Effective Thickness and Resistance of the Air Boundary Layer Adjacent to Spherical Plant Parts

Using existing heat transfer data a rather simple expressionis developed for the effective thickness of the boundary layerof air adjacent to spheres. The calculated boundary layer thicknessagreed fairly well with that determined for water vapour diffusionfrom a wet spherical surface 5.90 cm in diameter for wind velocitiesfrom 10 to 1000 cm s^–1. At a turbulence intensity representativeof field conditions (0.5), the measured boundary layer thicknessaveraged 11 per cent less than for a relatively low turbulenceintensity of 0.01. For wind velocities up to 100 cm s^–1,the resistance of the boundary layer for water vapour diffusionwas actually greater than the resistance of the basidiocarpsof Lycoperdon perlatum, L. polymorphum, and Scleroderma australe.The tissue resistance for water vapour movement from the fruitingbodies of other members of the Basidiomycetes was also low (below10 s cm^–1), while the resistance of 20 different sphericalfruits ranged from 30 to over 5000 s cm^–1. The thicknessof the air boundary layer for spheres can depend on the squareroot of a characteristic dimension divided by the wind velocity,as has already been shown for cylindrical and flat plant material.     

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a K_d for DNA of 9.2 nm, with an off-rate constant of 0.013 s⁻¹and an on-rate constant of 14 μm⁻¹ s⁻¹. dNTP binding is rapid with K_d values of 20 and 476 μm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a K_d value for PP_i of 3.7 mm and a maximal rate constant of 11 s⁻¹. Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s⁻¹; rapid phosphodiester bond formation occurs with a K_eq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s⁻¹, for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency.     

Aftereffects of low and high temperature pretreatment on leaf resistance, transpiration, and leaf temperature in xanthium

Leaf resistance for water vapor (total diffusion resistance minus boundary layer resistance), transpiration, and leaf temperature were measured in attached leaves of greenhouse-grown Xanthium strumarium L. plants that had been pretreated for 72 hours with high (40 C day, 35 C night), or low (10 C day, 5 C night) air temperatures. Measurements were made in a wind tunnel at light intensity of 1.15 cal cm⁻² min⁻¹, air temperatures between 5 and 45 C, and wind speed of 65 cm sec⁻¹. Leaf resistances in low temperature pretreated plants were higher (8 to 27 sec cm⁻¹) than in controls or high temperature pretreated plants (0.5 to 3 sec cm⁻¹) at leaf temperatures between 5 and 25 C. Thus, the pretreatment influenced stomatal aperture.     

Exclusion of Protein from High Polymer Media: I. Derivation of Probability Distribution for the Number of Fiber Centers Within Any Sphere of Radius r

Ogston's (1958) fiber model based on Poisson's distribution function gives the average number of fibers making contact and no contact inside a sphere of radius r. The probability of penetration of spherical particles within a fibrous network was derived from the moment generating function [Formula: see text] A is the number of particles that intrude into a sphere of radious r. α(μ) is the probability that a particle, whose center is μ units away from the origin, intrudes into a sphere of radius r. A has a Poisson distribution with a mean value E(A) = 4πνα(μ)μ² dμ. The theoretical derivation of the distribution function of A gives Ogston's fiber model.     

Laser activation of rapid absorption changes in spinach chloroplasts and chlorella

The kinetics of the 520 mμ absorption change in spinach chloroplasts and Chlorella vulgaris following a flash from the ruby laser have been determined as follows: rise halftime ≤ 0.3 × 10⁻⁶ second; rapid recovery halftime = 5 to 6 × 10⁻⁶ second; intermediate recovery halftime = 4 × 10⁻⁴ second (spinach chloroplasts only); slow recovery halftime = 12 to 170 × 10⁻³ second, dependent on the measuring light intensity and aerobicity of the suspension.

The rapid phase of the 520 mμ reaction is approximately independent of temperature, from 295° to 77° Absolute.

With increasing oxygenation of the sample, the extent of the rapid phase decreases, the extent of the slow phase increases, while the extent of the intermediate phase in spinach chloroplasts remains constant.

In spinach chloroplasts, no recovery halftime of the 3 recovery phases for the 520 mμ absorption change was observed to correspond to the halftime for oxidation of cytochrome f (t_½ = 1.3 × 10⁻³ second).

     

FLOW‐INDUCED MORPHOLOGICAL VARIATIONS AFFECT DIFFUSION BOUNDARY‐LAYER THICKNESS OF MACROCYSTIS PYRIFERA (HETEROKONTOPHYTA,LAMINARIALES)1

In slow mainstream flows (<4–6 cm · s^?1), the transport of dissolved nutrients to seaweed blade surfaces is reduced due to the formation of thicker diffusion boundary layers (DBLs). The blade morphology of Macrocystis pyrifera (L.) C. Agardh varies with the hydrodynamic environment in which it grows; wave‐exposed blades are narrow and thick with small surface corrugations (1 mm tall), whereas wave‐sheltered blades are wider and thinner with large (2–5 cm) edge undulations. Within the surface corrugations of wave‐exposed blades, the DBL thickness, measured using an O₂ micro‐optode, ranged from 0.67 to 0.80 mm and did not vary with mainstream velocities between 0.8 and 4.5 cm · s^?1. At the corrugation apex, DBL thickness decreased with increasing seawater velocity, from 0.4 mm at 0.8 cm · s^?1 to being undetectable at 4.5 cm · s^?1. Results show how the wave‐exposed blades trap fluid within the corrugations at their surface. For wave‐sheltered blades at 0.8 cm · s^?1, a DBL thickness of 0.73 ± 0.31 mm within the edge undulation was 10‐fold greater than at the undulation apex, while at 2.1 cm · s^?1, DBL thicknesses were similar at <0.07 mm. Relative turbulence intensity was measured using an acoustic Doppler velocimeter (ADV), and overall, there was little evidence to support our hypothesis that the edge undulations of wave‐sheltered blades increased turbulence intensity compared to wave‐exposed blades. We discuss the positive and negative effects of thick DBLs at seaweed surfaces.     

Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics

KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 μm⁻¹ s⁻¹, followed by rate-limiting ADP release at 12.8 s⁻¹. ATP binding at 7.5 μm⁻¹ s⁻¹ was followed by an ATP-promoted isomerization at 84 s⁻¹ to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s⁻¹. ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s⁻¹. The dissociation step showed an apparent affinity for ATP that was very weak (K_½,ATP at 133 μm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 μm⁻¹ s⁻¹, which is inconsistent with fast ATP binding at 7.5 μm⁻¹ s⁻¹ and a K_d,ATP at 6.1 μm. These results suggest that ATP binding per se cannot account for the apparent weak K_½,ATP at 133 μm. The steady-state ATPase K_m,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins.     

Viscoelastic Properties of the Human Red Blood Cell Membrane: I. Deformation, Volume Loss, and Rupture of Red Cells in Micropipettes

Single human red blood cells suspended in buffered Ringer's solution were rapidly drawn, at recorded pressures, into glass micropipettes of diameter 0.6-3.2 μm. Cells could enter micropipettes of diameter ≥ 2.9 μm with minimal pressure. In micropipettes of 0.9-2.9 μm, the pressure required increased linearly with decreasing diameter. For diameters 2.5-2.9 μm, pressures ranged up to 7 cm Hg, and the cells returned to normal biconcave shape on release. For diameters 1.9-2.5 μm, the required pressures ranged from 7 to 17 cm Hg. The released cells were crenated. In micropipettes 0.9-1.9 μm, the pressures required ranged from 17 to 34 cm Hg. The cells hemolyzed on entry. As diameter decreased from 0.9 to 0.6 μm, cells were drawn into dumbbell shapes and parts of the cells were pinched off without complete hemolysis of the cell. Using an accepted value of 138 μm² for the mean cell area, the mean volume of the human red cell was calculated to be 94 μm³. Under mechanical stress, about 12% of this volume is rapidly exchangeable with the external medium. The cell volume may further decrease by 20% which is not reversible.     

DNA polymerases ν and θ are required for efficient immunoglobulin V gene diversification in chicken

The chicken DT40 B lymphocyte line diversifies its immunoglobulin (Ig) V genes through translesion DNA synthesis–dependent point mutations (Ig hypermutation) and homologous recombination (HR)–dependent Ig gene conversion. The error-prone biochemical characteristic of the A family DNA polymerases Polν and Polθ led us to explore the role of these polymerases in Ig gene diversification in DT40 cells. Disruption of both polymerases causes a significant decrease in Ig gene conversion events, although POLN^−/−/POLQ^−/− cells exhibit no prominent defect in HR-mediated DNA repair, as indicated by no increase in sensitivity to camptothecin. Polη has also been previously implicated in Ig gene conversion. We show that a POLH^−/−/POLN^−/−/POLQ^−/− triple mutant displays no Ig gene conversion and reduced Ig hypermutation. Together, these data define a role for Polν and Polθ in recombination and suggest that the DNA synthesis associated with Ig gene conversion is accounted for by three specialized DNA polymerases.     

Water transport in the midrib tissue of maize leaves : direct measurement of the propagation of changes in cell turgor across a plant tissue

Water movement across plant tissues occurs along two paths: from cell-to-cell and in the apoplasm. We examined the contribution of these two paths to the kinetics of water transport across the parenchymatous midrib tissue of the maize (Zea mays L.) leaf. Water relations parameters (hydraulic conductivity, Lp; cell elastic coefficient, ε; half-time of water exchange for individual cells, T_½) of individual parenchyma cells determined with the pressure probe varied in different regions of the midrib. In the adaxial region, Lp = (0.3 ± 0.3)·10⁻⁵ centimeters per second per bar, ε = 103 ± 72 bar, and T_½ = 7.9 ± 4.8 seconds (n = seven cells); whereas, in the abaxial region, Lp = (2.5 ± 0.9)·10⁻⁵ centimeters per second per bar, ε = 41 ± 9 bar, and T_½ = 1.3 ± 0.5 seconds (n = 7). This zonal variation in Lp, ε, and T_½ indicates that tissue inhomogeneities exist for these parameters and could have an effect on the kinetics of water transport across the tissue.

The diffusivity of the tissue to water (D_t) obtained from the sorption kinetics of rehydrating tissue was D_t = (1.1 ± 0.4)·10⁻⁶ square centimeters per second (n = 6). The diffusivity of the cell-to-cell path (D_c) calculated from pressure probe data ranged from D_c = 0.4·10⁻⁶ square centimeters per second in the adaxial region to D_c = 6.1·10⁻⁶ square centimeters per second in the abaxial region of the tissue. D_t D_c suggests substantial cell-to-cell transport of water occurred during rehydration. However, the tissue diffusivity calculated from the kinetics of pressure-propagation across the tissue (D_t′) was D_t′ = (33.1 ± 8.0)·10⁻⁶ square centimeters per second (n = 8) and more than 1 order of magnitude larger than D_t. Also, the hydraulic conductance of the midrib tissue (Lp_m per square centimeter of surface) estimated from pressure-induced flows across several parenchyma cell layers was Lp_m = (8.9 ± 5.6)·10⁻⁵ centimeters per second per bar (n = 5) and much larger than Lp.

These results indicate that the preferential path for water transport across the midrib tissue depends on the nature of the driving forces present within the tissue. Under osmotic conditions, the cell-to-cell path dominates, whereas under hydrostatic conditions water moves primarily in the apoplasm.

     

Mobilization of Genomic Islands of Staphylococcus aureus by Temperate Bacteriophage

The virulence of Staphylococcus aureus, in both human and animal hosts, is largely influenced by the acquisition of mobile genetic elements (MGEs). Most S. aureus strains carry a variety of MGEs, including three genomic islands (νSaα, νSaβ, νSaγ) that are diverse in virulence gene content but conserved within strain lineages. Although the mobilization of pathogenicity islands, phages and plasmids has been well studied, the mobilization of genomic islands is poorly understood. We previously demonstrated the mobilization of νSaβ by the adjacent temperate bacteriophage ϕSaBov from strain RF122. In this study, we demonstrate that ϕSaBov mediates the mobilization of νSaα and νSaγ, which are located remotely from ϕSaBov, mostly to recipient strains belonging to ST151. Phage DNA sequence analysis revealed that chromosomal DNA excision events from RF122 were highly specific to MGEs, suggesting sequence-specific DNA excision and packaging events rather than generalized transduction by a temperate phage. Disruption of the int gene in ϕSaBov did not affect phage DNA excision, packaging, and integration events. However, disruption of the terL gene completely abolished phage DNA packing events, suggesting that the primary function of temperate phage in the transfer of genomic islands is to allow for phage DNA packaging by TerL and that transducing phage particles are the actual vehicle for transfer. These results extend our understanding of the important role of bacteriophage in the horizontal transfer and evolution of genomic islands in S. aureus.     

Elastic-Mathematical Theory of Cells and Mitochondria in Swelling Process: II. Effect of Temperature upon Modulus of Elasticity of Membranous Material of Egg Cells of Sea Urchin, Strongylocentrotus purpuratus, and of Oyster, Crassostrea virginica

The elastic behavior of the cell wall as a function of the temperature has been studied with particular attention being given to the swelling of egg cells of Strongylocentrotus purpuratus and Crassostrea virginica in different sea water concentrations at different temperatures. It was found that the modulus of elasticity is a nonlinear function of temperature. At about 12-13°C the modulus of elasticity (E) is constant, independent of the stress (σ) and strain (ε_ν) which exist at the cell wall; the membranous material follows Hooke's law, and E ≈ 3 × 10⁷ dyn/cm² for S. purpuratus and C. virginica. When the temperature is higher or lower than 12-13°C, the modulus of elasticity increases, and the membranous material does not follow Hooke's law, but is almost directly proportional to the stresses existing at the cell wall. On increasing the stress, the function E_σ = E(σ) approaches saturation. The corresponding stress-strain diagrams, σ = σ(ε_ν), and the graphs, E_σ = E(σ) and E_σ = E(t) are given. The cyto-elastic phenomena at the membrane are discussed.     

Turbulence Model Selection for Low Reynolds Number Flows

One of the major flow phenomena associated with low Reynolds number flow is the formation of separation bubbles on an airfoil’s surface. NACA4415 airfoil is commonly used in wind turbines and UAV applications. The stall characteristics are gradual compared to thin airfoils. The primary criterion set for this work is the capture of laminar separation bubble. Flow is simulated for a Reynolds number of 120,000. The numerical analysis carried out shows the advantages and disadvantages of a few turbulence models. The turbulence models tested were: one equation Spallart Allmars (S-A), two equation SST K-ω, three equation Intermittency (γ) SST, k-kl-ω and finally, the four equation transition γ-Re_θ SST. However, the variation in flow physics differs between these turbulence models. Procedure to establish the accuracy of the simulation, in accord with previous experimental results, has been discussed in detail.     

Resonance Raman Studies of Co—O2 and O—O Stretching Vibrations in Oxy-Cobalt Hemes

Strong evidence suggests that the stretching vibration of the bound oxygen can be perturbed by an accidentally degenerate porphyrin ring mode, resulting in two split frequencies. In the Co(II)(TpivPP) (pyridine) ¹⁸O₂ complex, we demonstrate that the ν(¹⁸O—¹⁸O) mode, after being shifted from its ν(¹⁶O—¹⁶O) value at 1,156 cm^-1, undergoes a resonance interaction with the 1,080 cm^-1 porphyrin mode, giving rise to two lines at 1,067 and 1,089 cm^-1. In the O₂ complex of Co(II) mesoporphyrin IX-substituted sperm whale myoglobin, we observed a dramatic intensity increase at 1,132 cm^-1 upon ¹⁶O₂ → ¹⁸O₂ substitution, which is due to the reappearance of the 1,132-cm^-1 porphyrin mode after the removal of resonance conditions. A decrease in O₂ binding affinity, caused by the proximal base tension, corresponds to an increase in the Co—O₂ stretching frequency. The ν(Co—O₂) at 527 cm^-1 for the low affinity Co(II)(TpivPP)(1,2-Me₂Im) O₂ complex is 11 cm^-1 higher than the 516-cm^-1 value for the high affinity complex (with N-MeIm replacing 1,2-Me₂Im). However, in the corresponding iron complexes the reverse behavior is observed, i.e., the ν(Fe—O₂) decreases for the (1,2-Me₂Im) complex. There is a 24-cm^-1 difference in the Co—O₂ stretching frequencies between Co(II)(TpivPP)(N-MeIm)O₂ (at 516 cm^-1) and oxy meso CoMb (at 540 cm^-1), suggesting a protein induced distortion of the Co—O—O linkage. However, the values for ν(Fe—O₂) are nearly identical between Fe(II)(TpivPP)(N-MeIm)O₂ (at 571 cm^-1) and oxy Mb (at 573 cm^-1), indicating that O₂ binds to myoglobin in the same manner as in the sterically unhindered “picket fence” complex. Evidence is presented that suggests the presence of two dioxygen stretching frequencies due to two different conformers in each of the N-MeIm and 1,2-Me₂Im complex of oxy Co(II)(TpivPP).     

Single-Base-Pair Discrimination of Terminal Mismatches by Using Oligonucleotide Microarrays and Neural Network Analyses

The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (T_d) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5′ terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the T_d and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the T_d and signal intensity, and it decreased the variability of the signal. Although T_ds of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower T_ds than those with mismatches in the first or second position. The trained NNs predicted the T_d with high accuracies (R² = 0.93). However, the NNs predicted the signal intensity only moderately accurately (R² = 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in T_ds, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5′ terminus plays a greater role in determining the T_d and signal intensity of duplexes than the type of mismatch.     

An analysis of unstirred layers in series with "tight" and "porous" lipid bilayer membranes

The present experiments were designed to evaluate the effective thickness of the unstirred layers in series with native and porous (i.e., in the presence of amphotericin B) lipid bilayer membranes and, concomitantly, the respective contributions of membranes and unstirred layers to the observed resistances to the diffusion of water and nonelectrolytes between aqueous phases. The method depended on measuring the tracer permeability coefficients for the diffusion of water and nonelectrolytes (P_DDi, cm sec^-1) when the aqueous phase viscosity (η) was increased with solutes having a unity reflection coefficient, such as sucrose or dextran. The effective thickness of the unstirred layers (α^t, cm) and the true, or membrane, permeability coefficients for diffusion of water and nonelectrolytes (P_mmi, cm sec^-1) were computed from, respectively, the slope and intercept of the linear regression of 1/P_DDi on η. In both the native and porous membranes, α^t was approximately 110 x 10^-4 cm. The ratio of P_f, the osmotic water permeability coefficient (cm sec^-1) to P_mmH2O was 1.22 in the native membranes and 3.75 in the porous membranes. For the latter, the effective pore radius, computed from Poiseuille's law, was approximately 5.6 A. A comparison of P_mmi and P_DDi, indicated that the porous membranes accounted for 16, 25, and 66% of the total resistance to the diffusion of, respectively, H₂O, urea, and glycerol, while the remainder was referable to the unstirred layers.     

The inhibition of β-lactamases from Gram-negative bacteria by clavulanic acid

The β-lactamase from Klebsiella pneumoniae E70 behaved in a similar fashion to the TEM-2 plasmid mediated enzyme on reaction with clavulanic acid. Both enzymes produced two types of enzyme–clavulanate complex, a transiently stable species (t_½=4min at pH7.3 and 37°C) and irreversibly inhibited enzyme. In the initial rapid reaction (2.5min) the enzymes partitioned between the transient and irreversible complexes in the ratios 3:1 for TEM-2 β-lactamase and 1:1 for Klebsiella β-lactamase. Biphasic inactivation was observed for both enzymes and the slower second phase was rate limited by the decay of the transiently stable complex. This decay released free enzyme for further reaction with fresh clavulanic acid, the products again partitioning between transiently stable and irreversibly inhibited enzyme. This cycle continued until all the enzyme had been irreversibly inhibited. A 115 molar excess of inhibitor was required to achieve complete inactivation of TEM-2 β-lactamase. Hydrolysis of clavulanic acid with product release appeared to occur with the inhibition reaction, which explained this degree of clavulanic acid turnover. The stoichiometry of the interaction with Klebsiella β-lactamase was not examined. The penicillinase from Proteus mirabilis C889 was rapidly inhibited by low concentrations of clavulanic acid. The major product was a moderately stable complex (t_½=40min at pH7.3 and 37°C); the proportion of the enzyme that was irreversibly inactivated was small. The cephalosporinase from Enterobacter cloacae P99 had low affinity for the inhibitor and only reacted with high concentrations of clavulanic acid (k=4.0m⁻¹·s⁻¹) to produce a relatively stable complex (t_½=180min at pH7.3 and 37°C). No irreversible inactivation of this enzyme was detected. The rates of decay of the clavulanate–enzyme complexes produced in reactions with Proteus and Enterobacter enzymes were markedly increased at acid pH.     

A Genetic Polymorphism in Coumarin 7-Hydroxylation: Sequence of the Human CYP2A Gnes and Identification of Variant CYP2A6 Alleles

A group of human cytochrome P450 genes encompassing the CYP2A, CYP2B, and CYP2F subfamilies were cloned and assembled into a 350-kb contig localized on the long arm of chromosome 19. Three complete CYP2A genes—CYP2A6, CYP2A7, and CYP2A13—plus two pseudogenes truncated after exon 5, were identified and sequenced. A variant CYP2A6 allele that differed from the corresponding CYP2A6 and CYP2A7 cDNAs previously sequenced was found and was designated CYP2A6ν2. Sequence differences in the CYP2A6ν2 gene are restricted to regions encompassing exons 3, 6, and 8, which bear sequence relatedness with the corresponding exons of the CYP2A7 gene, located downstream and centromeric of CYP2A6ν2, suggesting recent gene-conversion events. The sequencing of all the CYP2A genes allowed the design of a PCR diagnostic test for the normal CYP2A6 allele, the CYP2A6ν2 allele, and a variant—designated CYP2A6ν1—that encodes an enzyme with a single inactivating amino acid change. These variant alleles were found in individuals who were deficient in their ability to metabolize the CYP2A6 probe drug coumarin. The allelic frequencies of CYP2A6ν1 and CYP2A6ν2 differed significantly between Caucasian, Asian, and African-American populations. These studies establish the existence of a new cytochrome P450 genetic polymorphism.     

Partial Purification and Properties of an Alkaline alpha-Galactosidase from Mature Leaves of Cucurbita pepo

A fourth molecular from of α-galactosidase, designated L_IV, an alkaline α-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-α-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. K_m determinations in McIlvaine buffer (200 millimolar Na₂-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-α-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. L_IV was partially inhibited by Ca²⁺, Mg²⁺, and Mn²⁺, more so by Ni²⁺, Zn²⁺, and Co²⁺, and highly so by Cu²⁺, Ag²⁺, Hg²⁺ and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-α-d-galactoside or by myo-inositol, but α-d-galactose was a strong inhibitor. As observed for most other forms of α-galactosidase, L_IV only catalyzed the hydrolysis of glycosides possessing the α-d-galactose configuration at C₁, C₂, and C₄, and did not hydrolyze p-nitrophenyl-α-d-fucoside (α-d-galactose substituted at C₆). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-α-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of L_IV in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of L_IVin vivo. It was possible that the results reflected the ability of these anions, and ethylene-diaminetetraacetate, to restore L_IV activity through coordination with some toxic cation introduced as a buffer contaminant.     

Raman dispersion spectroscopy probes heme distortions in deoxyHb-trout IV involved in its T-state Bohr effect

The depolarization ratios of heme protein Raman lines arising from vibrations of the heme group exhibit significant dependence on the excitation wavelength. From the analysis of this depolarization ratio dispersion, one obtains information about symmetry-lowering distortions δQ^Γ of the heme group that can be classified in terms of the symmetry races Γ = A_1g, B_1g, B_2g, and A_2g in D_4h symmetry. The heme-protein interaction can be changed by the protonation of distinct amino acid side chains (i.e., for instance the Bohr groups in hemoglobin derivates), which gives rise to specific static heme distortions for each protonation state. From the Raman dispersion data, it is possible to obtain parameters by fitting to a theoretical expression of the Raman tensor, which provide information on these static distortions and also about the pK values of the involved titrable side chains. We have applied this method to the ν₄ (1,355 cm^-1) and ν₁₀ (1,620 cm^-1) lines of deoxygenated hemoglobin of the fourth component of trout and have measured their depolarization ratio dispersion as a function of pH between 6 and 9. From the pH dependence of the thus derived parameters, we obtain pK values identical to those of the Bohr groups, which were earlier derived from the corresponding O₂-binding isotherms. These are pK_α1 = pK_α2 = 8.5 for the α and pK_β1 = 7.5, pK_β2 = 7.4 for the β chains. We also obtain the specific distortion parameters for each protonation state. As shown in earlier studies, the ν₄ mode mainly probes distortions from interactions between the proximal histidine and atoms of the heme core (i.e., the nitrogens and the C_α atoms of the pyrroles). Group theoretical argumentation allows us to relate specific changes of the imidazole geometry as determined by its tilt and azimuthal angle and the iron-out-of-plane displacement to distinct variations of the normal distortions δQ^Γ derived from the Raman dispersion data. Thus, we found that the pH dependence of the heme distortions δQ^A1g (totally symmetric) and δQ^B1g (asymmetric) is caused by variations of the azimuthal rather than the tilt angle of the Fe-His (F8) bond. In contrast to this, the ν₁₀ line mainly monitors changes resulting from the interaction between peripheral substituents of the porphyrin macrocycle (vinyl). From the pH dependence of the parameters, it is possible to separately identify distortions δQ^Γ affecting the hemes in the α and β chains, respectively. From this, we find that in the α subunit structural changes induced on protonation of the corresponding Bohr groups are mainly transferred via the Fe—N_ε bond and give rise to changes in the azimuthal angle. In the β subunit, however, in addition, structural changes of the heme pocket arise, which most probably result from protonation of the imidazole of the COOH-terminal His (HC3 β). This rearranges the net of H bonds between His HC3 β, Ser (F9 β), and Glu (F7 β).