBeauveria bassiana from adults of coffee berry borer (Hypothenemus hampei) from 10 countries in Latin America, Africa, Asia and the Pacific. Thirteen formed an homogeneous group with very similar electrophoretic and physiological profiles. Two isolates differed in esterase and acid phosphatase band patterns, one of which was also deficient in chitin-degrading enzyme production, but both were still relatively similar to the major group compared with the total variability within the species. A third isolate was abnormal and may have degenerated in storage. These results suggest a distinct population of the fungus on this host. The implications for selection of pathogenic isolates for development as biological pesticides are discussed.     

The nerve impulse in the axon — a new theory

The Classical Theory of function in the nervous system postulates that the nerve impulse is the result of a sequential reversal of the membrane potential due to an increased permeability of the membrane, first to sodium ions, then to potassium ions. The new theory presents a bio-physical model which depicts the nerve impulse as an event involving the motions of electrons and waves, and their interactions with sodium and potassium atoms and ions. The velocity of the nerve impulse (the most important parameter of nerve function) is determined by the product of two constants: c = the speed of light, which is a constant for all nerves; k =a constant for each nerve and is believed to be a specific property of nerve matter related in some way to the atomic process. The theory proposes that the nerve impulse in the axon is dualistic in nature (particles and waves play equally significant roles). The dualistic nature accounts for the three most fundamental characteristics of conduction of the nerve impulse: periodicity (conduction of a nerve impulse over long distances with constant velocity and form); non-summing (two nerve impulses cannot be in the same place at the same time); quantum nature of each nerve impulse — i.e., the unit message of the nerve impulse is an indivisible unit.     

Kinetic characteristics of monoamine oxidase and serum cholinesterase in several related rat strains

This study was performed in order to delineate differences in kinetic enzyme characteristics of brain monoamine oxidase (MAO) and plasma cholinesterase (ChE) derived from the Walker-Walker (Fawn Hooded, FH) rat and from its putative ancestors, the Wistar (W) and Long-Evans (LE). As compared with the enzyme isolated from the other two strains, brain MAO from FH has both a higher V _max and increased reaction rate at lower substrate concentrations. It may thus be described as a “more efficient” enzyme. This study confirms previous work which shows that plasma ChE activity of females is higher than that of males. Fluoride ion is a noncompetitive inhibitor of the Wistar ChE, is a competitive inhibitor of the FH enzyme, and has no effect on the LE enzyme. Dibucaine is a competitive inhibitor in all cases except one: ChE derived from the FH female is uncompetitively inhibited. A comparison of the inhibitor constants shows that FH ChE is more resistant to Dibucaine than is that of W, and that LE is the most sensitive. FH cholinesterase is twice as resistant to the action of fluoride as is the Wistar enzyme.     

A theory of ion transport across cell surfaces by a process analogous to electron transport across liquid-solid interfaces

A kinetic theory of ion transport across cell surfaces has been developed in a form analogous to the kinetic theory of electron transport across solid-liquid interfaces of biological particles. The ionic theory is based on the observation that, at least in one instance, the voltage-current behavior for ion conduction across a cell surface is describable by the Tafel equation, in analogy to the conduction of electrons across solid-liquid interfaces. The theory predicts that the kinetics of ion transport across cell surfaces should conform to the Elovich rate equation, which is shown to be true for various experimental data. The opinions and conclusions contained in this report are those of the author. They are not to be construed as necessarily reflecting the views or the endorsement of the Navy Department.     

Structural states of the flexible catalytic loop of M. tuberculosis tyrosyl-tRNA synthetase in different enzyme–substrate complexes

Tyrosyl-tRNA synthetase from Mycobacterium tuberculosis (MtTyrRS) is an enzyme that belongs to class I of aminoacyl-tRNA synthetases, which catalyze the attachment of l-tyrosine to its cognate tRNATyr in the preribosomal step of protein synthesis. MtTyrRS is incapable of cross-recognition and aminoacylation of human cytoplasmic tRNATyr, so this enzyme may be a promising target for development of novel selective inhibitors as putative antituberculosis drugs. As a class I aminoacyl-tRNA synthetase, MtTyrRS contains the HIGH-like and KFGKS catalytic motifs that catalyze amino acid activation with ATP. In this study, the conformational mobility of MtTyrRS catalytic KFGKS loop was analyzed by 100-ns all-atoms molecular dynamics simulations of the free enzyme and its complexes with different substrates: tyrosine, ATP, and the tyrosyl–adenylate intermediate. It was shown that in the closed state of the active site, the KFGKS loop, readily adopts different stable conformations depending on the type of bound substrate. Molecular dynamics simulations revealed that the closed state of the loop is stabilized by dynamic formation of two antiparallel β-sheets at flanking ends which hold the KFGKS fragment inside the active center. Prevention of β-sheet formation by introducing point mutations in the loop sequence results in a rapid (<20 ns) transition of the loop from its functional “closed” M-like structure to an inactive “open” O-like structure, i.e. rapid diffusion of the catalytic loop outside the active site. The flexibility and rapid dynamics of the wild-type aaRS catalytic loop structure are crucial for formation of protein–substrate interactions and subsequently for overall enzyme functional activity.     

Method for the stabilization and immobilization of enzymatic extracts and its application to the decolorization of textile dyes

Peroxidases from Pleurotus eryngii have been investigated for their ability to degrade recalcitrant, phenolic pollutants. The use of crude enzymatic extracts can reduce the high costs associated with enzyme purification, and enzyme immobilization can enhance enzyme stability and recovery. The present study tests the effectiveness of various conditions for crude enzyme stabilization in polyethylene glycol and glycine solutions, and immobilization on monofunctional and heterofunctional agarose solid supports. Glycine at 0.5 M at 4 °C and pH 4 was most effective stabilization agent for the crude enzymatic extracts, and enzyme immobilization efficiency was greatest for heterofunctional supports. MANA-glyoxyl heterofunctional supports were demonstrated to have the greatest enhancement of decolorization (1.3-fold) and velocity of substrate consumption (fivefold). Therefore, the application of crude enzymatic extracts to industrial processes, such as dye decolorization, represents a cost-effective alternative to purified enzymes.     

Characterization of distinct sub-cellular location of transglutaminase type II: changes in intracellular distribution in physiological and pathological states

Transglutaminase type II (TG2) is a pleiotropic enzyme that exhibits various activities unrelated to its originally identified functions. Apart from post-translational modifications of proteins (peculiar to the transglutaminase family enzymes), TG2 is involved in diverse biological functions, including cell death, signaling, cytoskeleton rearrangements, displaying enzymatic activities, G-protein and non-enzymatic biological functions. It is involved in a variety of human diseases such as celiac disease, diabetes, neurodegenerative diseases, inflammatory disorders and cancer. Regulatory mechanisms might exist through which cells control multifunctional protein expression as a function of their sub-cellular localization. The definition of the tissue and cellular distribution of such proteins is important for the determination of their function(s). We investigate the sub-cellular localization of TG2 by confocal and immunoelectron microscopy techniques in order to gain an understanding of its properties. The culture conditions of human sarcoma cells (2fTGH cells), human embryonic kidney cells (HEK293^TG) and human neuroblastoma cells (SK-n-BE(2)) are modulated to induce various stimuli. Human tissue samples of myocardium and gut mucosa (diseased and healthy) are also analyzed. Immuno-gold labeling indicates that TG2 is localized in the nucleus, mitochondria and endoplasmic reticulum under physiological conditions but that this is not a stable association, since different locations or different amounts of TG2 can be observed depending on stress stimuli or the state of activity of the cell. We describe a possible unrecognized location of TG2. Our findings thus provide useful insights regarding the functions and regulation of this pleiotropic enzyme.     

L-Arabinose isomerase and its use for biotechnological production of rare sugars

L-Arabinose isomerase (AI), a key enzyme in the microbial pentose phosphate pathway, has been regarded as an important biological catalyst in rare sugar production. This enzyme could isomerize L-arabinose into L-ribulose, as well as D-galactose into D-tagatose. Both the two monosaccharides show excellent commercial values in food and pharmaceutical industries. With the identification of novel AI family members, some of them have exhibited remarkable potential in industrial applications. The biological production processes for D-tagatose and L-ribose (or L-ribulose) using AI have been developed and improved in recent years. Meanwhile, protein engineering techniques involving rational design has effectively enhanced the catalytic properties of various AIs. Moreover, the crystal structure of AI has been disclosed, which sheds light on the understanding of AI structure and catalytic mechanism at molecular levels. This article reports recent developments in (i) novel AI screening, (ii) AI-mediated rare sugar production processes, (iii) molecular modification of AI, and (iv) structural biology study of AI. Based on previous reports, an analysis of the future development has also been initiated.     

昆虫碱性磷酸酶的研究进展

碱性磷酸酶存在于昆虫的头、唾液腺（唾液）、肠道、马氏管、表皮、血淋巴、脂肪体、生殖系统、附肢等部位,广泛参与了昆虫的发育、神经传导、激素合成、物质代谢、滞育、社会型昆虫亚种形成等过程。同时碱性磷酸酶与昆虫抗性有关,特别涉及到对Bt制剂的阻滞作用,其本身也是某些农药的靶标酶,某些生物源化合物及病毒、真菌也可以影响其活性。昆虫碱性磷酸酶的研究,将有助于提高对昆虫生化机制及代谢过程的认识,并为害虫治理和资源昆虫饲养提供新的思路。本文综述了国内外对昆虫碱性磷酸酶的研究状况,并描述了昆虫碱性磷酸酶的生化性质及其与生理功能的关系。