Horse heart ferricytochromec: Conformation and heme configuration of low ionic strength acidic forms

Resonance Raman, absorption and circular dichroism spectroscopic studies of the stable forms of horse heart ferricytochromec in thepH range 6–0.8 and at the lowest possible ionic strengths, in water, and at 30°C are reported. The neutralpH form, state III, changes to the acidicpH form, state I, through a three-step process: state III ? state IIIa ? state II ? state I, with pKa's of 3.6±0.3, 2.7±0.2, and 1.2±0.2, depending on the monitoring probe, respectively. State IIIa ferricytochromec is like state III (i.e., with the Met-80-sulfur-iron linkage and a closed heme crevice) but with a higher degree of folding and a slightly larger porphyrin core. State II ferricytochromec is an unfolded form with an open heme crevice and no Met-80-sulfur-iron linkage. The heme iron is high-spin and hexacoordinated with weak ligand-field groups, water, and nitrogen of the protonated/hydrogen-bonded imidazole of the His-18 residue at the axial positions. The state I form also lacks the Met-80-sulfur-iron linkage and has an open heme crevice like the state II form; however, it is less unfolded and has a high-spin pentacoordinated heme iron, with the nitrogen of the imidazole of His-18 as the axial ligate, which is out of the porphyrin plane by about 0.45 Å.     

Horse heart ferricytochrome c: conformation and heme configuration of high ionic strength acidic forms.

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochromec in the presence of 0.2 M KCl, 0.1 M NaClO₄, and 0.2 M KNO₃, in thepH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990)J. Protein Chem.,9, 379–387), the change from neutral to low acidicpH's in the presence of salts is a three-step process: state III _s state III _s,a state II _s state I _s , withpK _a 's of 3.5±0.2, 2.2±0.2, and 1.1±0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutralpH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III _s,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II _s and I _s are also essentially folded forms, but with a smaller degree of protein secondary structure. State II _s has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I _s has a high-spin pentacoordinated heme iron, which is about 0.49 Å out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III _a state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).     

Effect of temperature and guanidine hydrochloride on ferrocytochrome<Emphasis Type="Italic"> c</Emphasis> at neutral pH

Thermally denatured horse heart ferrocytochrome c (ferrocyt c) has been characterized using absorption spectroscopy, differential scanning calorimetry (DSC) and viscometry at pH 7.0. DSC experiments have yielded the transition temperature of denaturant-free ferrocyt c unfolding as 100.6±0.3 °C, indicating an extremely high stability of the protein. The presence of guanidine hydrochloride (GdnHCl) facilitated estimation of the structural features of thermally unfolded ferrocyt c. The stability of the protein, expressed by G _D at 25 °C, is 59±5 kJ mol^–1 (DSC) and 65±6 kJ mol^–1 (absorption spectroscopy). An absorption spectrum of ferrocyt c demonstrates that the heme occurs in the high-spin state at extreme denaturing conditions (94 °C, 6.6 M GdnHCl). Absorption spectroscopy, using heme as a probe, shows that thermal denaturation of ferrocyt c occurs as a transition from a native low-spin (Met80/His18) to a high-spin disordered state with involvement of non-native, low-spin (bis-His) species.Abbreviations CD circular dichroism - cyt c cytochrome c - DSC differential scanning calorimetry - ferricyt c ferricytochrome c - ferrocyt c ferrocytochrome c - GdnHCl guanidine hydrochloride - NHE normal hydrogen electrode     

Investigation of ion binding to the cytoplasmic binding sites of the Na,K-pump

A dual-wavelength fluorimeter was constructed, which used two light emitting diodes (LEDs) to excite the fluorescence dye RH 421 alternately with two different wavelengths. The ratio of the emissions at the two excitation wavelengths provided a drift-insensitive signal, which allowed detection of very small changes of the fluorescence intensity. Those small changes were induced by ion binding and release in conformation E₁ of the Na,K-ATPase. Titration experiments were performed to determine equilibrium dissociation constants (± standard deviation) for each step in the complete binding and release sequence: 0.12 ± 0.01 mM (E₂(K₂) KE₁), 0.08 ± 0.01 mM (KE₁ E₁), 3.0 ± 0.2 mM (NaE₁ E₁), 5.2 ± 0.4 mM (Na₂E₁ NaE₁) and 6.5 ± 0.4 m_M (Na₃E₁ Na₂E₁) at pH 7.2 and T=16°C. These numbers show that the affinities of the binding sites exposed to the cytoplasm, are higher for K⁺ than for Na⁺ ions, similar to what was found on the extracellular side. The physiological requirement for extrusion of Na⁺ from the cytoplasm, and for import of K⁺ from the extracellular medium seems to be facilitated not by favorable binding affinities in state E₁ but by the two ATP-driven reaction steps of the cycle, E₂(K₂) + ATP K₂E₁ · ATP and Na₃E₁ · ATP (Na₃) E_l-P, which border the ion exchange reactions at the binding sites in conformation E₁. Correspondence to: H.-J. Apell     

Mutational specificity of a conditional Escherichia coli mutator, mutD5

Summary MutD5, a conditional mutator in Escherichia coli, causes the stimulation of mutation frequencies 50 to 100 fold in minimal medium. In rich medium mutation frequencies are further increased 50 to 100 fold. We show here that all possible base-pair mutations are increased in a mutD5 strain grown in rich medium. A:TG:C transitions as well as A:TC:G, A:TT:A aud G:CC:G transversions are stimulated. Transitions occur more frequently than transversions. MutD5 also increases the reversion frequencies of three trpA frameshift mutations by causing base-pair additions, and, possibly, base-pair deletions.     

Diverse polymorphism within a short coding region of the human aldehyde dehydrogenase-5 (ALDH5) gene

Human aldehyde dehydrogenase-5 gene (originally named as ALDHX) is expressed in liver and testis. The ALDH5 does not contain introns in the coding sequence for 517 amino acid residues. Within a short nucleotide region of the gene, the following three nucleotide changes were found in high frequencies, i.e., a silent CT at nucleotide (nt) 183, CT at nt 257 associated with a ValAla substitution, and TG at nt 320 associated with a ArgLeu substitution. The frequency of C at nt 183 is 81% in Caucasians and 65% in Japanese, and the difference is statistically not significant. The frequency of C at nt 257 is 76% in Caucasians and 55% in Japanese, and the difference is statistically significant (P = 0.02). The frequency of T at nt 320 is 71% in Caucasians, while it is only 27% in Japanese. The racial difference at nt 320 is highly significant (P < 0.001). No significant difference was found in the genotypes of the three nucleotide positions between alcoholic and nonalcoholic Caucasians within the limited numbers of subjects examined.     

The continuum of pure and hybrid myosin heavy chain-based fibre types in rat skeletal muscle

The myosin heavy chain (MHC)-based fibre composition of adult rat adductor magnus (AM) and tibialis anterior (TA) muscles was investigated using single fibre analysis. Microelectrophoresis performed on single fibre fragments demonstrated a predominance of pure fast MHC-based fibre types (expressing only one fast MHC). Most of the fibres analysed from both the AM (72%) and TA (50%) were pure type IIB (expressing only MHCIIb). Pure type IID fibres (expressing only MHCIId) were also abundant in AM (20%) and TA (18%). In addition, hybrid fibres coexpressing MHCIIb and MHCIId in varying proportions (fibre types IIBD and IIDB) were found, as well as fibres coexpressing MHCIId and MHCIIa with a predominance of MHCIId (type IIDA) and some C fibres (coexpressing MHCI and MHCIIa in varying proportions). Considered altogether, these data reflect the dynamic nature of adult skeletal muscle fibres and indicate a continuum of MHC-based fibre types in normal rat muscle with transitions in the order IIB IIBD IIDB IID IIDA IIAD IIA IIC IC I.     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

Active-site sulfhydryl chemistry plays a major role in the misfolding of urea-denatured rhodanese

Unfolded bovine rhodanese, a sulfurtransferase, does not regain full activity upon refolding due to the formation of aggregates and disulfide-linked misfolded states unless a large excess of reductant such as 200 mM -ME and 5 mg/ml detergent are present [Tandon and Horowitz (1990), J. Biol. Chem. 265, 5967]. Even then, refolding is incomplete. We have studied the unfolding and refolding of three rhodanese forms whose crystal structures are known: ES, containing the transferred sulfur as a persulfide; E, without the transferred sulfur, and carboxymethylated rhodanese (CMR), in which the active site was blocked by chemical modification. The X-ray structures of ES, E, and CMR are virtually the same, but their tertiary structures in solution differ somewhat as revealed by near-UV CD. Among these three, CMR is the only form of rhodanese that folds reversibly, requiring 1 mM DTT. A minimum three-state folding model of CMR (NIU) followed by fluorescence at 363 nm, (NI) by fluorescence at 318 nm, and CD (IU) is consistent with the presence of a thermodynamically stable molten globule intermediate in 5–6 M urea. We conclude that the active-site sulfhydryl group in the persulfide form is very reactive; therefore, its modification leads to the successful refolding of urea-denatured rhodanese even in the absence of a large excess of reductant and detergent. The requirement for DTT for complete reversibility of CMR suggests that oxidation among the three non-active-site SH groups can represent a minor trap for refolding through species that can be easily reduced.     

Equilibrium amide hydrogen exchange and protein folding kinetics

The classical Linderstrøm-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (IUN). On the other hand, in an on-pathway three-state system (UIN), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments.     

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium conformational states at low temperatures

Absorption spectra of highly purified liver microsomal cytochrome P-450 in non-equilibrium states were obtained at 77 K by reduction with trapped electrons, formed by gamma-irradiation of the water-glycerol matrix. In contrast to the equilibrium form of ferrous cytochrome P-450 with the heme iron in the high-spin state the non-equilibrium ferrous state has a low-spin heme iron. The absorption spectrum of the non-equilibrium ferrous cytochrome P-450 is characterized by two bands at 564 (-band) and 530 nm (-band). When the temperature is increased to about 278 K this non-equilibrium form of the reduced enzyme is relaxed to the corresponding equilibrium form with a single absorption band at 548 nm in the visible region characteristic for a high-spin heme iron.     

Ferricytochromec induces monophasic kinetics of ferrocytochromec oxidation in cytochromec oxidase

The kinetics of ferrocytochromec oxidation by reconstituted cytochromec oxidase (COX) from bovine heart was followed by a spectrophotometric method, using on-line data collection and subsequent calculation of reaction rates from a function fitted to the progress curve. When reaction rates were calculated at increasing reaction times, the multiphasic kinetics of ferrocytochromec oxidation gradually changed into monophasic Michaelis-Menten kinetics. The same phenomenon was observed when ferrocytochromec oxidation was followed in the presence of increasing amounts of ferricytochromec. From these results we conclude that ferricytochromec shifts the multiphasic kinetics of ferrocytochromec oxidation by COX into monophasic kinetics, comparable to high ionic strength conditions. Furthermore, we show that ferricytochromec inhibits the high affinity phase of ferrocytochromec oxidation in an apparently competitive way, while inhibition of the low affinity phase is noncompetitive. These findings are consistent with a regulatory site model where both the catalytic and the regulatory site bind ferro- as well as ferricytochromec.     

Methionine-oxidized horse heart cytochromes c. II. Conformation and heme configuration

The two products from the reaction of horse heart ferricytochrome c with Chloramine-T, the F^III and F^II CT-cytochromes, contain modification of the methionines to methionine sulfoxides, but they are distinct in their physiological functions. Conformational and heme-configurational characterization of the two CT-cytochromes has been carried out by using absorption, circular dichroism, fluorescence, proton magnetic resonance, and resonance Raman spectroscopy. The pH-absorption spectroscopic behavior, thermal stability, and ionization of the phenolic hydroxyls have also been reported. Spectroscopic studies of the heme c fragment, H8, in the presence of dimethylsulfoxide, as a model for CT-cytochrome heme configuration, were also conducted. The ferric and the ferrous CT-cytochromes above pH 7.5 have similar, yet distinct, spectroscopic properties, absorption, CD, resonance Raman, and PMR spectra, typical of low-spin hexacoordinated hemes, but distinct from those of the unmodified protein. The ferric spectrum lacks the 695-nm band, and the reduced spectrum contains an additional inflection at about 400 nm, a feature also observed in the spectra of ferrous H8-DMSO systems. The CD, resonance Raman, and PMR spectra are typical of a cytochrome with a loosened heme crevice and altered coordination configuration. The Methionine-80 proton resonances are absent in the uupfield PMR spectra of both the CT-ferricytochromes. The ferrous spectra, on the other hand, contain all the Met-80 resonances, but with smaller upfield shifts than those of the native protein. Both CT-ferric cytochromes are less stable in the acid region and convert to high-spin forms with a two-step transition and with a distinct set of pK _a values. The overall conformation is nearly identical to that of the native protein, but it is less stable to thermal unfolding. All the factors differentiating the modified preparations from the unmodified protein are more pronunced in the case of F^II, with F^III being the closest to the unmodified form. The two functionally distinct CT-cytochromes are two conformational isomers; conformationally and heme configurationally, they are spectroscopically very similar, yet distinct. Both contain an altered heme iron coordination configuration. The sulfur of Met-80 is repalced by the oxygen of Met-80 sulfoxide of a different configuration, R or S. Both contain a loosened heme crevice and are conformationally less stable than the native protein, F^II CT-cytochrome c being the most deranged.     

Plant distribution in relation to the length of the growing season in a snow-bed in the Taisetsu Mountains,northern Japan

The distribution pattern of plants was studied in an alpine snow-bed in six plots along a snow-melting gradient. Each plot consisted of two habitats with respect to the microtopography; the flat habitat and the mound habitat. The number of species per plot decreased with the shortened snow-free period. In the flat habitat, the dominant growth forms changed from the early exposed plots to the late exposed ones as follows; lichens evergreen and deciduous shrubs forbs graminoids bryophytes. In the mound habitat, evergreen and deciduous shrubs prevailed widely along the gradient because of the ability to exploit new habitat by creeping over exposed rocks. For shrubs, the existence of mounds contributed to the expansion of the distribution ranges. Forbs and graminoids shifted their distribution modes to the late exposed plots where shrubs decreased in cover. Deciduous shrubs and forbs completely disappeared in the latest exposed plot.     

Horse heart ferricytochromec: Conformation and heme configuration of high ionic strength acidic forms

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochromec in the presence of 0.2 M KCl, 0.1 M NaClO₄, and 0.2 M KNO₃, in thepH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990)J. Protein Chem.,9, 379–387), the change from neutral to low acidicpH's in the presence of salts is a three-step process: state III _s ?state III _s,a ?state II _s ?state I _s , withpK _a 's of 3.5±0.2, 2.2±0.2, and 1.1±0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutralpH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III _s,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II _s and I _s are also essentially folded forms, but with a smaller degree of protein secondary structure. State II _s has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I _s has a high-spin pentacoordinated heme iron, which is about 0.49 Å out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III _a →state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).     

Cytochromes of the non-thiosulfate-utilizing green sulfur bacterium Chlorobium limicola

Flavocytochrome c-553 of the non-thiosulfateutilizing green sulfur bacterium Chlorobium limicola strain 6330 was partially purified by ion exchange column chromatography and ammonium sulfate fractionation (highest purity index obtained: A ₂₈₀/A _{417 red}=0.96). It is autoxidizable and located in the soluble fraction. This hemoprotein contains a flavin component and one heme per molecule. The dithionite reduced spectrum reveals the typical maxima of a c-type cytochrome: =553,5 nm; =523 nm; =417 nm, while the oxidized form shows a -band at 410 nm and two shoulders at 440 nm and 480 nm indicating the flavin component. The flavocytochrome is a basic protein with an isoelectric point at pH 9.0 (± 0.5), a redox potential of 65 mV, a molecular weight of 56,000. It participates in sulfide oxidation and shows neither adenylylsulfate reductase nor sulfite reductase activity. C. limicola further contains a soluble cytochrome c-555 (highest purity index obtained: A ₂₈₀/A _{412 ox}=0.13; isoelectric point between pH 9.5 and 10) and the non-heme iron-containing proteins rubredoxin and ferredoxin, but lacks cytochrome c-551. Besides these soluble electron transfer proteins a membrane-bound c-type cytochrome (=554,5 nm) can be detected spectrophotometrically.Non-common abbreviations HIPIP high-potential iron sulfur protein - APS adenylylsulfate     

Dissimilatory hexaheme c nitrite reductase of “Spirillum” strain 5175: purification and properties

When grown with nitrate as terminal electron acceptor both the soluble (periplasm, cytoplasm) and the membrane fraction of Spirillum strain 5175 exhibited high nitrite reductase activity. The nitrite reductase obtained from the soluble fraction was purified 76-fold to electrophoretical homogeneity. The enzyme reduced nitrite to ammonia with a specific activity of 723 mol NO _inf2 ^sup- × (mg protein × min)^-1. The molecular mass was 58±1 kDa by SDS-PAGE compared to 59±2 kDa determined by size exclusion chromatography under nondenaturing conditions. The enzyme (as isolated) contained 5.97±0.15 heme c molecules/M_r 58 kDa. The absorption spectrum was typical for c-type cytochrome with maxima at 280, 408, 532 and 610 nm (oxidized) and at 420, 523 and 553 nm (dithionite-reduced). The enzyme (as isolated) exhibited a complex set of high-spin and lowspin ferric heme resonances with g-values at 9.82, 3,85, 3.31, 2.95, 2.30 and 1.49 in agreement with data reported for electron paramagnetic resonance spectra of nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli.Abbreviations DNRA dissimilatory nitrate reduction to ammonia - EPR electron paramagnetic resonance - PAGE polyacrylamide gel electrophoresis - NaP_i sodium phosphate - SDS sodium dodecylsulfate     

Effects of axial methionine coordination on the in-plane asymmetry of the heme electronic structure of cytochrome<Emphasis Type="Italic"> c</Emphasis>

The paramagnetic susceptibility () tensors of the oxidized forms of thermophile Hydrogenobacter thermophilus cytochrome c₅₅₂ (Ht cyt c₅₅₂) and a quintuple mutant (F7A/V13 M/F34Y/E43Y/V78I; qm) of mesophile Pseudomonas aeruginosa cytochrome c₅₅₁ (Pa cyt c₅₅₁) have been determined on the basis of the redox-dependent ¹H NMR shift changes of the main-chain NH and CH proton resonances of non-coordinated amino acid residues and the NMR structures of the reduced forms of the corresponding proteins (J. Hasegawa, T. Yoshida, T. Yamazaki, Y. Sambongi, Y. Yu, Y. Igarashi, T. Kodama, K. Yamazaki, Y. Kyogoku, Y. Kobayashi (1998) Biochemistry 37:9641–9649; J. Hasegawa, S. Uchiyama, Y. Tanimoto, M. Mizutani, Y. Kobayashi, Y. Sambongi,Y. Igarashi (2000) J Biol Chem 275:37824–37828). From the tensors determined, we obtained the contact shifts for heme methyl proton resonances, which provided the heme electronic structures of the oxidized forms of Ht cyt c₅₅₂ and qm. We also characterized the heme electronic structure of the cyanide adducts of the proteins, where the axial Met was replaced by an exogenous cyanide ion, through the analysis of ¹H NMR spectra. The results indicated that the heme electronic structures of both the proteins in their oxidized forms with axial His and Met coordination are largely different to each other, while those in their cyanide adducts are similar to each other. These results demonstrated that the orientation of the axial Met sulfur lone pair, with respect to heme, predominantly contributes to the spin delocalization into the porphyrin- system of heme in the oxidized proteins with axial His and Met coordination.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations COSY correlation spectroscopy - DQF-COSY double quantum filtered COSY - TOCSY total correlation spectroscopy - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect correlated spectroscopy - Cyt c cytochrome c - Pa cyt c₅₅₁ Pseudomonas aeruginosa cytochrome c₅₅₁ - Ht cyt c₅₅₂ Hydrogenobacter thermophilus cytochrome c₅₅₂ - _obs observed shift - _para paramagnetic shift - _dia diamagnetic shift - _con contact shift - _pc pseudo-contact shift     

Inheritance and linkage relationships of glutamate oxaloacetate transaminase isoenzymes in apple

Summary Four zones of enzymatic activity for glutamate oxaloacetate transaminase (GOT) were found in apple tissue. A dimeric gene, GOT-1, determining the fastest migrating zone, was identified. Six alleles were found, including a near null allelle which produced detectable heterodimeric bands but not homodimeric bands. A marked deficit or absence of certain geno-types in all backcrosses and in some crosses between unrelated varieties was attributed to the close linkage (r=0.02±0.005) of GOT-1 with the incompatibility S locus. GOT-1 was also closely linked with the isocitrate dehydrogenase locus IDH-1 (0.03±0.01). Proposed incompatibility genotypes for four cultivars, and the linked GOT-1 alleles are Cox: S ₁ b/S ₂ d, Idared: S ₃ a/S ₄ c, Fiesta: S ₃ a/S ₂ d and Kent: S ₃ a/S ₁ b.The results reported in this paper are part of a PhD Thesis by the first author