NH2-terminal Segments Research Articles

Regions of Melanocortin 2 (MC2) Receptor Accessory Protein Necessary for Dual Topology and MC2 Receptor Trafficking and Signaling

MRAP, melanocortin 2 (MC2) receptor accessory protein, is required for trafficking by the MC2 (ACTH) receptor. MRAP is a single transmembrane protein that forms highly unusual antiparallel homodimers. We used molecular complementation to ask where MRAP achieves dual topology. Fragments of yellow fluorescent protein (YFP) were fused to the NH2 or COOH terminus of MRAP such that YFP fluorescence could occur only in antiparallel homodimers; fluorescence was present in the endoplasmic reticulum. MRAP retained dual topology after deletion of most of the amino terminus. In contrast, deletion of residues 31-37, just NH2-terminal to the transmembrane domain, forced MRAP into a single Nexo/Ccyt orientation and blocked its ability to promote MC2 receptor trafficking and homodimerize. When the transmembrane domain of MRAP was replaced with the corresponding region from RAMP3, dual topology was retained but MRAP was inactive. Insertion of MRAP residues 29-37 conferred dual topology to RAMP3, normally in an Nexo/Ccyt orientation. When expressed with MRAPDelta1-30, MRAPDelta10-20, or MRAPDelta21-30, MC2 receptor was localized on the plasma membrane but unable to respond to ACTH. Residues 18-21 of MRAP were critical; MC2 receptor expressed with MRAP(18-21A) localized to the plasma membrane but did not bind 125I-ACTH or increase cAMP in response to ACTH. A newly identified MRAP homolog, MRAP2, lacks amino acids 18LDYI21 of MRAP and, like MRAP(18-21A), allows MC2 receptor trafficking but not signaling. MRAP2 with an LDYI insertion functions like MRAP. These results demonstrate that MRAP not only facilitates MC2 receptor trafficking but also allows properly localized receptor to bind ACTH and consequently signal.

Amino acids in the COOH-terminal region of the oxytocin receptor third intracellular domain are important for receptor function

Previously, residue K6.30 in the COOH-terminal region of the third intracellular domain (3iC) of the oxytocin (OT) receptor (OTR) was identified as important for receptor function leading to phospholipase C activation in both OTR and the vasopressin V(2) receptor (V(2)R) chimera V(2)ROTR3iC. Substitution of either A6.28K or V6.30K in wild-type V(2)R did not recapitulate the increase in phosphatidylinositide (PI) turnover observed in V(2)ROTR3iC. Hence, the role of K6.30 may be context-specific. Deletion of two NH(2)-terminal OTR3iC segments in the V(2)ROTR3iC chimera did not diminish vasopressin-stimulated PI turnover, whereas deletion of RVSSVKL (residues 6.19-6.25) reduced receptor expression. Deletion of this sequence in wild-type OTR reduced expression by 50% without affecting affinity for [(3)H]OT. This OTR mutant was unable to activate PI turnover or extracellular signal-regulated kinase 1/2 phosphorylation. The effects of alanine substitution for individual residues in RVSSVKL indicated differential importance for OTR function. The R6.19A substitution lost high-affinity sites for [(3)H]OT and the ability to stimulate PI turnover. Affinity for [(3)H]OT and membrane expression was not affected by any other substitutions. OTR-V6.20A and OTR-K6.24A mutants functioned as well as wild-type OTR, whereas OTR S6.21A, S6.22A, and V6.23A mutants exhibited impaired abilities to activate PI turnover (20-40% of OTR), and the OTR-L6.25A mutant exhibited constitutive activity. In conclusion, specific amino acids in the RVSSVKL segment in the COOH-terminal region of the third intracellular domain of OTR influence the ability of OTR to activate G protein-mediated actions.

Isolation of Cholesterol-requiring Mutant Chinese Hamster Ovary Cells with Defects in Cleavage of Sterol Regulatory Element-binding Proteins at Site 1

The synthesis and uptake of cholesterol requires transcription factors designated sterol regulatory element-binding proteins (SREBPs). SREBPs are bound to membranes in a hairpin orientation with their transcriptionally active NH2-terminal segments facing the cytosol. The NH2-terminal segments are released from membranes by two-step proteolysis initiated by site 1 protease (S1P), which cleaves in the luminal loop between two membrane-spanning segments. Next, site 2 protease (S2P) releases the NH2-terminal fragment of SREBP. The S2P gene was recently isolated by complementation cloning using Chinese hamster ovary cells that require cholesterol for growth, due to a mutation in the S2P gene. A similar approach cannot be used for S1P because all previous cholesterol auxotrophs manifest defects in S2P, which is encoded by a single copy gene. To circumvent this problem, in the current studies we transfected Chinese hamster ovary cells with the S2P cDNA, assuring multiple copies. We mutagenized the cells, selected for cholesterol auxotrophy, and identified two mutant cell lines (SRD-12A and -12B) that fail to cleave SREBPs at site 1. Complementation analysis demonstrated that the defects in both cell lines are recessive and noncomplementing, indicating a mutation in the same gene. These cells should now be useful for expression cloning of the sterol-regulated S1P gene.

Characterization of the Secondary Structure and Membrane Interaction of the Putative Membrane Anchor Domains of Prostaglandin I2Synthase and Cytochrome P450 2C1

Prostaglandin I2synthase (PGIS) produces prostaglandin I2(PGI2) which has opposite actions on platelet aggregatory and vasoconstrictive properties compared to thromboxane A2(TXA2) produced from the same substrate by another P450 enzyme, thromboxane A2synthase (TXAS). PGIS and TXAS have only 16% amino acid sequence identity. Hydropathy analysis suggests that the putative NH2-terminal membrane anchor domain of PGIS is similar to many other membrane-bound microsomal P450s, which are believed to be anchored by a single transmembrane segment, and thus different from the TXAS anchor, which appears to have two transmembrane segments. To characterize the membrane anchor function of the PGIS NH2-terminal region, we have used the peptidoliposome reconstitution assay to identify the membrane anchor segment in the PGIS NH2-terminal domain and compared it with the anchor segment of P450 2C1. Four peptides, mimicking putative NH2-terminal membrane anchor segments of PGIS and P450 2C1, containing residues 1–28 (PGIS-LP1 and P450 2C1-LP1) or residues 25–54 (PGIS-LP2 and P450 2C1-LP2), were synthesized and their ability to insert in a lipid bilayer was evaluated. The results indicated that both LP1 peptides of PGIS and P450 2C1 became bound to the lipid bilayer, whereas both LP2 peptides did not bind the lipid. The two LP1 peptides were further characterized as to their conformation using CD spectroscopy. Helical structure induced in these peptides by addition of trifluoroethanol, dodecylphosphocholine, or incorporation into liposomes indicated that these segments tend to adopt a helical structure in a hydrophobic environment and thus could function as membrane anchor segments. These results support the hypothesis that PGIS and TXAS interact with the endoplasmic reticulum membrane in different ways, in which the NH2-terminal anchor domain of PGIS, as with P450 2C1, appears to have a single transmembrane segment.

Cleavage Site for Sterol-regulated Protease Localized to a Leu-Ser Bond in the Lumenal Loop of Sterol Regulatory Element-binding Protein-2

A sterol-regulated protease initiates release of the NH2-terminal segments of sterol regulatory element-binding proteins (SREBPs) from cell membranes, thereby allowing them to enter the nucleus and to stimulate transcription of genes involved in the uptake and synthesis of cholesterol and fatty acids. Using SREBP-2 as a prototype, we here identify the site of sterol-regulated cleavage as the Leu522-Ser523 bond in the middle of the 31-residue hydrophilic loop that projects into the lumen of the endoplasmic reticulum and nuclear envelope. This site was identified through use of a vector encoding an SREBP-2/Ras fusion protein with a triple epitope tag that allowed immunoprecipitation of the cleaved COOH-terminal fragment. The NH2 terminus of this fragment was pinpointed by radiochemical sequencing after replacement of selected codons with methionine codons and labeling the cells with [35S]methionine. Alanine scanning mutagenesis revealed that only two amino acids are necessary for recognition by the sterol-regulated protease: 1) the leucine at the cleavage site (leucine 522), and 2) the arginine at the P4 position (arginine 519). These define a tetrapeptide sequence, RXXL, that is necessary for cleavage. Cleavage was not affected when the second transmembrane helix of SREBP-2 was replaced with the membrane-spanning region of the low density lipoprotein receptor, indicating that this sequence is not required for regulation. Glycosylation-site insertion experiments confirmed that leucine 522 is located in the lumen of the endoplasmic reticulum. We conclude that the sterol-regulated protease is a novel enzyme whose active site faces the lumen of the nuclear envelope, endoplasmic reticulum, or another membrane organelle to which the SREBPs may be transported before cleavage.

Molecular biology of the amiloride-sensitive epithelial Na+ channel.

The amiloride-sensitive epithelial Na+ channel is formed by the assembly of three homologous subunits, alpha, beta and gamma. The channel is characterized by its sensitivity to amiloride and to some amiloride derivatives, such as phenamil and benzamil, by its small unitary conductance (approximately 5 pS), by its high selectivity for lithium and sodium, and by its slow kinetics. The alpha-, beta-, and gamma-proteins share significant identity with degenerins, a family of proteins found in the mechanosensory neurons and interneurons of the nematode Caenorhabditis elegans. They are also homologous to FaNaCh, a protein from Helix aspersa nervous tissues, which corresponds to a neuronal ionotropic receptor for the Phe-Met-Arg-Phe-NH2 peptide. All these proteins contain a large extracellular loop, located between two transmembrane alpha-helices. The NH2 and COOH terminal segments are cytoplasmic and contain potential regulatory segments that are able to modulate the activity of the channel. Accordingly, in Liddle syndrome, in which patients develop a form of genetic hypertension, mutations within the cytoplasmic COOH terminal of the beta- and gamma-chains of the epithelial Na+ channel lead to a hyperactivity of the channel. Epithelial Na+ channel activity is tightly controlled by several distinct hormonal systems, including corticosteroids and vasopressin. In kidney and colon, aldosterone is the major sodium-retaining hormone, acting by stimulation of Na+ reabsorption through the epithelium. In the distal colon from steroid-treated animals, a large increase in beta- and gamma-subunit transcription is observed, whereas the alpha-subunit remains constitutively transcribed. In kidney, RNA levels of the three subunits are not altered by aldosterone, suggesting that other mechanisms control Na+ channel activity in that tissue. In lung, the glucocorticoids are positive regulators of the channel activity, especially around birth, and act via an increased transcription of the three subunits.

The Amino Terminus of Apolipoprotein B Is Necessary but Not Sufficient for Microsomal Triglyceride Transfer Protein Responsiveness

Human apolipoprotein (apo) B mediates the formation of neutral lipid-containing lipoproteins in the liver and intestine. The association of apoB with lipid is thought to be promoted by the microsomal triglyceride transfer protein complex. We have reconstituted lipoprotein assembly in an insect cell line that normally does not support this process. Expression of human microsomal triglyceride transfer protein (MTP) and apolipoprotein B48 (apoB48) together enabled Sf-21 insect cells to secrete approximately 60-fold more lipoprotein-associated triacylglycerol than control cells. This dramatic effect demonstrates that effective partitioning of triacylglycerol into the secretory pathway requires an endoplasmic reticulum-associated neutral lipid transporter (provided by MTP) and an apolipoprotein to shuttle the lipid through the pathway. Expression of the human apoB48 gene in insect cells resulted in secretion of the protein product. Including both MTP subunits with apoB48 and oleic acid specifically increased apoB48 secretion 8-fold over individual subunits alone. To assess whether specific regions of apoB are necessary for MTP responsiveness, nine apoB segments were expressed. These included NH2-terminal segments as well as internal and COOH-terminal regions of apoB fused with a heterologous signal sequence. ApoB segments containing the NH2-terminal 17% of the protein were secreted and responded to MTP activity; however, a segment containing only the NH2-terminal 17% of the protein was not significantly responsive to MTP. Segments lacking the NH2 terminus were not MTP-responsive, and five of six of these proteins were trapped intracellularly but, in certain cases, could be rescued by fusion to apoB17. These results suggest that the NH2 terminus of apoB is necessary but not sufficient for MTP responsiveness.

Cloning and characterization of a Kv1.5 delayed rectifier K+ channel from vascular and visceral smooth muscles

We have cloned and characterized the expression of a Kv1.5 K+ channel (cKv1.5) from canine colonic smooth muscle. The amino acid sequence displayed a high level of identity to other K+ channels of the Kv1.5 class in the core region between transmembrane segments S1-S6; however, identity decreased to between 74 and 82% in the NH2 and COOH terminal segments, suggesting that cKv1.5 is a distinct isoform of the Kv1.5 class. Functional expression of cKv1.5 in oocytes demonstrated a channel highly selective for K+, which activates in a voltage-dependent manner on depolarization to membrane potentials positive to -40 mV. At room temperature the channel showed fast activation (time to half of peak current, 5.5 ms) and slow inactivation that was incomplete after 20-s depolarizations. Single channel analysis of the channel expressed in oocytes displayed a linear current-voltage curve and had a slope conductance of 9.8 +/- 1.1 pS. Northern blot analysis demonstrated differential expression of cKv1.5 in smooth muscles of the gastrointestinal tract and abundant expression in several vascular smooth muscles. We propose that cKv1.5 represents a component of the delayed rectifier current in both vascular and visceral smooth muscles.

Liver microsomes contain two distinct NADPH-monooxygenases with NH 2-terminal segments homologous to the flavin containing NADPH monooxygenase of [formula omitted][formula omitted

Two NADPH-reductase preparations (FAD-containing monooxygenases) were isolated from rabbit liver microsomes, referred to as form 1 and form 2. Purification was achieved by means of anion-exchange, cation-exchange and hydroxylapatite chromatography in the presence of cholate and Nonidet P-40. Affinity chromatography on 2′, 5′-ADP Sepharose was used to increase the purity and to concentrate the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, form 1 exhibited a single band at Mr 58,500 and form 2 at Mr 58,000. The NH 2- terminus of form 1 is blocked, whereas the NH 2-terminus of form 2 is homologous to the NADPH-p-hydroxybenzoate hydrolase from Pseudomonas fluorescens . The latter and the form 2 enzyme share 11 identical residues in the NH 2-terminal segment of 15 residues. Both forms were subjected to tryptic cleavages and peptide mapping. Sequence analysis of the peptides obtained indicated that forms 1 and 2 are similar but not identical proteins. A tryptic peptide, homologous to residues 3 to 32 of form 2 enzyme was isolated from the form 1 protein. This segment has 24 residues that are identical to the form 2 and contains the consensus sequence Gly-X-Gly-X-X-Gly, found in most FAD binding proteins. These results indicate that the NADPH-monooxygenase system consists of at least two distinct proteins representing different gene products.

On the membrane topology of vertebrate cytochrome P-450 proteins.

Hydropathy profiles of 34 aligned cytochrome P-450 sequences were compared to identify potential transmembrane segments. Eleven regions with the potential to cross a membrane in at least some P-450 sequences were detected. The known sidedness of several residues and peptides was used to eliminate some of these regions from consideration. Further arguments based on the location and orientation of the heme relative to the membrane excluded others. This process of elimination was continued until only two regions remained. These two segments, present in the first 66 amino acids of the P-450 NH2 termini, are proposed as the only transmembrane peptides of vertebrate microsomal P-450s. Mitochondrial P-450s may have a different membrane association. The three-dimensional structure of cytochrome P-450cam was examined for the location of conserved charged residues. These residues occurred mainly on the opposite surface from the substrate-binding site and along the edges of the flat triangular P-450cam. A model is proposed for vertebrate microsomal P-450s that is similar to P-450cam. The substrate-binding site faces the membrane, the heme is parallel to the membrane surface, and two NH2-terminal transmembrane segments anchor the protein to the bilayer.

Structural similarity between legumin and vicilin storage proteins from legumes.

The primary structures for several members of both the vicilin and legumin families of storage proteins were examined using a computer routine based on amino acid physical characteristics. The comparison algorithm revealed that sequences from the two families could be aligned and share a number of predicted secondary structural features. The COOH-terminal half of the subunits in both families displayed a highly conserved core region that was largely hydrophobic and in which a high proportion of the residues were predicted to be in beta-sheet conformations. The central region of the molecules which contained mixed areas of predicted helical and sheet conformations showed more variability in residue selection than the COOH-terminal regions. The NH2-terminal segments of subunits from the two different families could not be aligned though they characteristically had a high proportion of residues predicted to be in helical conformations. The feature which most clearly distinguished subunits between the two families was an inserted span in the legumin group with a high proportion of acidic amino acids located between the central and COOH-terminal domains. Residues in this insertion were predicted to exist mainly in helical conformation. Since considerable size variation occurs in this area amongst the legumin subunits, alterations in this region may have a minimal detrimental effect on the structure of the proteins.

Characterization of the procollagen IV cleavage products produced by a specific tumor collagenase.

The specific mammalian collagenase isolated from cultures of metastatic mouse PMT sarcoma cells cleaves murine procollagen IV into two segments, of approximate mass ratio 3:1. These fragments were separated by velocity sedimentation, visualized by electron microscopy, and analyzed. The longer COOH-terminal procollagen segment has a 270-nm collagenous portion with a knob at one end. This knob consists of the three previously identified, noncollagenous carboxyl propeptides, of approximately 30,000 daltons each. These carboxyl propeptides are chain-specific, and the three chains of each segment have the same amino to carboxyl orientation. The collagenase cuts through all three chains at one site, and the three-component chains of both the longer COOH-terminal procollagen segment and the shorter NH2-terminal procollagen segment are linked by interchain disulfide bridges. The enzyme cuts off the same COOH-terminal procollagen segment from procollagen IV monomers and tetramers, and the flexibility of this segment is similar to that of interstitial collagen helices. The amino ends of the NH2-terminal procollagen segments derived from tetramers remain joined as the 32-nm long "7 S collagen" junctional complex, and the remaining 89-nm long projecting threads are significantly more flexible than the COOH-terminal procollagen segment. The electrophoretic mobilities of the enzyme cleavage products are consistent with a heterotrimeric composition of this procollagen IV.

Rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis.

We have developed a rapid and general technique for purification of a protein encoded by a cistron contained in a recombinant DNA clone. The technique consists of fusing the target cistron DNA in the correct reading frame to a marker cistron via a piece of DNA that codes for a linker peptide. The target cistron in the example presented here is the replication initiator cistron of the plasmid R6K. The linker is a DNA fragment encoding 60 amino acids from the triple helical region of chicken pro alpha-2 collagen, and the marker cistron encodes the beta-galactosidase protein of Escherichia coli. The tripartite hybrid protein was rapidly purified by selective binding to and elution from a beta-galactosidase specific-affinity column. The hybrid protein was then digested with a purified microbial collagenase to cleave the linker, and high-pressure liquid chromatography allowed the rapid isolation of the target protein from the marker protein. Using this technique, we have purified the highly labile R6K replication initiator to homogeneity, and we have resolved the protein into NH2-terminal and COOH-terminal segments. We have further shown, by in vitro binding, that the COOH-terminal segment has at least one DNA-binding domain. The domain binds to the same restriction fragments of the R6K chromosome as the intact or beta-galactosidase-tagged initiator protein.

Mitochondrial aspartate aminotransferase 27/32-410. Partially active enzyme derivative produced by limited proteolytic cleavage of native enzyme.

Native mitochondrial aspartate aminotransferase (AATase) is cleaved selectively by trypsin at the peptide bonds after Arg 26 or after Lys 31 yielding two shortened enzyme derivatives, AATase 27-410, and AATase 32-410. Recent x-ray crystallographic determination of the spatial structure of AATase has shown that the NH2-terminal segments of the two polypeptide chains of this dimeric enzyme pass in front of the active site clefts and form two separate junctions with the neighboring subunit which are not contiguous with the main subunit interface (Eichele, G., Ford, G. C., Glor, M., Jansonius, J. N., Mavrides, C., and Christen, P. (1979) J. Mol. Biol. 133, 161-180). The peptide bonds cleaved by trypsin are situated in the following stretch of the polypeptide chain which runs in exposed position on the surface of the subunit. The split-off peptide is lost during gel filtration. The molecular activity of AATase 27/32-410 (a mixture of about equal amounts of the two not readily separable derivatives) is about 3% of that of the native enzyme. In contrast, the K'm values for aspartate and 2-oxoglutarate are unchanged, indicating an unaltered geometry of the substrate binding site. A substantially diminished syncatalytic response of the reactivity of Cys 166 toward 5,5'-dithiobis-(2-nitrobenzoate) suggests that the decrease in catalytic activity is due to an interference with the syncatalytic conformational dynamics observed previously in AATase (Gehring, H., and Christen, P. (1978) J. Biol. Chem. 253, 3158-3163). Consonant with a role of the NH2-terminal segment in propagating the syncatalytic conformational rearrangements the rate of the tryptic cleavage is retarded 4-fold in the presence of the transaminating substrate pair aspartate and oxalacetate.

Hepatic microsomal epoxide hydrase. Chemical evidence for a single polypeptide chain.

Highly purified hepatic microsomal epoxide hydrase, which had been purified in the presence of proteolytic enzyme inhibitors, was subjected to carboxypeptidase Y digestion, automated Edman degradation, and carbohydrate analysis. Carboxypeptidase Y digestion resulted in the near stoichiometric release of leucine, the COOH-terminal amino acid. Automated Edman degradation permitted the identification of the first 20 amino acid residues of epoxide hydrase. Methionine was identified as the NH2-terminal residue. The NH2-terminal region of epoxide hydrase is similar in hydrophobicity to the NH2-terminal precursor segments of several secretory proteins and the NH2-terminal regions of several microsomal cytochromes P-450. Carbohydrate analyses of the enzyme revealed the presence of 0.5 to 1.0 mol of mannose/50,000 g of protein. These results provide evidence for the presence of a single polypeptide chain in our purified enzyme preparations and suggest that there may be only one enzymic form of epoxide hydrase in microsomes from phenobarbital-treated rats.

Amino acid sequence of the NH2-terminal extra piece segments of the precursors of mouse immunoglobulin lambda1-type and kappa-type light chains.

The mRNA molecules coding for mouse immunoglobulin light (L) chains direct the cell-free synthesis of precursors in which extra peptide segments precede the amino termini of the mature proteins. The results of amino acid sequence analyses of two precursors labeled with 20 radioactive amino acids enabled unambiguous determination of the complete primary structure of the extra piece segments. The complete sequences (and sizes) of the NH2-terminal extra pieces are: in MOPC-104E lambda1 L-chain precursor, Met-Ala-Trp-Ile-Ser-Leu-Ile-Leu-Ser-Leu-Leu-Ala-Leu-Ser-Ser-Gly-Ala-Ile-Ser (19 residues); in MOPC-41 kappa L-chain precursor, Met-Asp-Met-Arg-Ala-Pro-Ala-Gln-Ile-Phe-Gly-Phe-Leu-Leu-Leu-Leu-Phe-Pro-Gly-Thr-Arg-Cys (22 residues). The extra pieces in the precursors of MOPC-104E (lambda1), MOPC-41 (kappa), and MOPC-321 (kappa) L-chains differ extensively from each other in their amino acid sequence (65-73%). In addition to this sequence heterogeneity, the extra pieces are characterized by a high percentage of hydrophobic residues: 69% in the MOPC-104E lambda1 L-chain precursor (this report), 73-75% in the kappa L-chain precursors [Schechter, I. & Burstein, Y. (1976) Proc, Natl. Acad. Sci. USA 73, 3273-3277]. The marked hydrophobicity of the extra piece suggests that it may favor interaction of the precursor with cell membranes, in a manner similar to the function of the "hydrophobic domain" of membrane-bound proteins. We propose two possible targets for interaction: (i) the endoplasmic membranes, where the NH2-terminal extra piece is cleaved from the precursor to yield mature protein destined for secretion; (ii) the cell surface membrane, where the intact precursor is anchored by virtue of the hydrophobic extra piece to serve as the antigen-recognizing receptor.

Multiple Hemoglobins of Catostomid Fish: I. ISOLATION AND CHARACTERIZATION OF THE ISOHEMOGLOBINS FROM CATOSTOMUS CLARKII

Abstract Multiple hemoglobins from a fish (Catostomus clarkii) were separated and characterized by electrophoresis, column chromatography, ultracentrifugation, end group analysis, and oxygen equilibria studies. The hemoglobins were all tetramers of α and β chains. Two different α chains were detected in unfractionated hemoglobins. Two major β chains were isolated from the hemoglobin components which migrated electrophoretically toward the anode at pH 8.6. Two additional β chains, which were constituents of hemoglobins that migrated toward the cathode, could not be separated from the α chains. The β chains of the anodal and cathodal hemoglobin components of an individual fish were subjected to amino end group analyses with a sequenator. The partial sequences of the βa and βb chains of the anodal components differed by l10%, a finding consistent with the presence of mixed α2βaβb tetramers among the hemoglobins. The NH2-terminal segments of the two β chains of the cathodal hemoglobins were also similar to each other, but differed from the β chains in the anodal components by the same order of magnitude (40 to 45%) as the α and β chains of mammalian hemoglobins. Unlike the anodal components, the cathodal hemoglobins showed no Bohr effect. The chemical groups responsible for most of the Bohr effect were absent in the α and β chains of the cathodal components. These observations were used to propose a possible role for the cathodal hemoglobins in helping to maintain intrastream niche segregation of sympatric catostomid species.