Evidence for the existence of two isocolipases in horse pancreas

The N-terminal amino acid sequences of two forms of colipase isolated from horse pancreas have been compared. Four sequence differences were found in the first 51 amino acids. This lead us to conclude that there are two distinct colipases in the horse pancreas.     

Evidence for the existence of an inner membrane anion channel in mitochondria

Mitochondria normally exhibit very low electrophoretic permeabilities to physiologically important anions such as chloride, bicarbonate, phosphate, succinate, citrate, etc. Nevertheless, considerable evidence has accumulated which suggests that heart and liver mitochondria contain a specific anion-conducting channel. In this review, a postulated inner membrane anion channel is discussed in the context of other known pathways for anion transport in mitochondria. This anion channel exhibits the following properties. It is anion-selective and inhibited physiologically by protons and magnesium ions. It is inhibited reversibly by quinine and irreversibly by dicyclohexylcarbodiimide. We propose that the inner membrane anion channel is formed by inner membrane proteins and that this pathway is normally latent due to regulation by matrix Mg2+. The physiological role of the anion channel is unknown; however, this pathway is well designed to enable mitochondria to restore their normal volume following pathological swelling. In addition, the inner membrane anion channel provides a potential futile cycle for regulated non-shivering thermogenesis and may be important in controlled energy dissipation.     

Determinants of conductance of a bacterial voltage-gated sodium channel

Through molecular dynamics (MD) and free energy simulations in electric fields, we examine the factors influencing conductance of bacterial voltage-gated sodium channel Na_vMs. The channel utilizes four glutamic acid residues in the selectivity filter (SF). Previously, we have shown, through constant pH and free energy calculations of pKa values, that fully deprotonated, singly protonated, and doubly protonated states are all feasible at physiological pH, depending on how many ions are bound in the SF. With 173 MD simulations of 450 or 500 ns and additional free energy simulations, we determine that the conductance is highest for the deprotonated state and decreases with each additional proton bound. We also determine that the pKa value of the four glutamic residues for the transition between deprotonated and singly protonated states is close to the physiological pH and that there is a small voltage dependence. The pKa value and conductance trends are in agreement with experimental work on bacterial Na_v channels, which show a decrease in maximal conductance with lowering of pH, with pKa in the physiological range. We examine binding sites for Na⁺ in the SF, compare with previous work, and note a dependence on starting structures. We find that narrowing of the gate backbone to values lower than the crystal structure's backbone radius reduces the conductance, whereas increasing the gate radius further does not affect the conductance. Simulations with some amount of negatively charged lipids as opposed to purely neutral lipids increases the conductance, as do simulations at higher voltages.     

The saxitoxin receptor of the sodium channel from rat brain. Evidence for two nonidentical beta subunits

The saxitoxin receptor of the sodium channel purified from rat bran contains three types of subunits: alpha with Mr approximately 270,000, beta 1 with Mr approximately 39,000, and beta 2 with Mr approximately 37,000. These are the only polypeptides which quantitatively co-migrate with the purified saxitoxin receptor during velocity sedimentation through sucrose gradients. beta 1 and beta 2 are often poorly resolved by gel electrophoresis in sodium dodecyl sulfate (SDS), but analysis of the effect of beta-mercaptoethanol on the migration is covalently attached to the alpha subunit by disulfide bonds while the beta 1 subunit is not. The alpha and beta subunits of the sodium channel were covalently labeled in situ in synaptosomes using a photoreactive derivative of scorpion toxin. Treatment of SDS-solubilized synaptosomes with beta-mercaptoethanol decreases the apparent molecular weight of the alpha subunit band without change in the amount of 125I-labeled scorpion toxin associated with either the alpha or beta subunit bands. These results indicate that the alpha and beta 1 subunits are labeled by scorpion toxin whereas beta 1 is not and that the beta 2 subunit is covalently attached to alpha by disulfide bonds in situ as well as in purified preparations.     

Evidence for existence of two types of massive obesity.

The responses of growth hormone, cortisol, and prolactin to symptomatic hypoglycaemia during an intravenous insulin tolerance test were measured in 20 massively obese subjects and six lean volunteers. In 11 subjects, who had been obese since early childhood, an impaired growth-hormone response and an absent prolactin response were found. In the nine other obese subjects, however, the growth-hormone and prolactin responses were not significantly impaired. Seven of these subjects had become obese either as a teenager or during adult life. These findings suggest the existence of two types of human obesity similar to those found in rodent models. In one the disorder of hypothalamic function may be due to a basic, possibly genetic abnormality, while in the other it is acquired.     

A theoretical analysis of pH dependence of sodium channel conductance

A new theory termed tunnel-acid-group-potential (TAGPT), which explains the effects of pHo and pHi on the ion conductance through different membrane channels, is presented. It is suggested that shifts in pHo and pHi change the values of negative charges generated by acid groups of side chains of some polar (Glu, Asp) amino acid residues lining the tunnel part of the channel. The resulting electrostatic field modification affects the heights of rate-limiting energy barriers (for ion transport) in the transition zones between the tunnel and the vestibules, which changes the channel conductance.     

Evidence for two photoreceptors controlling growth in de-etiolated seedlings

The effect of blue light on hypocotyl extension in de-etiolated seedlings of lettuce, cucumber and tomato was investigated under conditions which precluded the involvement of phytochrome. Small but highly inhibitory amounts of blue light were added to a high intensity background illumination from low pressure sodium lamps. A log-linear response for inhibition of hypocotyl extension against the blue light fluence rate was obtained for lettuce and cucumber, and inhibition in tomato was also related to the blue light fluence rate. The added blue light did not alter phytochrome photostationary state and its effect was independent of the total fluence rate. Growth inhibition by Pfr could be demonstrated in tomato and cucumber but not in lettuce. The results indicate that two photoreceptors may normally be involved in the control of seedling growth but their relative importance varies greatly between species.Abbreviations HIR high irradiance reaction - Pfr far red absorbing form of phytochrome - Pr red absorbing form of phytochrome     

Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

Evidence from high-pressure liquid chromatography for the existence of two ferredoxins in plants

Crude ferredoxin preparations were obtained from blue-green algae, green algae, ferns, and higher plants. We analyzed the preparations by high-pressure liquid chromatography using two different types of columns, a hydrophobic phenyl-5PW column and an ion-exchange DEAE-5PW column. Two ferredoxins were detected in all plants analyzed. The ferredoxins from some plants were separated by use of both columns and those from others were separated by one of the two columns. Thus, there were three possible ways in which pairs of ferredoxins from a single species of plant could be separated. We suspect that there are two different ferredoxins in most if not all species of plants.     

Evidence suggesting the existence of two H-Y antigens in the mouse

An exceptional (C57BL/6 × BALB/c)F₁ male mouse, from an X-irradiated father, and lacking the H-Y (male) antigen, on the basis of skin-graft testing, was found. Conventional serological tests for H-Y antigen, however, were positive. His karyotype contained only 39 chromosomes, the Y chromosome apparently being absent. Although the testes were small, lacked germ cells, and were essentially Leydig cell tumors, the mutant was normal with respect to external male phenotype, accessory glands, and sexual behavior. The data suggest that the histogenic and serological tests for H-Y antigen may detect two different antigens, paralleling findings with mutants of theH-2 complex, where histoincompatibility can be generated without accompanying serological changes.     

Comparison of the gramicidin a potassium/sodium permeability and single channel conductance ratio

If the ion concentration is low enough that most channels are unoccupied, then the ‘independence relations’ should be satisfied and the permeability ratio should equal the conductance ratio. It has been previously reported that for the gramicidin A channel these ratios for Na⁺ and K⁺ were not equal at concentrations as low as 10 mM. However, these ratios were not measured at the same applied potential, as is required by the theory. Instead, the conductance ratio was measured at 100 mV and corrected using calculated current-voltage relations. In this report the comparison between permeability and conductance ratios is reexamined using data obtained at the correct potential. There is no significant difference in the ratios at 10 mM when they are measured at the same voltage. This implies that most channels are not occupied by sodium or potassium ions at 10 mM.     

Evidence for the existence of two assembly domains within the sea urchin fertilization envelope.

The sea urchin fertilization envelope (FE) is a complex, macromolecular aggregate assembled by the addition of cortical granule secretions to the vitelline layer. The completed, trilaminar structure has a dense layer sandwiched between surface coats of paracrystalline material. Two cortical granule enzymes, ovoperoxidase and protease, and a cell surface transglutaminase are required for the assembly process. We have examined, by quick-freeze, deep-etch, rotary-shadow electron microscopy, the effects of inhibiting each of these enzymes upon FE assembly. These experiments reveal two domains within the FE, distinguishable by their enzymatic requirements for proper maturation. The first domain consists of the microvillar casts which require both protease and transglutaminase activities to obtain a normal paracrystalline coat. The second domain comprises the regions between casts and appears to mature by ovoperoxidase-mediated cross-linking of paracrystalline material to the envelope.     

Spectroscopic investigations on the binding site of bovine hepatic fatty acid binding protein. Evidence for the existence of a single binding site for two fatty acid molecules

The hydrophobic region of the binding site of a bovine fatty acid binding protein (pI 7.0-FABP) has been characterized using fluorescence and circular dichroism (CD) spectroscopy. Blue-shifts of fluorescence emission maxima and increased lifetimes of naphthylamine dyes, anthroyloxy-fatty acids, pyrene nonanoic acid and trans-parinaric acid indicated a hydrophobic interaction with FABP. The fluorescence quenching of various anthroyloxy-fatty acids by iodide and acrylamide showed lower accessibility to the fluorophore linked to the carbon adjacent to the carbonyl group and towards the methyl end of the fatty acid. Binding stoichiometries were different for fatty acids and their bulky fluorescent analogues. trans-Parinaric acid when bound to FABP showed a complex induced CD-spectrum, which is explained by a close proximity of two ligands in the same binding site. Fluorescent derivatives of phosphatidylcholine with trans-parinaric acid and cholesteryl trans-parinarate did not bind to FABP. Thus, the binding site appears to be constructed for high affinity binding of long chain fatty acids.     

Molecular evidence supporting the existence of two major groups in uropathogenic Escherichia coli

Abstract Molecular methods allow an extremely fine strain typing that can be used to establish the population structure of bacterial species. This methodology has been used to characterize a collection of 74 uropathogenic Escherichia coli obtained from three hospitals located in geographically distant towns in Spain, some representatives of the ECOR collection and other reference strains. Genomic DNA was analyzed by RAPD (Random Amplified Polymorphic DNA) that can characterize a bacterial strain to the level of defining individual clones. The 16S rDNA-23S rDNA spacers were amplified by PCR and submitted to restriction analysis. Finally, the presence or absence of G adhesins in Escherichia coli as well as the type of adhesin (three types are known) have been shown by PCR amplification followed by digestion with restriction enzymes. As expected a wide diversity was shown by RAPD and identical patterns were only found in the case of strains isolated from the same individual, an obvious case of relapse. Analysis of the spacers' restriction patterns showed the presence of two markedly differentiated clusters that we have named α and ß. Both RAPD and spacer restriction patterns originated similar clusters of strains showing a consistency in the evolution of the global genome with the sequence variation of the ribosomal spacers. Furthermore, most of the strains having G-adhesin, with only a few exceptions, corresponded to the α rRNA spacer group. The two spacer types detected were also consistent with some phenotypic markers such as sucrose and raffinose utilization. The α and β clusters could be intraspecific groups produced by partial sexual isolation or other barriers that are originating a divergent evolution.     

Evidence for the existence of two soluble NAD(+) kinase isoenzymes in Euglena gracilis Z

Two soluble NAD(+) kinase isoenzymes (isoenzymes 1 and 2) from Euglena gracilis were separated by preparative electrophoresis and characterized. They display several similar properties: both have an identical apparent molecular weight of 68 kDa and their activities are independent on calmodulin, insensitive to 2-mercaptoethanol but inhibited by p-chloromercurybenzoate, 5, 5'-dithiobis(2-nitrobenzoate) and, surprisingly, by low dithiothreitol concentrations, the inhibition by dithiothreitol being irreversible for isoenzyme 1 but reversible for isoenzyme 2. Nevertheless, the two isoenzymes mainly differ by their specificities towards triphosphate nucleotides and their catalytic mechanisms. Isoenzyme 1 is as active in the presence of ATP as of GTP and acts by a ping-pong mechanism with a k(M) for NAD(+) of 0.26 mM and a k(M) for low MgATP(2-)concentrations of 0.03 mM. Isoenzyme 2 is three-fold more active in the presence of GTP than of ATP and operates by a sequential mechanism with k(M)s for NAD(+) and MgGTP(2-) of 1.03 and 0.20 mM, respectively. This study shows the evidence for the existence of two structurally similar but catalytically different NAD(+) kinase isoenzymes in E. gracilis. One resembles the enzyme previously described in bacteria. The other displays a catalytic mechanism identical to that of NAD(+) kinase from other organisms but remains unique among all the NAD(+) kinases studied to-date regarding its specificity towards GTP.