Mapping of the functional determinants of the integrin beta 1 cytoplasmic domain by site-directed mutagenesis.

We describe here the expression of deletion mutants of the cytoplasmic domain of the avian integrin beta 1 subunit. These mutants, which contain termination codons at positions 767, 776, 791, and 800, were transfected into mouse 3T3 cells to determine which sequences were essential for localization of integrins into focal contact sites. In all cases, high-level expression of the truncated avian integrins was obtained. Heterodimers were formed between the exogenous truncated avian beta 1 subunits and endogenous mouse alpha subunits, and these heterodimers were efficiently exported to the cell surface. The longest truncated beta 1 subunit tested, which is only four amino acids shorter than the wild type, does localize to focal contacts. In contrast, beta 1 subunits with moderately long truncations of the cytoplasmic domain failed to localize to focal contacts, including one which contains the consensus sequence for tyrosine phosphorylation. Surprisingly, a mutant subunit in which the bulk of the cytoplasmic domain was missing (but the segment nearest the membrane including the dibasic residues (RR) remained) did localize weakly to focal contacts. These results implicate the peptide segment nearest to the transmembrane region in focal contact localization. In addition, mutant subunits that included this segment together with a larger portion of the cytoplasmic domain did not localize as well as the shorter form, suggesting that these cytoplasmic domain segments are defective, presumably because of abnormal folding.     

Amino acid sequence of a novel integrin beta 4 subunit and primary expression of the mRNA in epithelial cells.

Using the polymerase chain reaction, we have isolated cDNA clones that encode a new integrin beta subunit--beta 4. Its cDNA, which is 5676 bp in length, has one long coding sequence (5256 bp), a polyadenylation signal and a poly(A) tail. The deduced sequence of 1752 amino acids is unique among the integrin beta subunits. It contains a putative signal sequence as well as a transmembrane domain that divides the molecule into an extracellular domain at the N-terminal side and a cytoplasmic domain at the C-terminal side. The extracellular domain exhibits a 4-fold repeat of cysteine-rich motif similar to those of other integrin beta subunits. Certain features of the extracellular domain, however, are unique to the beta 4 subunit sequence. Of the 56 conserved cysteine residues found within the extracellular domain of other mature beta subunits, eight such residues are deleted from the beta 4 subunit sequence. The cytoplasmic domain is much larger (approximately 1000 amino acids) than those of other beta subunits (approximately 50 amino acids) and has no significant homology with them. A protein homology search revealed that the beta 4 subunit cytoplasmic domain has four repeating units that are homologous to the type III repetition exhibited by fibronectin. The beta 4 subunit mRNA was expressed primarily in epithelial cells. The restricted expression and the new structural features distinguish the integrin beta 4 subunit from other integrin beta subunits.     

Rapid-quench and isotope-trapping studies on fructose-1,6-bisphosphatase

Rapid-quench kinetic measurements yielded presteady-state rate data for rabbit liver fructose-1,6-bisphosphatase (FBPase) (a tetramer of four identical subunits) that are triphasic: the rapid release of Pi (complete within 5 ms), followed by a second reaction phase liberating additional Pi that completes the initial turnover of two or four subunits of the enzyme (requiring 100-150 ms), and a steady-state rate whose magnitude depends on the [alpha-Fru-1,6-P2]/[FBPase] ratio. With Mg2+ in the presence of excess alpha-fructose 1,6-bisphosphate (alpha-Fru-1,6-P2) all four subunits turn over in the pre steady state; with Mn2+ only two of the four are active. Thus the expression of half-site reactivity is a consequence of the nature of the metal ion and not a subunit asymmetry. In the presence of limiting alpha-anomer concentrations only two of the four subunits now remain active with Mg2+ as well as with Mn2+ in the pre steady state. However, so that the amount of Pi released can be accounted for, a beta leads to alpha anomerization or direct beta utilization is required at the active site of one subunit. Such behavior is consistent with the two-state conformational hysteresis displayed by the enzyme and altered affinities manifested within these states for alpha and beta substrate analogues. Under these limiting conditions the subsequent steady-state rate is limited by the beta leads to alpha solution anomerization. These data in combination with pulse--chase experiments permit evaluation of the internal equilibrium, which in the case of Mg2+ is unequivocally higher in favor of product complexes and represents a departure from balanced internal substrate-product complexes.     

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits.     

The role of the specificity-determining loop of the integrin beta subunit I-like domain in autonomous expression, association with the alpha subunit, and ligand binding

Integrin beta subunits contain a highly conserved I-like domain that is known to be important for ligand binding. Unlike integrin I domains, the I-like domain requires integrin alpha and beta subunit association for optimal folding. Pactolus is a novel gene product that is highly homologous to integrin beta subunits but lacks associating alpha subunits [Chen, Y., Garrison, S., Weis, J. J., and Weis, J. H. (1998) J. Biol. Chem. 273, 8711-8718] and a approximately 30 amino acid segment corresponding to the specificity-determining loop (SDL) in the I-like domain. We find that the SDL is responsible for the defects in integrin beta subunit expression and folding in the absence of alpha subunits. When transfected in the absence of alpha subunits into cells, extracellular domains of mutant beta subunits lacking SDL, but not wild-type beta subunits, were well secreted and contained immunoreactive I-like domains. The purified recombinant soluble beta1 subunit with the SDL deletion showed an elongated shape in electron microscopy, consistent with its structure in alphabeta complexes. The SDL segment is not required for formation of alpha5beta1, alpha4beta1, alphaVbeta3, and alpha6beta4 heterodimers, but is essential for fomation of alpha6beta1, alphaVbeta1, and alphaLbeta2 heterodimers, suggesting that usage of subunit interface residues is variable among integrins. The beta1 SDL is required for ligand binding and for the formation of the epitope for the alpha5 monoclonal antibody 16 that maps to loop segments connecting blades 2 and 3 of beta-propeller domain of alpha5, but is not essential for nearby beta-propeller epitopes.     

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.     

Synthesis of a repeating unit and the dimer of the repeating unit of the major chain of Shigella flexneri O-antigen polysaccharide

Methyl glycoside of the tetrasaccharide GlcNAc(beta 1-2)Rha(alpha 1-2)Rha(alpha 1-3)Rha, which represents a repeating unit of the basic chain of Shigella flexneri O-antigenic polysaccharides, was synthesized using acylated monosaccharide synthons. A dimer of the repeating unit, octasaccharide [GlcNAc(beta 1-2)Rha(alpha 1-2) Rha(alpha 1-3)Rha(alpha 1-3)]2-OMe was obtained by TrClO4-catalyzed condensation of two tetrasaccharide blocks.     

Multistep assembly of chloroplast NADH dehydrogenase-like subcomplex A requires several nucleus-encoded proteins, including CRR41 and CRR42, in Arabidopsis

Chloroplast NADH dehydrogenase-like complex (NDH) mediates photosystem I cyclic electron transport and chlororespiration in thylakoids. Recently, substantial progress has been made in understanding the structure of NDH, but our knowledge of its assembly has been limited. In this study, a series of interactive proteomic analyses identified several stroma-localized factors required for the assembly of a stroma-protruding arm of NDH (subcomplex A). In addition to further characterization of the previously identified CHLORORESPIRATORY REDUCTION1 (CRR1), CRR6, and CRR7, two novel stromal proteins, CRR41 and CRR42, were discovered. Arabidopsis thaliana mutants lacking these proteins are specifically defective in the accumulation of subcomplex A. A total of 10 mutants lacking subcomplex A, including crr27/cpn60β4, which is specifically defective in the folding of NdhH, and four mutants lacking NdhL-NdhO subunits, were extensively characterized. We propose a model for subcomplex A assembly: CRR41, NdhO, and native NdhH, as well as unknown factors, are first assembled to form an NDH subcomplex A assembly intermediate (NAI500). Subsequently, NdhJ, NdhM, NdhK, and NdhI are incorporated into NAI500 to form NAI400. CRR1, CRR6, and CRR42 are involved in this process. CRR7 is likely to be involved in the final step, in which the fully assembled NAI, including NdhN, is inserted into thylakoids.     

An extensive survey of CK2 alpha and beta subunits in Arabidopsis: multiple isoforms exhibit differential subcellular localization

Casein kinase 2 (CK2) is a ubiquitous enzyme essential for the viability of eukaryotic cells. In the present work we analyzed the Arabidopsis thaliana genome in a search for the genes coding for all CK2 alpha and beta subunits. We found four alpha subunit and four beta subunit genes. Expression analysis showed that all CK2 subunit genes are expressed in inflorescences, stems, leaves and roots. The level of expression of these genes is very similar, except for the one that codes for an alpha subunit harboring a putative chloroplastic destination peptide (alphacp), which shows a slightly higher expression level in all tissues. Using transgenic plants and agroinfiltration, we have also characterized the subcellular localization of all proteins encoded by CK2 genes. Our results show that all alpha subunits are localized in the nucleus, with the exception of alphacp, which is only found in the chloroplasts. On the other hand, beta subunits have a more diverse distribution, with some of them localizing both to the nucleus and to the cytosol, while others are exclusively located in one of these compartments. Remarkably, no CK2beta subunit was found in the chloroplasts. Finally, by directly measuring its activity, we have demonstrated that purified Arabidopsis chloroplasts have active CK2 that can be regulated by external addition of CK2beta. This study represents a complete survey of the CK2 gene family in Arabidopsis and the first step for future studies on CK2 cellular function in this species.     

Subunit arrangement of gamma-aminobutyric acid type A receptors

The GABA(A) receptors are ligand-gated chloride channels. The subunit stoichiometry of the receptors is controversial; four, five, or six subunits per receptor molecule have been proposed for alphabeta receptors, whereas alphabetagamma receptors are assumed to be pentamers. In this study, alpha-beta and beta-alpha tandem cDNAs from the alpha1 and beta2 subunits of the GABA(A) receptor were constructed. We determined the minimal length of the linker that is required between the two subunits for functional channel expression for each of the tandem constructs. 10- and 23-amino acid residues are required for alpha-beta and beta-alpha, respectively. The tandem constructs either alone or in combination with each other failed to express functional channels in Xenopus oocytes. Therefore, we can exclude tetrameric or hexameric alphabeta GABA(A) receptors. We can also exclude proteolysis of the tandem constructs. In addition, the tandem constructs were combined with single alpha, beta, or gamma subunits to allow formation of pentameric arrangements. In contrast to the combination with alpha subunits, the combination with either beta or gamma subunits led to expression of functional channels. Therefore, a pentameric arrangement containing two alpha1 and three beta2 subunits is proposed for the receptor composed of alpha and beta subunits. Our findings also favor an arrangement betaalphagammabetaalpha for the receptor composed of alpha, beta, and gamma subunits.     

CRR23/NdhL is a subunit of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

The chloroplast NAD(P)H dehydrogenase (NDH) complex functions in PSI cyclic and chlororespiratory electron transport in higher plants. Eleven plastid-encoded and three nuclear-encoded subunits have been identified so far, but the entire subunit composition, especially of the putative electron donor-binding module, is unclear. We isolated Arabidopsis thaliana crr23 (chlororespiratory reduction) mutants lacking NDH activity according to the absence of a transient increase in Chl fluorescence after actinic light illumination. Although CRR23 shows similarity to the NdhL subunit of cyanobacterial NDH-1, it has three transmembrane domains rather than the two in cyanobacterial NdhL. Unlike cyanobacterial NdhL, CRR23 is essential for stabilizing the NDH complex, which in turn is required for the accumulation of CRR23. Furthermore, CRR23 and NdhH, a subunit of chloroplast NDH, co-localized in blue-native gel. All the results indicate that CRR23 is an ortholog of cyanobacterial ndhL in Arabidopsis, despite its diversity of structure and function.     

A eukaryotic factor required for accumulation of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

The NAD(P)H dehydrogenase (NDH) complex in chloroplasts mediates photosystem I cyclic and chlororespiratory electron transport. Eleven chloroplast genes and three nuclear genes have been identified as encoding Ndh subunits, but the entire subunit composition is still unknown. An Arabidopsis (Arabidopsis thaliana) chlororespiratory reduction (crr3) mutant was isolated based on its lack of transient increase in chlorophyll fluorescence after actinic light illumination; this was due to a specific defect in accumulation of the NDH complex. The CRR3 gene (At2g01590) encodes a novel protein containing a putative plastid-targeting signal and a transmembrane domain. Consistent with the gene structure, CRR3 localized to the membrane fraction of chloroplasts. In addition to the essential function of CRR3 in stabilizing the NDH complex, the NDH complex is also required for the accumulation of CRR3. These results suggest that CRR3 interacts with the NDH complex in the thylakoid membrane. In contrast to other subunits in the chloroplast NDH complex, CRR3 is not conserved in cyanobacteria from which the chloroplast NDH complex is believed to have originated. We propose that CRR3 is a subunit of the NDH complex, which is specific to the chloroplast.     

Expression of a functional recombinant C‐type lectin‐like protein lebecetin in the human embryonic kidney cells

Lebecetin is an anticoagulant C‐type lectin‐like protein that was previously isolated from Macrovipera lebetina venom and described to consist of two subunits (alpha and beta). It was reported to potently prevent platelet aggregation by binding to glycoprotein Ib and to exhibit a broad spectrum of inhibitory activities on various integrin‐mediated functions of tumor cells, including adhesion, proliferation, and cell migration. This study aimed to investigate the structure‐function of lebecetin. Accordingly, the cDNA of each subunit was cloned and separately or jointly expressed in the human embryonic kidney cells using two vectors with different selectable tags. The immunofluorescence analysis of transfected cells revealed significant expression levels and co‐localization of the two lebecetin subunits. The recombinant proteins were efficiently secreted and purified using metal‐chelating affinity chromatography. We found that the Lebecetin alpha and beta subunits were produced as a mixture of homodimers and heterodimers and that the heterodimerization represents a prerequisite for functioning. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Phylogenetic relationships and chromosomal location of five distinct glycine receptor subunit genes in the teleost Danio rerio

Glycine receptors mediating synaptic inhibition are heteromeric proteins constituted of alpha and beta subunits. The mammalian GlyR subunits constitute a subgroup in the superfamily of ligand-gated ionic channels. To compare the evolutionary events in the mammalian and teleostean lineages for the receptor family, we first undertook systematic cloning of the constitutive subunits of the zebrafish glycine receptor. The isolation of two alpha subunits (alphaZ1 and alphaZ2) and one beta subunit (betaZ) has been reported previously and we report here the characterization of two novel alpha subunits, alphaZ3 and alphaZ4, increasing the known zebrafish subunits number to four alpha and one beta. Establishment of phylogenetic relationships reveals that alphaZ1, alphaZ3 and betaZeta are orthologous to mammalian alpha1, alpha3 and beta subunits. However, two zebrafish GlyRalpha subunit genes are orthologous to the unique avian and mammalian alpha4 subunit revealing a duplication of the alpha4 gene in zebrafish. Whole-mount in situ hybridization in 24-hours post fertilization (hpf) and 52-hpf embryos of the daughter gene products display very different expression patterns indicating distinct functions of the duplicated genes. Gene mapping reveals that the two duplicated genes are localized on two different linkage groups (LG5 and LG22) as would be daughter genes resulting from a large-scale duplication of the ancestral genome. Finally, we report that a linked pair of genes on human chromosome 4 (alpha3 and beta) is also linked on linkage group 1 in zebrafish (alphaZ3 and betaZ) as a consequence of a mosaic conserved syntheny.     

Subunit interactions involved in the assembly of pyridine nucleotide transhydrogenase in the membranes of Escherichia coli.

The pyridine nucleotide transhydrogenase (PNT) of Escherichia coli consists of two different subunits (alpha and beta) and assembles as a tetramer (alpha 2 beta 2) in the inner membrane. The pnt genes from E. coli have been cloned on a multicopy plasmid resulting in high level expression of the enzyme activity. We have studied the influence of the different segments of the polypeptide chains of the alpha and beta subunits on the assembly and function of the enzyme by constructing a series of deletion mutants for both of the subunits. Our results show that the assembly of the beta subunit is contingent upon the insertion of the alpha subunit into the membrane, while the alpha subunit can assemble independently of the beta subunit. All deletions constructed for the cytosolic portion of the alpha subunit gave no incorporation of the alpha subunit and, as a consequence, of the beta subunit, also. Of the four membrane-spanning regions of the alpha subunit, the last two were indispensable, while the deletion of the first two still allowed the association of alpha as well as of the beta subunit with the membrane. However, the enzyme was not functional. The two subunits were also loosely associated as mild detergent treatment released them from the membrane in contrast with the wild-type enzyme. Deletions within the beta subunit had little effect on the assembly of the alpha subunit, although less was incorporated. All deletions involving the cytosolic portion of the beta subunit resulted in loss of incorporation into the membrane. Of the eight membrane-spanning regions of the beta subunit, the deletion of regions 2-3, 2-4, 2-6, and 2-7 yielded significant association of both the subunits with the membrane. However, none of these mutants assembled a functional enzyme, and again the two subunits were loosely associated with the membrane. Based on the stringent requirement of the cytosolic portions of alpha and beta subunits for assembly, a model is proposed that suggests interactions between these two regions must occur prior to assembly.     

Affinity chromatographic isolation of the melanoma adhesion receptor for the IIICS region of fibronectin and its identification as the integrin alpha 4 beta 1

Eukaryotic cells adhere to at least two different regions of the fibronectin molecule: a central domain present in all fibronectin isoforms, and the type III connecting segment domain (IIICS), the expression of which is controlled by complex alternative splicing of precursor mRNA. Using affinity chromatography on a matrix containing a synthetic peptide ligand (CS1) representing the strongest active site within the IIICS, we have isolated the human melanoma cell receptor recognizing this region of fibronectin. The receptor is a complex of two polypeptides with subunit molecular masses of 145 and 125 kDa. This heterodimeric structure resembles that of receptors for other extracellular matrix proteins. Immunological analysis with specific antibodies identified these polypeptides as the integrin subunits alpha 4 and beta 1. In addition, antifunctional monoclonal antibodies directed against either alpha 4 or beta 1, but not against other integrin subunits, were potent inhibitors of CS1-mediated melanoma cell spreading. Furthermore, when the function of the central cell-binding domain was blocked, anti-alpha 4 and anti-beta 1 antibodies abolished spreading of A375-M cells on fibronectin, indicating that alpha 4 beta 1 is an authentic fibronectin receptor. Taken together, these results identify the human fibronectin IIICS receptor as the integrin heterodimer alpha 4 beta 1.     

Regulation of maximal open probability is a separable function of Ca(v)beta subunit in L-type Ca2+ channel, dependent on NH2 terminus of alpha1C (Ca(v)1.2alpha)

beta subunits (Ca(v)beta) increase macroscopic currents of voltage-dependent Ca2+ channels (VDCC) by increasing surface expression and modulating their gating, causing a leftward shift in conductance-voltage (G-V) curve and increasing the maximal open probability, P(o,max). In L-type Ca(v)1.2 channels, the Ca(v)beta-induced increase in macroscopic current crucially depends on the initial segment of the cytosolic NH2 terminus (NT) of the Ca(v)1.2alpha (alpha1C) subunit. This segment, which we term the "NT inhibitory (NTI) module," potently inhibits long-NT (cardiac) isoform of alpha1C that features an initial segment of 46 amino acid residues (aa); removal of NTI module greatly increases macroscopic currents. It is not known whether an NTI module exists in the short-NT (smooth muscle/brain type) alpha(1C) isoform with a 16-aa initial segment. We addressed this question, and the molecular mechanism of NTI module action, by expressing subunits of Ca(v)1.2 in Xenopus oocytes. NT deletions and chimeras identified aa 1-20 of the long-NT as necessary and sufficient to perform NTI module functions. Coexpression of beta2b subunit reproducibly modulated function and surface expression of alpha1C, despite the presence of measurable amounts of an endogenous Ca(v)beta in Xenopus oocytes. Coexpressed beta2b increased surface expression of alpha1C approximately twofold (as demonstrated by two independent immunohistochemical methods), shifted the G-V curve by approximately 14 mV, and increased P(o,max) 2.8-3.8-fold. Neither the surface expression of the channel without Ca(v)beta nor beta2b-induced increase in surface expression or the shift in G-V curve depended on the presence of the NTI module. In contrast, the increase in P(o,max) was completely absent in the short-NT isoform and in mutants of long-NT alpha1C lacking the NTI module. We conclude that regulation of P(o,max) is a discrete, separable function of Ca(v)beta. In Ca(v)1.2, this action of Ca(v)beta depends on NT of alpha1C and is alpha1C isoform specific.     

Staggering of subunits in NMDAR channels

Functional N-methyl-D-aspartate receptors (NMDARs) are heteromultimers formed by NR1 and NR2 subunits. The M3 segment, as contributed by NR1, forms the core of the extracellular vestibule, including binding sites for channel blockers, and represents a critical molecular link between ligand binding and channel opening. Taking advantage of the substituted cysteine accessibility method along with channel block and multivalent coordination, we studied the contribution of the M3 segment in NR2C to the extracellular vestibule. We find that the M3 segment in NR2C, like that in NR1, contributes to the core of the extracellular vestibule. However, the M3 segments from the two subunits are staggered relative to each other in the vertical axis of the channel. Compared to NR1, homologous positions in NR2C, including those in the highly conserved SYTANLAAF motif, are located about four amino acids more externally. The staggering of subunits may represent a key structural feature underlying the distinct functional properties of NMDARs.     

Comparison of the NH2-terminal amino acid sequences of the four non-identical subunits of the NAD-linked hydrogenases from Nocardia opaca 1b and Alcaligenes eutrophus H16

The cytoplasmic, NAD-linked hydrogenase of the Gram-positive hydrogen-oxidizing bacterium Nocardia opaca 1b was compared with the analogous enzyme isolated from the Gram-negative bacterium Alcaligenes eutrophus H16. The hydrogenase of N. opaca 1b was purified by a new procedure applying chromatography on phenyl-Sepharose and DEAE-Sephacel with two columns in series. A homogeneous enzyme preparation with a specific activity of 74 mumol H2 oxidized.min-1.mg protein-1 and a yield of 32% was isolated. The A. eutrophus enzyme was purified as previously published. Both enzymes are tetrameric proteins composed of four non-identical subunits (alpha, beta, gamma, delta). The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the alpha, beta, gamma, and delta subunits of one organism were serologically related to the analogous subunits of the other organism. Among themselves, the four subunits do not have any serological relationship. The eight individual polypeptides were also compared with respect to the NH2-terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions. Strong sequence similarities exist between the analogous subunits isolated from the two bacteria. Within the established N-terminal sequences the similarities between both alpha, beta, gamma and delta subunits amount to 63%, 79%, 80% and 65%, respectively. No similarities exist between the different, non-analogous subunits alpha, beta, gamma and delta.