Intracellular O2 gradients in cardiac myocytes. Lack of a role for myoglobin in facilitation of intracellular O2 diffusion

The P₅₀ for oxygenation of myoglobin in intact cells was very high relative to that for isolated myoglobin, and was changed by addition of agents that altered respiratory rate. The P₅₀ for cytochrome $\text{a}$ oxidation in cells was very high relative to that for isolated mitochondria, but was unaffected by oxidation of myoglobin to metmyoglobin. These results demonstrate the existence of a substantial intracellular O₂ gradient in myocytes and indicate that myoglobin does not have a significant role in facilitation of O₂ diffusion to mitochondria.     

Distal heme pocket regulation of ligand binding and stability in soybean leghemoglobin

Leghemoglobins facilitate diffusion of oxygen through root tissue to a bacterial terminal oxidase in much the same way that myoglobin transports oxygen from blood to muscle cell mitochondria. Leghemoglobin serves an additional role as an oxygen scavenger to prevent inhibition of nitrogen fixation. For this purpose, the oxygen affinity of soybean leghemoglobin is 20-fold greater than myoglobin, resulting from an 8-fold faster association rate constant combined with a 3-fold slower dissociation rate constant. Although the biochemical mechanism used by myoglobin to bind oxygen has been described in elegant detail, an explanation for the difference in affinity between these two structurally similar proteins is not obvious. The present work demonstrates that, despite their similar structures, leghemoglobin uses methods different from myoglobin to regulate ligand affinity. Oxygen and carbon monoxide binding to a comprehensive set of leghemoglobin distal heme pocket mutant proteins in comparison to their myoglobin counterparts has revealed some of these mechanisms. The "distal histidine" provides a crucial hydrogen bond to stabilize oxygen in myoglobin but has little effect on bound oxygen in leghemoglobin and is retained mainly for reasons of protein stability and prevention of heme loss. Furthermore, soybean leghemoglobin uses an unusual combination of HisE7 and TyrB10 to sustain a weak stabilizing interaction with bound oxygen. Thus, the leghemoglobin distal heme pocket provides a much lower barrier to oxygen association than occurs in myoglobin and oxygen dissociation is regulated from the proximal heme pocket.     

Spin state equilibria in soybean ferric leghemoglobin and its complexes with fomate and acetate

The interactions of fluoride, acetate and formate with soybean ferric leghemoglobin $\text{a}$ have been investigated by ¹H NMR spectroscopy. In the presence of fluoride or acetate leghemoglobin is locked into a high spin ferric conformation whilst the formate complex exists as an equilibrium mixture of high and low spin states. Both formate and acetate ligate directly to the iron and the different magnetic properties of the complexes are attributed to steric constraints within the heme pocket.     

Analysis of the kinetics of electron transfer reactions of hemoglobin and myoglobin with inorganic complexes.

The kinetics of methemoglobin reduction by Fe(EDTA)^2? have been studied and found to follow a second order rate law with k = 29.0 $\text{M}$^?1 s^?1 [25°C, μ = 0.2 $\text{M}$, pH 7.0 (phosphate)], $\text{ΔH}{}^3\text{= 5.5 ± 0.7 kcal/mol}$, and $\text{ΔS}{}^2\text{= ?33 ± 2 e.u.}$. The electrostatics-corrected self-exchange rate constant (k₁₁^corr) for hemoglobin based on the Fe(EDTA)^2? cross-reaction is 2.79×10^?3$\text{M}$^?1 s^?1. This rate constant is compared with others reported for a water-soluble iron porphyrin and calculated from published data for the reactions of myoglobin and hemoglobin with Fe(EDTA)^2? and Fe(CDTA)^2?/?. The k₁₁^corr values for these systems range over ten orders of magnitude with heme ? myoglobin > hemoglobin.     

Comparison of the three basic scattering functions of myoglobin in solution with those from the known structure in crystalline state

X-ray small-angle scattering of myoglobin in aqueous solutions enhances the probability that the structure of myoglobin is conserved on passing to the dissolved state. The agreement of measured and calculated results could be extended up to $\text{k = 1.2}\text{A}\text{?}{}^{\mathrm{?1}}$ which roughly corresponds to 5 Å resolution.     

An enzymic analysis of a nuclear envelope fraction

A rat liver nuclear envelope fraction isolated essentially by the technique of Monneron et al. (J. Cell Biol. 55, 104–125 (1972)) is characterized by high levels of glucose-6-phosphatase and 5′-nucleotidase. A broadly specific nucleoside triphosphatase activity is present. Cytochromes $\text{b}{}_5$ and $\text{P-450}$ as well as NADPH- and NADH-cytochrome $\text{c}$ reductase activities are present but at lower levels than found in microsomes. Cytochrome $\text{c}$ oxidase activity is low. RNA polymerase activity is absent from the nuclear envelope fraction. Cytochemistry shows that glucose-6-phosphatase activity is strong and restricted to the nuclear envelope of nuclei. 5′-Nucleotidase shows weak reaction deposit in whole nuclei but in contrast gives clear reaction deposit in isolated nuclear envelopes. Cytochemical reaction deposit due to nucleoside trisphosphatase activity is not restricted to the nuclear envelope but is found to a larger extent within the nucleus.     

Progesterone-induced inactivation of nuclear estrogen receptor in the hamster uterus is mediated by acid phosphatase

Inactivation of hamster uterine estrogen receptor was assayed in nuclear KCl extracts (30 min, 37°C, pH 7.5) after progesterone treatment $\text{in}\text{vivo}$ for 2h. At very low concentrations ($\text{>}$0.05 mM), molybdate and vanadate blocked the progesterone-induced increase in receptor inactivation. In contrast, only high concentrations ($\text{>}$10 mM) of the inhibitors blocked receptor inactivation in extracts from untreated hamsters. Gel electrophoresis and inhibition curves for phosphatases in nuclear extract demonstrated that acid, rather than alkaline, phosphatase activity is most likely responsible for these effects. These data suggest that progesterone antagonizes estrogen action in the hamster uterus by promoting estrogen receptor dephosphorylation leading to inactivation.     

Expression of mitochondrial DNA in Saccharomyces cerevisiae: the construction of sets of isonuclear haploid strains containing different specified mitochondrial genomes.

In order to perform systematic biochemical studies of the expression of the mitochondrial genome in different strains of $\text{Saccharomyces}\text{cerevisiae}$, we have constructed a series of haploid strains in which a specified mitochondrial genome has been placed in cells containing a particular nuclear genome. The desired strains were obtained from crosses between $\text{rho}{}^{\mathrm{+}}$ and $\text{rho}{}^{\mathrm{o}}$ haploids, one of which carried the $\text{karl}$-l mutation which interferes with nuclear fusion. These newly constructed strains provide the basis for studying mitochondrial gene expression as a function of (i) the nucleotide sequence differences that are apparent in the mitochondrial DNAs from different strains, and (ii) the role played by different nuclear genomes in modulating the expression of mitochondrial genes.     

Tyrosine B10 inhibits stabilization of bound carbon monoxide and oxygen in soybean leghemoglobin

Detailed comparisons of the carbon monoxide FTIR spectra and ligand-binding properties of a library of E7, E11, and B10 mutants indicate significant differences in the role of electrostatic interactions in the distal pockets of wild-type sperm whale myoglobin and soybean leghemoglobin. In myoglobin, strong hydrogen bonds from several closely related conformations of the distal histidine (His(E7)) side chain preferentially stabilize bound oxygen. In leghemoglobin, the imidazole side chain of His(E7) is confined to a single conformation, which only weakly hydrogen bonds to bound ligands. The phenol side chain of Tyr(B10) appears to "fix" the position of His(E7), probably by donating a hydrogen bond to the Ndelta atom of the imidazole side chain. The proximal pocket of leghemoglobin is designed to favor strong coordination bonds between the heme iron and axial ligands. Thus, high oxygen affinity in leghemoglobin is established by a favorable staggered geometry of the proximal histidine. The interaction between His(E7) and Tyr(B10) prevents overstabilization of bound oxygen. If hydrogen bonding from His(E7) were as strong as it is in mammalian myoglobin, the resultant ultrahigh affinity of leghemoglobin would prevent oxygen transport in root nodules.     

Inhibition of cyclopropane fatty acid synthase by sinefungin and A9145C

Sinefungin and A9145C, antifungal antibiotic analogs of S-adeno-sylmethionine isolated from $\text{Streptomyces}\text{,}\text{griseolus}$, have been found to be very effective $\text{in}\text{,}\text{vitro}$ inhibitors of cyclopropane fatty acid synthase from $\text{Lactobacillus}\text{,}\text{plantarum}$. Both compounds exhibit linear competitive inhibition with a K_i for Sinefungin of 220 nM and a K_i for A9145C of 11 nM.     

Identification of NG, NG-dimethylarginine in a nuclear protein from the lower eukaryote physarum polycephalum homologous to the major proteins of mammalian 40S ribonucleoprotein particles

A nuclear protein apparently homologous to the two major proteins of 40S heterogeneous nuclear ribonucleoprotein particles from mammalian cells has been isolated from the lower eukaryote $\text{Physarum polycephalum}$, purified, and found to contain a substantial amount of the unusual amino acid N^G, N^G-dimethylarginine. The apparent homology is based on similar molecular weights, basic isoelectric points and amino acid compositions including the dimethylarginine and a high content of glycine. The implications of the presence of this protein in $\text{Physarum polycephalum}$ and the possible significance of the N^G, N^G-dimethylarginine are discussed.     

Erythroid specific nuclear antigen and globin gene binding protein

Using the procedure of Bekhor and Mirell (Biochem, $\text{18}$, 609, 1979), we isolated a nonhistone protein fraction tightly bound to DNA which putatively has a role in globin gene regulation in chicken reticulocytes. This fraction was tested by gel electrophoresis and microcomplement fixation and appears by these criteria very similar to the chicken nuclear antigen previously identified in reticulocyte chromatin and structurally altered erythrocyte chromatin. This antigen is tissue and species specific (Pumo $\text{et}\text{al}$, Biochem. $\text{19}$, 2362, 1980).     

Ribosomal proteins L1, L17 and L27 from Escherichia coli localized at single sites on the large subunit by immune electron microscopy

Three-dimensional locations have been determined for Escherichia coli ribosomal proteins L1, L17 and L27 by immune electron microscopy using antibodies directed against these proteins. From the positions of immunoglobulin G attachment, observed in two characteristic projections, it was determined that these three proteins are located at single sites in different regions on the surface of the large subunit. In the quasisymmetric projection, L1 maps on the side opposite the “$\text{L}\text{7}\text{L}\text{12}$ stalk,” named the L1 ridge; protein L17 maps at the base of the subunit opposite the “central protuberance” (toward the $\text{L}\text{7}\text{L}\text{12}$ side of the subunit); and protein L27 is found on the central protuberance (on the side distal to the $\text{L}\text{7}\text{L}\text{12}$ stalk). In the asymmetric projection, proteins L1 and L27 are found on the surface of the subunit contracting the small subunit and protein L17 is on the surface of the subunit distal to the small subunit; i.e. on the cytoplasmic surface of the large subunit. Antibody binding at all three sites was eliminated when the immunoglobulin G molecules were preabsorbed with their specific proteins.     

pH Dependence of oxy and deoxy cobalt-substituted leghemoglobin from soybean

In leghemoglobin a, which is the major hemoglobin component in soybean root nodules, the haem iron has been replaced by cobalt. The electron spin resonance (ESR) of frozen solutions of the cobalt-substituted leghemoglobin has been studied at 77 K in the deoxy and oxy forms respectively. Both ligation states exhibit rhombic g tensors. The hyperfine constants of ⁵⁹Co, ¹⁴N-imidazole (residue of the proximal histidine) and ¹⁴N-pyrroles are determined for the three principal directions of the g tensor. Both, the oxy and the deoxy state exhibit pH-dependent changes of the hyperfine structures. For oxy cobalt leghemoglobin a quantitative analysis of the pH titration and of the ESR parameters of the low and high-pH forms respectively are performed. The interconversion of the low and the high-pH forms is controlled by a proton-dissociating group with pK=6.4 which is most probably the distal histidine. g tensors and hyperfine constants are compared with those described for oxy cobalt myoglobin crystal spectra [34] allowing assignments of the low and high-pH species of leghemoglobin to stereoelectronic structures with non-equivalent and equivalent dioxygen atoms respectively. Hydrogen-bonding of the distal histidine with dioxygen favours the structure with equivalent oxygen atoms. The pH dependence of the deoxy form is interpreted as interaction of the proximal imidazole with the central cobalt atom.     

NMR studies of the conformations of leghemoglobins from soybean and lupin

Phase-sensitive two-dimensional NMR methods have been used to obtain extensive proton resonance assignments for the carbon monoxide complexes of lupin leghemoglobins I and II and soybean leghemoglobin a. The assigned resonances provide information on the solution conformations of the proteins, particularly in the vicinity of the heme. The structure of the CO complex of lupin leghemoglobin II in solution is compared with the X-ray crystal structure of the cyanide complex by comparison of observed and calculated ring current shifts. The structures are generally very similar but significant differences are observed for the ligand contact residues, Phe30, His63 and Val67, and for the proximal His97 ligand. Certain residues are disordered and adopt two interconverting conformations in lupin leghemoglobin II in solution. The proximal heme pocket structure is closely conserved in the lupin leghemoglobins I and II but small differences in conformation in the distal heme pocket are apparent. Larger conformational differences are observed when comparisons are made with the CO complex of soybean leghemoglobin. Altered protein-heme packing is indicated on the proximal side of the heme and some conformational differences are evident in the distal heme pocket. The small conformational differences between the three leghemoglobins probably contribute to the known differences in their O2 and CO association and dissociation kinetics. The heme pocket conformations of the three leghemoglobins are more closely related to each other than to sperm whale myoglobin. The most notable differences between the leghemoglobins and myoglobin are: (a) reduced steric crowding of the ligand binding site in the leghemoglobins, (b) different orientations of the distal histidine, and (c) small but significant differences in proximal histidine coordination geometry. These changes probably contribute to the large differences in ligand binding kinetics between the leghemoglobins and myoglobin.     

Catalysis by hemoproteins and their structural organization in reversed micelles of surfactants in octane

The cytochromes $\text{P-450}$ LM-2 and $\text{b}{}_5$ from rabbit liver microsomes have been entrapped into reversed micelles of surfactants in octane. The optimum conditions providing for the maximum stability of the hemoproteins have been found: pH and concentration of the buffer solution, the glycerol addition, the surfactant concentration, the [H₂O]/[surfactant] ratio and, finally, the reversed micelles composition including aerosol OT and its mixture with Triton X-45, Tween 20 and cetyltrimethylammonium bromide (CTAB). The transformation kinetics of the hemoproteins solubilized by detergents has been studied by monitoring the absorbance of hemoproteins in the Soret band region. Significant changes in tryptophan fluorescence of cytochrome $\text{b}{}_5$ and in CD spectra of myoglobin in reversed micelles and their dependence on the [H₂O]/[aerosol OT] ratio have been shown. The three hemoproteins in reversed micelles have been found to exhibit high catalytic activity with respect to their reaction with cumene hydroperoxide. The kinetic and spectral data reveal the structural transformations of the proteins entrapped into the micelles due to the interactions of the lumenal surface of the micelles and the protein molecule surface.     

Heme oxygenase provides α-selectivity to physiological heme degradation

The isomeric composition of biliverdin formed by the degradation of heme by purified NADPH-cytochrome $\text{c}$ reductase has been determined by high performance liquid chromatography. Methemalbumin heme yields a mixture of the four biliverdin IX isomers while myoglobin yields only the IX-α isomer of biliverdin. In both cases biliverdin is a minor product of the reaction. Addition of purified heme oxygenase to the methemalbumin NADPH-cytochrome $\text{c}$ reductase system confers α-selectivity on the reaction and allows stoichiometric conversion of heme to biliverdin. Thus the role of heme oxygenase in enzymatic heme degradation appears to be to provide a suitable environment for quantitative conversion of heme to biliverdin in addition to conferring α-selectivity on the reaction.     

Conditions for the measurement of nuclear estrogen receptor at low temperature

This study was undertaken to determine optium conditions for the extraction and measurement of uterine nuclear estrogen receptor at low temperature. We measured the influence of glycero, 0.5 M KCl, 10 mM pyridoxal 5′-phosphate, and 0.5 M NaSCN on the dissocation of estradiol from the receptor at 0°C. The half-time (${}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of estradiol dissociation from the receptor in 0.5 M KCl nuclear extracts containing 30% glycerol was very slow (greater than 250 h). Exclusion of glycerol from the extract (Tris buffer) increased the dissociation rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 35}\text{h}$). The inhibitory effect of glycerol on estradiol dissociation kinetics predominated over the mild stimulatory effect of KCl; and both effects were independent of the electrical conductivity of the buffer. When pyridoxal phosphate was added to a nuclear KCl extract (barbital fubber) lacking glycerol, dissociation of the estrogen-receptor complex increased such that the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) decreased from 20 to 7.6 h; the receptor extracted from nuclei with 10 mM pyridoxal phosphate exhibited these same rapid dissociation kinetics. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of estradiol dissociation from the receptor at 0°C in the presence of 0.5 M NaSCN was 5.6 h. Following extraction of uterine receptro by KCl, pyridoxal phosphate, or NaSCN, we measured the number of estradiol binding sites at each of two incubation temperatures: 30°C for 1 hr and 0°C for 24 h. We verified that unoccupied receptors was measured reliability in KCl extract during incubation at 0°C in the presence of glycerol. Total receptor can be determined using either pyridoxal phosphate extract or NaSCN extract at low temperature. However, the number of sites recovered in either pyridoxal phosphate or NaSCN extract was twice the number obtained with the KCl procedure at elevated temperature. It is noteworthy that pyridoxal phosphate and NaSCN increased the number of sites when added directly to nuclear KCl extract, and the effect of pyridoxal phosphate and NaSCN was reversed by treatment with L-lysine and dialysis against KCl, respectively. Thus, the lower receptor recovery with the KCl procedure is not due to the inability of KCl to extract these sites from the nucleus but rather is ascribable to the assay procedure itself. Although total receptor can be measured at low temperature with either NaSCN or pyridoxal phosphate, the pyridoxal phosphate method can be used to assay nuclear progesterone receptor in tha same extract.     

The linear cable theory as a model of gill blood flow

The vascular organization of the teleost gill suggests that blood flow distribution from the filamental artery to the respiratory lamellae is governed by relationships analogous to the cable conduction properties of a nerve axon. The space constant (λ) by definition is the distance along the gill filament at which the in-series resistance of the afferent filament artery equals the in-parallel resistance of the afferent lamellar arteriolar, lamellar, efferent lamellar arteriolar (ALA-L-ELA) segments. Constriction of the afferent filamental artery or uniform dilation of the ALA-L-ELA will decrease λ. As λ decreases, flow through the proximal (basal) lamellae greatly increases at the expense of distal lamellar perfusion. When λ increases in a system of finite length the flow profile must account for reflected pressures within the main vessel. The λ calculated from corrosion casts of gill vasculature is $\text{1}\text{4}$ to $\text{1}\text{2}$ the filament length. This favors blood flow through the proximal lamellae and when cardiac output increases, the proportion of cardiac output perfusing the proximal areas increases at the expense of distal lamellar blood flow. To offset these changes it is proposed that increased distal lamellar perfusion is achieved by simultaneous vasodilatation of distal and constriction of proximal ALA-L-ELA segments and dilation of the afferent filamental artery.