Probing the structure of membrane proteins with electron spin echo envelope modulation spectroscopy

A new approach has been developed to probe the structural properties of membrane peptides and proteins using the pulsed electron paramagnetic resonance technique of electron spin echo envelope modulation (ESEEM) spectroscopy and the α-helical M2δ subunit of the acetylcholine receptor incorporated into phospholipid bicelles. To demonstrate the practicality of this method, a cysteine-mutated nitroxide spin label (SL) is positioned 1, 2, 3, and 4 residues away from a fully deuterated Val side chain (denoted i + 1 to i + 4). The characteristic periodicity of the α-helical structure gives rise to a unique pattern in the ESEEM spectra. In the i + 1 and i + 2 samples, the 2H nuclei are too far away to be detected. However, with the 3.6 residue per turn pattern of an α-helix, the i + 3 and i + 4 samples reveal a strong signal from the 2H nuclei of the Val side chain. Modeling studies verify these data suggesting that the closest 2H-labeled Val to SL distance would in fact be expected in the i + 3 and i + 4 samples. This technique is very advantageous, because it provides pertinent qualitative structural information on an inherently difficult system like membrane proteins in a short period of time (minutes) with small amounts of protein (μg).     

The structure of bovine mitochondrial F1-ATPase: an example of rotary catalysis

There is now compelling evidence in support of a rotary catalytic mechanism in F1-ATPase, and, by extension, in the intact ATP synthase. Although models have been proposed to explain how protein translocation in F0 results in rotation of the gamma-subunit relative to the alpha 3/beta 3 assembly in F1 [22], these are still speculative. It seems likely that a satisfactory explanation of this mechanism will ultimately depend on structural information on the intact ATP synthase.     

Electron microscopy of beef heart mitochondrial F1-ATPase

The quaternary structure of isolated and membrane-bound F1-ATPase (submitochondrial particles) has been studied by electron microscopy. A model of the molecule has been proposed: six protein masses are arranged in two layers approximately at the vertices of a triangular antiprism. Computer averaging of the images showed that the frontal view of the molecule can be approximately characterized by mirror plane symmetry.     

On the location and function of the noncatalytic sites on the bovine heart mitochondrial F1-ATPase

Modification of Tyr-345 at a catalytic site in a single beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) by 5'-p-fluorosulfonylbenzoylinosine did not affect subsequent labeling of noncatalytic sites at Tyr-368 and His-427 in three copies of the beta subunit by 5'-p-fluorosulfonylbenzoyladenosine (FSBA). These results clearly show that the beta subunit contains at least parts of the catalytic and noncatalytic nucleotide binding sites. Inactivation of MF1 by 96% with FSBA was accompanied by a decrease in the endogenous ADP content from 1.86 to 0.10 mol per mol of MF1. Decrease in the endogenous ADP content during the inactivation of the enzyme with FSBA paralleled loss in activity in a manner which suggests that the reaction of FSBA with an open noncatalytic site promoted release of ADP from another noncatalytic site until the third site reacted with FSBA. Two pKa values of about 5.9 and 7.6 were observed on the acid side of the pH optimum in the pH-rate profile for ATP hydrolysis catalyzed by MF1 in neutral acid buffers. In contrast, a single pKa of 5.9 was present in the pH-rate profile for ITP hydrolysis catalyzed by the enzyme in the same buffers. The augmented rate observed for ATP hydrolysis at pH 8.0, over that observed at pH 6.5, was lost as the enzyme was inactivated by FSBA in a manner suggesting that modulation is lost as the third noncatalytic site is modified. This suggests that ATP hydrolysis by MF1 is modulated in a pH-dependent manner by ATP binding to an open noncatalytic site. Two other modulations associated with binding of adenine nucleotides to noncatalytic sites, ADP-induced hysteretic inhibition and apparent negative cooperativity reflected by the Hill coefficient for the hydrolysis of 50-3000 microM ATP at pH 8.0, also disappeared as the third noncatalytic site reacted with FSBA.     

Probing the iron-substrate orientation for taurine/alpha-ketoglutarate dioxygenase using deuterium electron spin echo envelope modulation spectroscopy

The structural relationship between substrate taurine and the non-heme Fe(II) center of taurine/alpha-ketoglutarate (alphaKG) dioxygenase (TauD) was measured using electron spin echo envelope modulation (ESEEM) spectroscopy. Studies were conducted on TauD samples treated with NO, cosubstrate alphaKG, and either protonated or specifically deuterated taurine. Stimulated echo ESEEM data were divided to eliminate interference from 1H and 14N modulations and accentuate modulations from 2H. For taurine that was deuterated at the C1 position (adjacent to the sulfonate group), 2H ESEEM spectra show features that arise from dipole-dipole and deuterium nuclear quadrupole interactions from a single deuteron. Parallel measurements taken for taurine deuterated at both C1 and C2 show an additional ESEEM feature at the deuterium Larmor frequency. Analysis of these data at field positions ranging from g = 4 to g = 2 have allowed us to define the orientation of substrate taurine with respect to the magnetic axes of the Fe(II)-NO, S = 3/2, paramagnetic center. These results are discussed in terms of previous X-ray crystallographic studies and the proposed catalytic mechanism for this family of enzymes.     

Oxidative phosphorylation in a hybrid system containing bovine heart membranes and pea mitochondrial F1-ATPase

Purified pea mitochondrial F1-ATPase reconstituted oxidative phosphorylation in both partially and completely F1-depleted bovine heart mitochondrial membranes. The isolated plant enzyme exhibited high rates of ATP synthesis when combined with bovine heart membranes, suggesting great evolutionary conservation of the ATP synthase complex in mitochondria.     

Conformational dynamics of the F1-ATPase beta-subunit: a molecular dynamics study

According to the different nucleotide occupancies of the F(1)-ATPase beta-subunits and due to the asymmetry imposed through the central gamma-subunit, the beta-subunit adopts different conformations in the crystal structures. Recently, a spontaneous and nucleotide-independent closure of the open beta-subunit upon rotation of the gamma-subunit has been proposed. To address the question whether this closure is dictated by interactions to neighbored subunits or whether the open beta-subunit behaves like a prestressed "spring," we report multinanosecond molecular dynamics simulations of the isolated beta-subunit with different start conformations and different nucleotide occupancies. We have observed a fast, spontaneous closure motion of the open beta(E)-subunit, consistent with the available x-ray structures. The motions and kinetics are similar to those observed in simulations of the full (alpha beta)(3)gamma-complex, which support the view of a prestressed "spring," i.e., that forces internal to the beta(E)-subunit dominate possible interactions from adjacent alpha-subunits. Additionally, nucleotide removal is found to trigger conformational transitions of the closed beta(TP)-subunit; this provides evidence that the recently resolved half-closed beta-subunit conformation is an intermediate state before product release. The observed motions provide a plausible explanation why ADP and P(i) are required for the release of bound ATP and why gamma-depleted (alpha beta)(3) has a drastically reduced hydrolysis rate.     

Exchange and hydrolysis of tightly bound nucleotides in normal and photolabelled bovine heart mitochondrial F1-ATPase

Treatment of F1 by threefold fast-column centrifugation or by single ammonium sulphate precipitation followed by fast-column centrifugation resulted in enzyme preparations containing 2.5-2.8 mol of bound nucleotides per mol of F1. Short incubations of such F1 preparations in the presence of relatively low concentrations of [14C]ATP and 2-azido[alpha-32P]ATP (100-250 microM), followed by ammonium sulphate precipitation and fast-column centrifugation, resulted in exchange of about 1 mol of the bound nucleotide per mol of F1 not affecting the total amount of bound nucleotides. Exchange of bound nucleotides with 2-azidoATP, followed by ultraviolet irradiation, results in inhibition of the enzyme activity, full inhibition being obtained (via extrapolation) when 1 mol of 2-nitreno-adenosine 5'-tri- or diphosphate (2-N-AT(D)P) is covalently bound to the presumably catalytic site on the enzyme (Van Dongen, M.B.M., De Geus, J.P., Korver, T., Harton, A.F. and Berden, J.A. (1986) Biochim. Biophys. Acta 850, 359-368). In agreement with this, it was found that incorporated [gamma-32P]ATP was hydrolysed by more than 80%. Newly incorporated, not covalently bound radioactive nucleotides could be rapidly exchanged again by the addition of non-radioactive nucleotides, but a higher concentration of nucleotides was needed to fully exchange the incorporated nucleotide. Also, when F1 was depleted of most of its bound nucleotides by repeated ammonium sulphate precipitation, part of the residual nucleotides was still rapidly exchangeable. The ability of F1 to exchange (and hydrolyse) one of the bound nucleotides was not lost when one catalytic and one non-catalytic binding site were occupied by covalently bound 8-N-ATP. Similar results were obtained with F1 containing 2-nitrenoATP covalently bound to one of the catalytic sites. Also, after photolabelling of up to four binding sites with 8-N[( 2-3H]AT(D)P, part of the two remaining non-covalently bound nucleotides could still be rapidly exchanged. In this case the exchanged nucleotide was also hydrolysed. It is concluded that one of the two bound nucleotides became exchangeable when all four other sites (i.e., two catalytic and two non-catalytic) were occupied with covalently bound nucleotides. The site involved showed catalytic properties suggestive of localisation on a beta-subunit.     

Is pyrophosphate an analog of adenosine diphosphate for beef heart mitochondrial F1-ATPase

Beef heart mitochondrial F1 possesses three pyrophosphate-binding sites, which comprises one high affinity binding site (Kd approximately equal to 1 microM) and two lower affinity sites (Kd approximately equal to 20 microM). High affinity pyrophosphate binding required the presence of Mg2+ in the incubation medium. Pyrophosphate competed with ADP, but not with Pi for binding to mitochondrial F1. Upon binding of 3 mol of pyrophosphate/mol of F1, one of the three tightly bound nucleotides present in native F1 was released. Like ADP and in contrast to Pi, pyrophosphate enhanced the fluorescence intensity of F1-bound aurovertin, and it prevented the photolabeling of F1 by 2-azido-ADP. As aurovertin and 2-azido-ADP are ligands of the beta subunit of F1, it is likely that pyrophosphate binds preferentially to the beta subunit. Whereas the binding affinity of F1 for Pi was increased by concentrations of pyrophosphate lower than 100 microM, it was decreased by a higher concentration of pyrophosphate. This biphasic effect of pyrophosphate on Pi binding was not observed with ADP, which, at all concentrations tested, inhibited Pi binding. Except for the effect of pyrophosphate on Pi binding to F1, for all the other effects, pyrophosphate mimicked ADP. It is suggested that pyrophosphate and ADP share the same binding site on F1 and that pyrophosphate interacts with the same amino acid residues as those interacting with the alpha and beta phosphate groups of ADP.     

Probing the local secondary structure of bacteriophage S21 pinholin membrane protein using electron spin echo envelope modulation spectroscopy

There have recently been advances in methods for detecting local secondary structures of membrane protein using electron paramagnetic resonance (EPR). A three pulsed electron spin echo envelope modulation (ESEEM) approach was used to determine the local helical secondary structure of the small hole forming membrane protein, S²¹ pinholin. This ESEEM approach uses a combination of site-directed spin labeling and ²H-labeled side chains. Pinholin S²¹ is responsible for the permeabilization of the inner cytosolic membrane of double stranded DNA bacteriophage host cells. In this study, we report on the overall global helical structure using circular dichroism (CD) spectroscopy for the active form and the negative-dominant inactive mutant form of S²¹ pinholin. The local helical secondary structure was confirmed for both transmembrane domains (TMDs) for the active and inactive S²¹ pinholin using the ESEEM spectroscopic technique. Comparison of the ESEEM normalized frequency domain intensity for each transmembrane domain gives an insight into the α-helical folding nature of these domains as opposed to a π or 3₁₀-helix which have been observed in other channel forming proteins.     

Electronic structure of the Mn-cofactor of modified bacterial reaction centers measured by electron paramagnetic resonance and electron spin echo envelope modulation spectroscopies

The electronic structure of a Mn(II) ion bound to highly oxidizing reaction centers of Rhodobacter sphaeroides was studied in a mutant modified to possess a metal binding site at a location comparable to the Mn₄Ca cluster of photosystem II. The Mn-binding site of the previously described mutant, M2, contains three carboxylates and one His at the binding site (Thielges et al., Biochemistry 44:389–7394, 2005). The redox-active Mn-cofactor was characterized using electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopies. In the light without bound metal, the Mn-binding mutants showed an EPR spectrum characteristic of the oxidized bacteriochlorophyll dimer and reduced quinone whose intensity was significantly reduced due to the diminished quantum yield of charge separation in the mutant compared to wild type. In the presence of the metal and in the dark, the EPR spectrum measured at the X-band frequency of 9.4 GHz showed a distinctive spin 5/2 Mn(II) signal consisting of 16 lines associated with both allowed and forbidden transitions. Upon illumination, the amplitude of the spectrum is decreased by over 80 % due to oxidation of the metal upon electron transfer to the oxidized bacteriochlorophyll dimer. The EPR spectrum of the Mn-cofactor was also measured at the Q-band frequency of 34 GHz and was better resolved as the signal was composed of the six allowed electronic transitions with only minor contributions from other transitions. A fit of the Q-band EPR spectrum shows that the Mn-cofactor is a high spin Mn(II) species (S = 5/2) that is six-coordinated with an isotropic g-value of 2.0006, a weak zero-field splitting and E/D ratio of approximately 1/3. The ESEEM experiments showed the presence of one ¹⁴N coordinating the Mn-cofactor. The nitrogen atom is assigned to a His by comparing our ESEEM results to those previously reported for Mn(II) ions bound to other proteins and on the basis of the X-ray structure of the M2 mutant that shows the presence of only one His, residue M193, that can coordinate the Mn-cofactor. Together, the data allow the electronic structure and coordination environment of the designed Mn-cofactor in the modified reaction centers to be characterized in detail and compared to those observed in other proteins with Mn-cofactors.     

Tyrosine alpha 244 is derivatized when the bovine heart mitochondrial F1-ATPase is inactivated with 5'-p-fluorosulfonylbenzoylethenoadenosine

The bovine heart mitochondrial F1-ATPase (MF1) is inactivated by 5'-p-fluorosulfonylbenzoylethenoadenosine (FSB epsilon A) with pseudo-first order kinetics. The dependence of the rate of inactivation on the concentration of FSB epsilon A revealed an apparent Kd of 0.25 mM. ATP and ADP, and to a lesser extent, ITP and IDP provide partial protection against inactivation by the reagent. Isolation and sequence analysis of major radioactive fragments in peptic or cyanogen bromide digests of MF1 inactivated with [3H]FSB epsilon A indicate that modification of Tyr-alpha 244 is associated with the loss of activity observed. Assessment of the amount of Tyr-alpha 244 derivatized with [3H]FSB epsilon A at specific points during inactivation of the ATPase indicates that maximal inactivation is achieved on modification of this residue in slightly greater than one copy of the alpha subunit. The following characteristics of inactivation of MF1 by FSB epsilon A have also been determined. (a) The rate of inactivation of ITPase activity by FSB epsilon A is 1.4 times greater than that observed for inactivation of ATPase activity under identical conditions. (b) After maximally inactivating the capacity of MF1 to hydrolyze saturating ATP with FSB epsilon A, the modified enzyme retained its capacity to hydrolyze substoichiometric ATP. (c) Inactivation of the ATPase by FSB epsilon A is accelerated by Pi. In each of the above characteristics, MF1 modified by FSB epsilon A resembles enzyme inactivated with 5'-p-fluorosulfonylbenzoyladenosine (FSBA) more than it does enzyme inactivated with 5'-p-fluorosulfonylbenzoylinosine (FSBI). Furthermore, prior inactivation of MF1 with FSBA completely prevents labeling of Tyr-alpha 244 with [3H]FSB epsilon A, whereas prior inactivation of the enzyme with FSBI does not. Since a single catalytic site is modified when FSBI inactivates MF1 whereas three noncatalytic sites are modified when it is maximally inactivated with FSBA, it is concluded that FSB epsilon A also modifies noncatalytic sites.     

An electron microscopic approach to the quaternary structure of mitochondrial F1-ATPase

The three-dimensional structure of F1-ATPase from beef heart mitochondria was investigated by electron microscopic techniques. The presence of high concentrations of nucleotides is essential for preservation of the quaternary structure. When investigated under such conditions, monodisperse F1-ATPase could not be distinguished from the membrane-bound enzyme. At low resolution, the particle shape resembles an oblate ellipsoid of revolution with an axial ratio of about 2:1. From several lines of evidence (including field micrographs at higher magnifications, Markham rotational analysis, and tilting experiments), two conclusions may be drawn concerning the three-dimensional fine structure of F1-ATPase. 1. At the periphery of the molecule, six globular protein masses are orientated in a way similar to the chair conformation of cyclohexane. This array is interpreted to be made up of an alternating sequence of alpha and beta subunits. 2. Part of the central space is occupied by a seventh protein mass, protrusions of which are likely to be in contact with some of the outer subunits. A gamma subunit is supposed to be constituent part of this central protein mass. As a consequence, this model favours a stoichiometry of alpha 3 beta 3 gamma for the large subunits of beef heart F1-ATPase.     

Development of electron spin echo envelope modulation spectroscopy to probe the secondary structure of recombinant membrane proteins in a lipid bilayer

Membrane proteins conduct many important biological functions essential to the survival of organisms. However, due to their inherent hydrophobic nature, it is very difficult to obtain structural information on membrane‐bound proteins using traditional biophysical techniques. We are developing a new approach to probe the secondary structure of membrane proteins using the pulsed EPR technique of Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. This method has been successfully applied to model peptides made synthetically. However, in order for this ESEEM technique to be widely applicable to larger membrane protein systems with no size limitations, protein samples with deuterated residues need to be prepared via protein expression methods. For the first time, this study shows that the ESEEM approach can be used to probe the local secondary structure of a ²H‐labeled d₈‐Val overexpressed membrane protein in a membrane mimetic environment. The membrane‐bound human KCNE1 protein was used with a known solution NMR structure to demonstrate the applicability of this methodology. Three different α‐helical regions of KCNE1 were probed: the extracellular domain (Val21), transmembrane domain (Val50), and cytoplasmic domain (Val95). These results indicated α‐helical structures in all three segments, consistent with the micelle structure of KCNE1. Furthermore, KCNE1 was incorporated into a lipid bilayer and the secondary structure of the transmembrane domain (Val50) was shown to be α‐helical in a more native‐like environment. This study extends the application of this ESEEM approach to much larger membrane protein systems that are difficult to study with X‐ray crystallography and/or NMR spectroscopy.     

Identification of the subunits of F1F0-ATPase from bovine heart mitochondria.

An oligomycin-sensitive F1F0-ATPase isolated from bovine heart mitochondria has been reconstituted into phospholipid vesicles and pumps protons. this preparation of F1F0-ATPase contains 14 different polypeptides that are resolved by polyacrylamide gel electrophoresis under denaturing conditions, and so it is more complex than bacterial and chloroplast enzymes, which have eight or nine different subunits. The 14 bovine subunits have been characterized by protein sequence analysis. They have been fractionated on polyacrylamide gels and transferred to poly(vinylidene difluoride) membranes, and N-terminal sequences have been determined in nine of them. By comparison with known sequences, eight of these have been identified as subunits beta, gamma, delta, and epsilon, which together with the alpha subunit form the F1 domain, as the b and c (or DCCD-reactive) subunits, both components of the membrane sector of the enzyme, and as the oligomycin sensitivity conferral protein (OSCP) and factor 6 (F6), both of which are required for attachment of F1 to the membrane sector. The sequence of the ninth, named subunit e, has been determined and is not related to any reported protein sequence. The N-terminal sequence of a tenth subunit, the membrane component A6L, could be determined after a mild acid treatment to remove an alpha-N-formyl group. Similar experiments with another membrane component, the a or ATPase-6 subunit, caused the protein to degrade, but the protein has been isolated from the enzyme complex and its position on gels has been unambiguously assigned. No N-terminal sequence could be derived from three other proteins. The largest of these is the alpha subunit, which previously has been shown to have pyrrolidonecarboxylic acid at the N terminus of the majority of its chains. The other two have been isolated from the enzyme complex; one of them is the membrane-associated protein, subunit d, which has an alpha-N-acetyl group, and the second, surprisingly, is the ATPase inhibitor protein. When it is isolated directly from mitochondrial membranes, the inhibitor protein has a frayed N terminus, with chains starting at residues 1, 2, and 3, but when it is isolated from the purified enzyme complex, its chains are not frayed and the N terminus is modified. Previously, the sequences at the N terminals of the alpha, beta, and delta subunits isolated from F1-ATPase had been shown to be frayed also, but in the F1F0 complex they each have unique N-terminal sequences.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of hydrogen bonds to the Rieske cluster through the weakly coupled nitrogens detected by electron spin echo envelope modulation spectroscopy

The interaction of the reduced[2Fe-2S] cluster of isolated Rieske fragment from the bc1 complex of Rhodobacter sphaeroides with nitrogens (14N and 15N) from the local protein environment has been studied by X- and S-band pulsed EPR spectroscopy. The two-dimensional electron spin echo envelope modulation spectra of uniformly 15N-labeled protein show two well resolved cross-peaks with weak couplings of approximately 0.3-0.4 and 1.1 MHz in addition to couplings in the range of 6-8 MHz from two coordinating Ndelta of histidine ligands. The quadrupole coupling constants for weakly coupled nitrogens determined from S-band electron spin echo envelope modulation spectra identify them as Nepsilon of histidine ligands and peptide nitrogen (Np), respectively. Analysis of the line intensities in orientation-selected S-band spectra indicated that Np is the backbone N-atom of Leu-132 residue. The hyperfine couplings from Nepsilon and Np demonstrate the predominantly isotropic character resulting from the transfer of unpaired spin density onto the 2s orbitals of the nitrogens. Spectra also show that other peptide nitrogens in the protein environment must carry a 5-10 times smaller amount of spin density than the Np of Leu-132 residue. The appearance of the excess unpaired spin density on the Np of Leu-132 residue indicates its involvement in hydrogen bond formation with the bridging sulfur of the Rieske cluster. The configuration of the hydrogen bond therefore provides a preferred path for spin density transfer. Observation of similar splittings in the 15N spectra of other Rieske-type proteins and [2Fe-2S] ferredoxins suggests that a hydrogen bond between the bridging sulfur and peptide nitrogen is a common structural feature of [2Fe-2S] clusters.     

Ca(2+) activation of heart mitochondrial oxidative phosphorylation: role of the F(0)/F(1)-ATPase

Ca²⁺ has been postulated as a cytosolicsecond messenger in the regulation of cardiac oxidativephosphorylation. This hypothesis draws support from the well-knowneffects of Ca²⁺ on muscle activity, which is stimulated inparallel with the Ca²⁺-sensitive dehydrogenases (CaDH). Theeffects of Ca²⁺ on oxidative phosphorylation were furtherinvestigated in isolated porcine heart mitochondria at the level ofmetabolic driving force (NADH or ) and ATPproduction rates (flow). The resulting force-flow (F-F) relationshipspermitted the analysis of Ca²⁺ effects on several putativecontrol points within oxidative phosphorylation, simultaneously. TheF-F relationships resulting from additions of carbon substrates aloneprovided a model of pure CaDH activation. Comparing this curve withvariable Ca²⁺ concentration([Ca²⁺]) effects revealed an approximatetwofold higher ATP production rate than could be explained by a simpleincrease in NADH or via CaDH activation. The half-maximal effectof Ca²⁺ at state 3 was 157 nM and was completely inhibitedby ruthenium red (1 µM), indicating matrix dependence of theCa²⁺ effect. Arsenate was used as a probe to differentiatebetween F₀/F₁-ATPase and adenylate translocaseactivity by a futile recycling of ADP-arsenate within the matrix,catalyzed by the F₀/F₁-ATPase. Ca²⁺increased the ADP arsenylation rate more than twofold, suggesting adirect effect on the F₀/F₁-ATPase. Theseresults suggest that Ca²⁺ activates cardiac aerobicrespiration at the level of both the CaDH andF₀/F₁-ATPase. This type of parallel control ofboth intermediary metabolism and ATP synthesis may provide a mechanismof altering ATP production rates with minimal changes in thehigh-energy intermediates as observed in vivo.

     

High performance liquid chromatography purification and amino terminal sequence analysis of the subunits of bovine heart mitochondrial F1-ATPase

All five subunits of bovine heart mitochondrial F1-ATPase have been isolated by reverse-phase HPLC and NH2-terminal sequences determined by gas phase Edman degradations. Bovine gamma exhibits 16 identities in the first 30 residues compared with the NH2-terminus of gamma from E.coli F1. Bovine delta exhibit about 27% identity with residues 28-59 of precursor delta from N.crassa and in the first six residues is identical with delta from S.cerevisiae. Approximately half of bovine epsilon has been sequenced. Possibly significant sequence similarities exist between bovine gamma and epsilon and kinase-related gene and oncogene products. The bovine alpha subunit has a blocked NH2-terminus.