Hemoglobins, XXX. The amino acid sequence of the monomeric hemoglobin from Lampetra fluviatilis (author's transl)]

The primary structure of the two main hemoglobin components of the river lamprey Lampetra fluviatilis was established. Having a chain length of 149 amino acids the molecular weight of the globin is 16272. The two main components differ only by an N-formyl residue at the N-terminal proline. The sequence was compared with those of two related species. The heterogeneity of Cyclostomata hemoglobins and their possible origin was discussed.     

The amino acid sequence of hemoglobin III from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated M_m of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem. 10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum.     

The amino acid sequence of hemoglobin III from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin III from the gill of the symbiont-harboring clamLucina pectinata consists of 152 amino acid residues, has a calculated M_m of 18,068, including heme, and has N-acetyl-serine as the N-terminal residue. Based on the alignment of its sequence with other vertebrate and nonvertebrate globins, it retains the invariant residues Phe45 at position CD1 and His98 at the proximal position F8, as well as the highly conserved Trp16 and Pro39 at positions A12 and C2, respectively. The most likely candidate for the distal residue at position E7 is Gln66.Lucina hemoglobin III shares 95 identical residues with hemoglobin II (J. D. Hockenhull-Johnsonet al., J. Prot. Chem. 10, 609–622, 1991), including Tyr at position B10, which has been shown to be capable of entering the distal heme cavity and placing its hydroxyl group within a 2.8 Å of the water molecule occupying the distal ligand position, by modeling the hemoglobin II sequence using the crystal structure of sperm whale metmyoglobin. The amino acid sequences of the twoLucina globins are compared in detail with the known sequences of mollusc globins, including seven cytoplasmic and 11 intracellular globins. Relative to 75% homology between the twoLucina globins (counting identical and conserved residues), both sequences have percent homology scores ranging from 36–49% when compared to the two groups of mollusc globins. The highest homology appears to exist between theLucina globins and the cytoplasmic hemoglobin ofBusycon canaliculatum.     

Myxinidin, A Novel Antimicrobial Peptide from the Epidermal Mucus of Hagfish, Myxine glutinosa L.

Fish epidermal mucus contains innate immune components that provide a first line of defense against various infectious pathogens. This study reports the bioassay-guided fractionation and characterization of a novel antimicrobial peptide, myxinidin, from the acidic epidermal mucus extract of hagfish (Myxine glutinosa L.). Edman sequencing and mass spectrometry revealed that myxinidin consists of 12 amino acids and has a molecular mass of 1,327.68 Da. Myxinidin showed activity against a broad range of bacteria and yeast pathogens at minimum bactericidal concentration (MBC) ranging from 1.0 to 10.0 μg/mL. Screened pathogens, Salmonella enterica serovar Typhimurium C610, Escherichia coli D31, Aeromonas salmonicida A449, Yersinia ruckeri 96-4, and Listonella anguillarum 02-11 were found to be highly sensitive to myxinidin at the MBC of 1.0–2.5 μg/mL; Staphylococcus epidermis C621 and yeast (Candida albicans C627) had an MBC of 10.0 μg/mL. The antimicrobial activity of myxinidin was found to be two to 16 times more active than a potent fish-derived antimicrobial peptide, pleurocidin (NRC-17), against most of the screened pathogens. The microbicidal activity of myxinidin was retained in the presence of sodium chloride (NaCl) at concentrations up to 0.3 M and had no hemolytic activity against mammalian red blood cells. These results suggest that myxinidin may have potential applications in fish and human therapeutics.     

Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric (α ₂ β ₂) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3′ and 5′ parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.     

Protozoan hemoglobin from Tetrahymena pyriformis. Isolation, characterization, and amino acid sequence

A hemoprotein that can be defined as hemoglobin based on oxygen binding was isolated from Tetrahymena pyriformis. The protein exists in monomeric form and is separated into four fractions (Ia, Ib, IIa, and IIb) on a CM-cellulose column. From examinations of the absorption spectra and the N-terminal sequence, fractions Ia and Ib were assigned to the oxy-form and its met-form, respectively, of the one protein, while IIa and IIb corresponded to those of the other one. The complete amino acid sequence was therefore determined of fractions I and II. The I was composed of 121 amino acid residues, with the N-terminal serine being blocked. The II, on the other hand, consisted of 119 amino acid residues, its sequence being exactly identical to that of the third residue, lysine, to the C-terminal lysine of the fraction I. Although the genomic multiplicity cannot be ruled out completely, we have concluded that fraction II is a degradation product of the fraction I by endogeneous proteases. The amino acid sequence of T. pyriformis hemoglobin is very unique and showed no notable degree of similarity with the other hemoglobins sequenced so far, but it was found to be 33.9% identical with Paramecium caudatum hemoglobin by a maximal alignment.     

The amino acid sequence of hemoglobin II from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin II from the gill of the clamLucina pectinata consists of 150 amino acid residues, has a calculatedM _m of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J.,J. Biol. Chem. 265, 16054–16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of theLucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 Å of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 inLucina Hb II.     

Two-domain hemoglobin from the blood clam,Barbatia lima. The cDNA-derived amino acid sequence

The blood clam,Barbatia lima, from Kochi, Japan, expresses a tetrameric ( _{2 2}) and a polymeric hemoglobin in erythrocytes. The latter hemoglobin is composed of unusual 34-kDa hemoglobin with a two-domain structure, and its molecular mass (about 430 kDa) is exceptionally large for an intracellular hemoglobin. The 3 and 5 parts of the cDNA ofB. lima two-domain globin have been amplified separately by polymerase chain reaction and the complete nucleotide sequence of 1147 bp was determined. The open reading frame is 930 nucleotides in length and encodes a protein with 309 amino acid residues, of which 73 amino acids were identified directly by protein sequencing. The mature protein begins with the acetylated Ser, and thus the N-terminus Met is cleaved. The molecular mass for the protein was calculated to be 35,244 Da. The cDNA-derived amino acid sequence ofB. lima two-domain globin shows 89% homology with that of two-domain globin fromB. reeveana, a North American species. The sequence homology between the two domains is 75%, suggesting that the two-domain globin resulted from the gene duplication of an ancestral 17-kDa globin.     

The amino acid sequence of hemoglobin II from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin II from the gill of the clamLucina pectinata consists of 150 amino acid residues, has a calculatedM _m of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J.,J. Biol. Chem. 265, 16054–16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of theLucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 Å of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 inLucina Hb II.     

The amino acid sequence of plastocyanin from Solanum tuberosum L. (potato)

The amino acid sequence of plastocyanin from potato was determined. It consists of a single polypeptide chain of 99 residues, of molecular weight 10332. The sequence was determined by using a Beckman 890c sequencer and by dansyl-Edman analysis of peptides derived from purified CNBr fragments. The sequence shows considerable similarity with that of Chlorella fusca, and also with the C-terminal region of bacterial azurins.     

The complete amino acid sequence of giant multisubunit hemoglobin from the polychaete Tylorrhynchus heterochaetus

The extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus is a "giant," multisubunit protein with an apparent molecular weight of 3.37 X 10(6), and consists of two types of subunits: a "monomeric" chain (chain I) and a disulfide-bonded "trimer" of chains IIA, IIB, and IIC. We reported the amino acid sequences of chains I, IIB, and IIC previously (Suzuki, T., Yasunaga, H., Furukohri, T., Nakamura, K., and Gotoh, T. (1985) J. Biol. Chem. 260, 11481-11487). The sequence of chain IIA has now been determined. Chain IIA consists of 146 amino acid residues with a heme group and has a molecular weight of 17,236. All of the constituent chains of Tylorrhynchus hemoglobin appear to be homologous with those of vertebrate hemoglobins and contain heme. Distal (E7) His, distal (E11) Val, and proximal (F8) His are all conserved in the four chains. Phylogenetically, chain IIA appears more closely related to the monomeric chain I than to either of the other "trimeric" chains IIB and IIC. This is the first giant extracellular hemoglobin to be sequenced completely.     

The amino acid sequence of plastocyanin from spinach. (Spinacia oleracea L.).

The amino acid sequence of spinach (Spinacia oleracea L.) plastocyanin was determined. It consists of a single polypeptide chain of 99 residues and has a sequence molecular weight of 10415. The sequence was determined by using a Beckman 890C automatic sequencer and by the dansyl--phenyl isothiocyanate analysis of peptides obtained by the enzymic digestion of purified CNBr fragments. Overlap through the two methionine residues was not shown. Sedimentation equilibrium in the ultracentrifuge gave a molecular weight for spinach plastocyanin of about 9000, in contrast with the value of 21000 reported previously by Katoh et al. (1962).     

Purification of chitinolytic protein from Rehmannia glutinosa showing N-terminal amino acid sequence similarity to thaumatin-like proteins.

We have purified a 21-kDa protein, designated as P1, from Rehmannia glutinosa to homogeneity by ammonium sulfate precipitation, anion exchange chromatography, hydrophobic interaction chromatography, and preparative native PAGE. The purified P1 had chitin degradation activity. The N-terminal amino acid sequence of P1 indicated that it is very similar to those of thaumatin and other reported thaumatin-like proteins.     

The amino acid sequence of component 7c, a type II intermediate-filament protein from wool.

Component 7c is one of the four homologous type II intermediate-filament proteins that, by association with the complementary type I proteins, form the microfibrils or intermediate filaments in wool. Component 7c was isolated as the S-carboxymethyl derivative from Merino wool and its amino acid sequence was determined by manual and automatic sequencing of peptides produced by chemical and enzymic cleavage reactions. It is an N-terminally blocked molecule of 491 residues and Mr (not including the blocking group) of 55,600; the nature of the blocking group has not been determined. The predicted secondary structure shows that component 7c conforms to the now accepted pattern for intermediate-filament proteins in having a central rod-like region of approximately 310 residues of coiled-coil alpha-helix flanked by non-helical N-and C-terminal regions. The central region is divided by three non-coiled-coil linking segments into four helical segments 1A, 1B, 2A and 2B. The N-and C-terminal non-helical segments are 109 and 71 residues respectively and are rich in cysteine. Details of procedures use in determining the sequence of component 7c have been deposited as a Supplementary Publication SUP 50152 (65 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257,5. The information comprises: (1) details of chemical and enzymic methods used for cleavage of component 7c, peptides CN1, CN2 and CN3, and various other peptides, (2) details of the procedures used for the fractionation and purification of peptides from (1), including Figures showing the elution profiles from the chromatographic steps used, (3) details of methods used to determine the C-terminal sequence of peptide CN3, and (4) detailed evidence to justify a number of corrections to the previously published sequence.