The structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. The amino acid sequence of the second largest cyanogen bromide fragment

Fourteen fragments have been isolated from hemocyanin component II of Limulus polyphemus by cleavage with CNBr. The amino acid sequence of the largest fragment, CNBr Ia has been reported (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749). The amino acid sequence of the 12 smaller fragments is reported in an accompanying paper (Moore, M. D., Behrens, P. Q., and Riggs, A. F. (1985) J. Biol. Chem. 261, 10511-10519). We have determined the amino acid sequence of the second largest fragment, CNBr Ib. The fragment contains 142 residues and has a molecular weight of 16,095.     

The structure of hemocyanin II from the horseshoe crab, Limulus polyphemus. The amino acid sequences of the smaller cyanogen bromide fragments

Fourteen fragments have been isolated from hemocyanin component II of Limulus polyphemus by cleavage with CNBr. The amino acid sequences of the two largest fragments, CNBr Ia and Ib, have been determined (Yokota, E., and Riggs, A. F. (1984) J. Biol. Chem. 259, 4739-4749; Behrens, P. Q., Nakashima, H., Yokota, E., and Riggs, A. F. (1986) J. Biol. Chem. 261, 10520-10525). We have determined the amino acid sequence of the remaining 12 smaller fragments.     

Amino acid sequence of the monomer subunit of the extracellular hemoglobin of Lumbricus terrestris

The giant extracellular hemoglobin (3,800 kDa) of the oligochaete Lumbricus terrestris consists of four subunits: a monomer (chain I), two subunits each of about 35 kDa (chains V and VI), and a disulfide-bonded trimer (50 kDa) of chains II, III, and IV. The complete amino acid sequence of chain I was determined: it consists of 142 amino acid residues and has a molecular weight of 16,750 including a heme group. Fifty-nine residues (42%) were found to be identical with those in the corresponding positions in Lumbricus chain II (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 257, 9005-9015); 45 (32%), 56 (40%), 44 (31%), and 45 (32%) residues were found to be in identical positions in the sequences of chains I, IIA, IIB, and IIC, respectively, of Tylorrhynchus heterochaetus hemoglobin (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267). When the sequences of all six annelid chains are compared, 18 invariant residues are found in the first 104 residues of the molecule; very little homology exists among the annelid chains in the carboxyl-terminal 38-residue region. Nine of the 18 invariant residues are also found in the human beta-globin chain.     

The amino acid sequences of chains a, b, and c that form the trimer subunit of the extracellular hemoglobin from Lumbricus terrestris

The extracellular hemoglobin of Lumbricus terrestris comprises four major heme-containing chains, a, b, c, and d in equal proportions. We have determined the amino acid sequences of chains a, b, and c which form a disulfide-linked trimer. Chains a, b, and c have 151, 145, and 153 residues and calculated molecular weights of 17,525, 16,254, and 17,289, respectively. The sequence of chain b, reported previously (Garlick, R. L., and Riggs, A. F. (1982) J. Biol. Chem. 287, 9005-9015) has been completely redetermined and found to contain 12 fewer residues than originally reported. Chains a and c both contain unusual, highly polar NH2-terminal extensions of 7 residues before the A helix. These segments must be close together because they are joined by a disulfide bond. We suggest that this structure, with seven negatively charged groups, may be part of a functionally important Ca2+-binding site in the trimer. Comparison of the sequences of chains a, b, and c with those of chain d (Shishikura, F., Snow, J. W., Gotoh, T., Vinogradov, S. N., and Walz, D. A. (1987) J. Biol. Chem. 262, 3123-3131) and the four chains of the hemoglobin of Tylorrhynchus heterochaetus (Suzuki, T., and Gotoh, T. (1986) J. Biol. Chem. 261, 9257-9267) shows that the number and positions of the cysteinyl residues are all conserved. This suggests that the extracellular hemoglobins from both the Oligochaeta and Polychaeta have the same number and configuration of disulfide bonds within the molecule. Phylogenetic analysis suggests that gene duplication first generated an intracellular hemoglobin branch and an extracellular hemoglobin branch. DNA coding for a signal peptide would have been acquired by the extracellular globin gene after this event. At least two further gene duplications are required to account for the present four polypeptide chains.     

Isolation and characterization of four heparin-binding cyanogen bromide peptides of human plasma apolipoprotein B

Apolipoprotein B-100 (apoB-100) is the major protein constituent of human plasma low-density lipoproteins (LDL). On the basis of its amino acid sequence [Chen, S.-H., Yang, C.-Y., Chen, P.-F., Setzer, D., Tanimura, M., Li, W.-H., Gotto, A. M., Jr., & Chan, L. (1986) J. Biol. Chem. 261, 12918-12921], apo B-100 is one of the largest monomeric proteins known with a calculated molecular weight of 512937. Heparin binds to the LDL surface by interacting with positively charged amino acid residues of apoB-100, forming soluble complexes in the absence of divalent metals and insoluble complexes in their presence. The purpose of this study was to isolate and characterize the heparin-binding domain(s) of apoB-100. Human plasma LDL were fragmented with cyanogen bromide (CNBr). After delipidation and reduction-carboxymethylation, the CNBr peptides were fractionated by sequential chromatography on DEAE-Sephacel, Mono S, and high reactive heparin (HRH) AffiGel-10; HRH was purified by chromatography of crude bovine lung heparin on LDL AffiGel-10. Heparin-binding peptides were further purified by reverse-phase high-performance liquid chromatography. Heparin-binding activity was monitored by a dot-blot assay with 125I-HRH. The amino-terminal sequences of four CNBr heparin-binding peptides (CNBr-I-IV) were determined. CNBr-I-IV correspond to residues 2016-2151, 3109-3240, 3308-3394, and 3570-3719, respectively, of the amino acid sequence of apoB-100. Each CNBr peptide contains a domain(s) of basic amino acid residues which we suggest accounts for their heparin-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the active-site serine in human lecithin: cholesterol acyltransferase

Purified human lecithin:cholesterol acyltransferase (LCAT) was covalently labeled by [3H]diisopropylflourophosphate with concomitant loss of enzymatic activity (M. Jauhiainen and P.J. Dolphin (1986) J. Biol. Chem. 261, 7023-7043). Some 60% of the enzyme was labeled in 1 h. Cyanogen bromide (CNBr) cleavage of the labeled, reduced, and carboxymethylated protein, followed by gel permeation chromatography yielded a 5- to 6-kDa peptide (LCAT CNBr-III) containing at least 60-70% of the incorporated label. Comparison of the amino acid composition of LCAT CNBr-III with that of the CNBr peptides predicted from the LCAT sequence (J. McLean et al. (1986) Proc. Natl. Acad. Sci. USA 83, 2335-2339) indicates that LCAT CNBr-III is peptide 168-220. In 22 cycles of automated Edman degradation of CNBr-III a radioactive derivative was only observed at cycle 14, and of the predicted CNBr fragments only peptide 168-220 contains a serine at position 14 from the amino terminus. Tryptic peptides predicted from the sequence should contain Ser181 at positions 22 and 23 from the N-terminus of fragments 160-199 and 159-199, respectively. On the other hand, Ser216 should be in position 15 from the N-terminus in fragment 202-238. Radiolabel sequencing of the tryptic digest of [3H]diisopropylphosphate-LCAT resulted in recovery of radioactivity in cycles 22 and 23, whereas cycle 15 yielded negligible radioactivity. These results establish that Ser181 is the major active site serine in human LCAT.     

Structure of the noncompetitive antagonist-binding site of the Torpedo nicotinic acetylcholine receptor. [3H]meproadifen mustard reacts selectively with alpha-subunit Glu-262.

[3H]Meproadifen mustard, an affinity label for the noncompetitive antagonist site of the nicotinic acetylcholine receptor (AChR), specifically alkylates the AChR alpha-subunit when the acetylcholine-binding sites are occupied by agonist (Dreyer, E. B., Hasan, F., Cohen, S. G., and Cohen, J. B. (1986) J. Biol. Chem. 261, 13727-13734). In this report, we identify the site of alkylation within the alpha-subunit as Glu-262. AChR-rich membranes from Torpedo californica electric organ were reacted with [3H]meproadifen mustard in the presence of carbamylcholine and in the absence or presence of nonradioactive meproadifen to define specific alkylation of the noncompetitive antagonist site. Alkylated alpha-subunits were isolated and subjected to chemical or enzymatic cleavage. When digests with CNBr in 70% trifluoroacetic acid or 70% formic acid were fractionated by gel filtration high performance liquid chromatography (HPLC), specifically labeled material was recovered in the void volume fractions. Based upon NH2-terminal sequence analysis, for both digests, the void volume fractions contained a fragment beginning at Gln-208 before the M1 hydrophobic sequence, whereas the sample from the digest in trifluoroacetic acid also contained as a primary sequence a fragment beginning at Thr-244 and extending through the M2 hydrophobic sequence. Sequence analysis revealed no release of 3H for the sample from digestion in formic acid, whereas for the trifluoroacetic acid digest, there was specific release of 3H in cycle 19, which would correspond to Glu-262. This site of alkylation was confirmed by isolation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase HPLC of a specifically labeled fragment from an endoproteinase Lys-C digest of the alkylated alpha-subunit. NH2-terminal amino acid sequencing revealed release of 3H at cycle 20 from a fragment beginning at Met-243 and extending into the M3 hydrophobic sequence. Because [3H]meproadifen mustard contains, as its reactive group, a positively charged quaternary aziridinium ion, Glu-262 of the alpha-subunit is identified as a contributor to the cation-binding domain of the noncompetitive antagonist-binding site and thus of the ion channel.     

Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Nucleotide sequence of the hdc gene and the corresponding amino acid sequence

The nucleotide sequence of a 1.3-kilobase NaeI fragment from Morganella morganii AM-15 that contains the gene for histidine decarboxylase has been determined. The gene was initially identified among total chromosomal digests using a mixed sequence oligonucleotide probe corresponding to amino acids 11-16 of histidine decarboxylase and then cloned on a 5.5-kilobase PstI fragment. The structural gene contains 1131 nucleotides and encodes 377 amino acids with the sequence: (sequence: in text). The independently determined NH2-terminal sequence of this enzyme (Tanase, S., Guirard, B. M., and Snell, E. E. (1985) J. Biol. Chem. 260, 6738-6746) and the amino acid sequences of two tryptic peptides reported in the accompanying paper (Hayashi, H., Tanase, S., and Snell, E. E. (1986) J. Biol. Chem. 261, 11003-11009) are localized in the sequence presented here; the lysine that binds pyridoxal phosphate is situated at residue 232, whereas the serine that binds the adduct formed between pyridoxal phosphate and the inhibitor alpha-fluoromethylhistidine is positioned at residue 322.     

Rat liver NAD(P)H: quinone reductase nucleotide sequence analysis of a quinone reductase cDNA clone and prediction of the amino acid sequence of the corresponding protein

We have determined the nucleotide sequence of a cDNA clone, pDTD55, complementary to rat liver quinone reductase mRNA (Williams, J.B., Lu, A.Y.H., Cameron, R.G., and Pickett, C.B. (1986) J. Biol. Chem. 261, 5524-5528). The cDNA clone contains an open reading frame of 759 nucleotides encoding a polypeptide comprised of 253 amino acids with a Mr = 28,564. To verify the predicted amino acid sequence of quinone reductase, we have been able to align the amino acid sequences of a cyanogen bromide digest of the purified enzyme to the sequence deduced from the cDNA clone. A comparison of the quinone reductase sequence with other known flavoenzymes did not reveal a significant degree of amino acid sequence homology. These data suggest that the quinone reductase gene has evolved independently from genes encoding other flavoenzymes.     

The structure of the hemocyanin from the horseshoe crab, Limulus polyphemus. The amino acid sequence of the largest cyanogen bromide fragment

The amino acid sequence of the largest CNBr fragment, CNBrIa, from hemocyanin II of Limulus polyphemus has been determined. This fragment contains 203 residues, has Mr = 23,600, and is the NH2-terminal segment of the molecule. Comparison of this sequence with the first 87 residues of the NH2-terminal sequence of the alpha chain of hemocyanin from the Japanese horseshoe crab, Tachypleus tridentatus, of the Western Pacific (Nemoto, T., and Takagi, T. (1981) Biochim. Biophys. Acta 670, 79-83) shows that 46% of the residues are identical. Residues 172-177, -His-His-Trp-His-Trp-His-, appear to constitute at least part of the copper-binding site. This conclusion is supported by our finding that 40% of the residues in the sequence 172-186 are identical to residues 188-202 in tyrosinase from Neurospora; residues 188 and 193 in tyrosinase are known to be copper-binding histidines (Pfiffner, E., and Lerch, K. (1981) Biochemistry 20, 6029-6035). Model building suggests that histidines 172 and 175 might bind one copper atom and histidines 173 and 177 might bind the second copper. These observations suggest that tyrosinase and hemocyanin may have a common evolutionary origin, but we have not so far seen other correspondences.     

Panulirus interruptus hemocyanin. The elucidation of the complete amino acid sequence of subunit a

As a final step in the elucidation of the primary structure of subunit a of Panulirus interruptus hemocyanin (657 residues, Mr 75696 excluding two copper ions and carbohydrate), the amino acid sequence of the largest fragment obtained by limited trypsinolysis was determined. The elucidation of the sequence of residues 176-657, comprising domains two and three, was mainly based on two digests, with CNBr and trypsin, respectively, from both of which a complete set of peptides was obtained. Additional sequence information was obtained from a digest with Staphylococcus aureus V8 protease and from one fragment obtained by cleaving subunit a with hydroxylamine. A block during Edman degradations indicated an Asn-Gly sequence at positions 597-598, although only aspartic acid was identified at position 597.     

Identification and primary structure of the cardiolipin-binding domain of mitochondrial creatine kinase

It was recently shown that the mitochondrial isozyme of heart creatine kinase binds to cardiolipin on the outer half of the inner membrane [Müller, M., et al. (1985) J. Biol. Chem. 260, 3839-3843]. The enzyme has now been extracted and purified to homogeneity from rat heart mitochondria, and cleaved with CNBr. The fragments have been separated on an FPLC system using a Mono Q HR 5/5 column. Only one of these binds to cardiolipin-containing liposomes and has thus been identified as the cardiolipin-binding domain of the enzyme. Its amino acid sequence has been determined. The fragment contains 25 amino acids and corresponds to the N-terminal region of the protein. The binding of the fragment of cardiolipin-containing liposomes was inhibited by adriamycin. Another and larger CNBr fragment could be specifically labelled with periodate-oxidized (di-aldehyde) ATP and has thus been identified as the ATP-binding domain. Chemical modification of the basic amino acids Lys and Arg of the enzyme abolished its binding to cardiolipin.     

Identification of two phosphorylated threonines in the tail region of Dictyostelium myosin II

We have previously purified and characterized a Dictyostelium myosin II heavy chain kinase which phosphorylates threonine residues (C?té, G. P., and Bukiejko, U. (1987) J. Biol. Chem. 262, 1065-1072). The phosphorylated threonines are located within a 34-kDa fragment which can be selectively cleaved from the carboxyl terminal end of the Dictyostelium myosin II tail. Tryptic and chymotryptic digests of the 34-kDa fragment phosphorylated with the kinase have now been performed and the resulting phosphopeptides isolated and sequenced. Two phosphorylated threonine residues have been identified, corresponding to residues 1833 and 2029 in the complete amino acid sequence of the Dictyostelium myosin II heavy chain. These amino acids are 87 and 283 residues, respectively, distant from the carboxyl terminus of the Dictyostelium myosin II heavy chain and are present in sections of the tail which seem to be alpha-helical coiled coils. In contrast, the three Acanthamoeba myosin II heavy chain phosphorylation sites are located within 10 residues of each other in a small globular domain at the carboxyl terminal tip of the tail (C?té, G. P., Robinson, E. A., Appella, E., and Korn, E. D. (1984) J. Biol. Chem. 259, 12781-12787). This suggests that the mechanism by which heavy chain phosphorylation inhibits the actin-activated ATPase activity and filament-forming properties of the two myosins may be quite different.     

Amino acid sequence of the calcium-binding light chain of myosin from the lower eukaryote, Physarum polycephalum

We have established a new method for preparing Physarum myosin whose actin-activated ATPase activity is inhibited by micromolar levels of Ca2+. This Ca2+-inhibition is mediated by the Ca2+ binding to the myosin rather than by the Ca2+-dependent modification of the phosphorylated state of the myosin (Kohama, K., and Kendrick-Jones, J. (1986) J. Biochem. (Tokyo) 99, 1433-1446). Ca2+-binding light chain (CaLC) has been suggested to be primary importance in this Ca2+ inhibition (Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, T., and Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). The amino acid sequence of CaLC was determined; it was composed of 147 amino acid residues and the N terminus was acetylated. The molecular weight was calculated to be 16,131. The homology of CaLC in the amino acid sequence with 5,5'-dithiobis-(2-nitrobenzoic acid) light chain and alkali light chain of skeletal muscle myosin were rather low, i.e., 25% and 30%, respectively. Interestingly, however, the CaLC sequence was 40% homologous with brain calmodulin. This amino acid sequence was confirmed by sequencing the cloned phage DNA accommodating cDNA coding CaLC. Northern and Southern blot analysis indicated that 0.8-kilobase pair mRNA was transcribed from a single CaLC gene. This is the first report on the amino acid sequence of myosin light chain of lower eukaryotes and nucleotide sequence of its mRNA.     

Covalent modification of the inhibitor-binding site(s) of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-AMP-incorporated peptides

The photoaffinity inhibitor analog [2-3H]8-azido-AMP is specifically and covalently incorporated into Escherichia coli ADP-glucose synthetase. The reaction site(s) of [2-3H]8-azido-AMP with the enzyme was identified by reverse phase high performance liquid chromatography isolation and chemical characterization of CNBr and mouse submaxillary arginyl protease-generated peptides containing the labeled analog. Three regions of modification, represented by six labeled peptides, accounted for over 85% of the covalently bound label. The major binding region of the azido analog, composed of residues 108-128, contained approximately 55% of the recovered covalently bound radioactivity. A single residue, Tyr-113, contained between 50 and 75% of the label found in the major binding region. This site is the same as the major binding region of the substrate site-specific probe, 8-azido-ADP-[14C]glucose (Lee, Y. M., and Preiss, J. (1986) J. Biol. Chem. 261, 1058-1064). Conformational analysis of this region predicts that it is a part of a Rossmann fold, the supersecondary structure found in many adenine nucleotide-binding proteins. Two minor reaction regions of the enzyme with [2-3H]8-azido-AMP were also identified by chemical characterization. One region, containing 20% of the covalently bound label, was composed of residues 11-68. This region contains Lys-38, the previously determined pyridoxal phosphate-modified allosteric activator site (Parsons, T. F., and Preiss, J. (1978) J. Biol. Chem. 253, 7638-7645). The third minor region of modification, residues 222-254, contained approximately 15% of the covalently bound label. The three modified peptide regions may be juxtaposed in the enzyme's tertiary structure.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora crassa. Isolation and sequences of several cyanogen bromide peptides from the NH2-terminal portion of the peptide chain

Previous studies of the amino acid sequence of the NAD-specific glutamate dehydrogenase of Neurospora crassa (EC 1.4.1.2) resulted in the assignments of peptides to four fragments, the longest being the COOH-terminal 669 residues of the protein. A further study of peptides derived by cyanogen bromide cleavage by different separation methods has yielded additional peptides that have provided new information concerning the sequence and has given overlaps of previously known sequences. This has permitted establishment of 313 residues in one sequence (fragment II). This is in addition to a sequence of 43 residues (fragment I) at the NH2-terminal end and a sequence of 669 residues (fragment III) previously established at the COOH-terminal end of the molecule. The present status of our knowledge of the overall sequence is given in the accompanying papers, together with some views regarding the conformation of the protein (Haberland, M.E., Chen, C.-W., and Smith, E.L. (1980) J. Biol. Chem. 255, 7993-8000, and Austen, B.M., Haberland, M.E., and Smith, E.L. (1980) J. Biol. Chem. 255, 8001-8004).     

Isolation and properties of a fragment of human serum albumin demonstrating the absence of a methionine residue from some of the albumin molecules.

1. A fragment designated D was isolated from human serum albumin degraded by CNBr. Its properties show that it is made up of the B and C fragments isolated by McMenamy et al. (1971) (J. Biol. Chem., 246, 4744-4750). 2. Reduction of fragment D gives rise to two chains, one of which consists of the second subfragment of reduced fragment B linked to fragment C by an amino acid different from methionine. It thus demonstrates the existence of albumin molecules from which the second methionine residue located between fragments B and C is missing.     

The arrangement of disulfide loops in human alpha 2-HS glycoprotein. Similarity to the disulfide bridge structures of cystatins and kininogens

The complete disulfide loop structure of human alpha 2-HS glycoprotein has been elucidated. alpha 2-HS glycoprotein isolated from human plasma was found to be a two-chain protein composed of a heavy and a light chain. The heavy chain comprises the A-chain of alpha 2-HS glycoprotein (Yoshioka, Y., Gejyo, F., Marti, T., Rickli, E. E., Bürgi, W., Offner, G. D., Troxler, R. F., and Schmid, K. (1986) J. Biol. Chem. 261, 1665-1676) and part of the connecting peptide which has been predicted from the corresponding cDNA sequence (Lee, C. C., Bowman, B. H., and Yang, F. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4403-4407), whereas the light chain corresponds to the beta-chain of alpha 2-HS glycoprotein (Gejyo, F., Chang, J. L., Bürgi, W., Schmid, K., Offner, G. D., Troxler, R. F., Van Halbeek, H., Dorland, L., Gerwig, G. J., Vliegenthart, J. F. G. (1983) J. Biol. Chem. 258, 4966-4971). Twelve half-cystine residues are present in the alpha 2-HS glycoprotein molecule, and 11 of them are positioned in the heavy chain and a single one in the light chain of the molecule; they form six disulfide bridges. The first and the last half-cystine residues of the amino acid sequence of alpha 2-HS glycoprotein are engaged in the formation of a loop spanning the extreme NH2- and COOH-terminal portions of the molecule, thereby connecting the heavy and light chains. The other 10 half-cystines residues are linked consecutively in the heavy chain and form five loops which span 4-19 amino acid residues. Among them are two pairs of loops which are characterized by mutual sequence homology. The particular arrangement of disulfide loops in alpha 2-HS glycoprotein is similar to the patterns of linearly arranged and tandemly repeated disulfide loops of cysteine proteinase inhibitors, i.e. the cystatins and the kininogens. It is concluded that alpha 2-HS glycoprotein represents a structural prototype of a novel family among the cystatin superfamily, characterized by the presence of two cystatin-like building blocks. Extensive similarity among the NH2-terminal sequences of alpha 2-HS glycoprotein and human histidine-rich glycoprotein suggest that the latter protein is another candidate protein of this new family.