Primary structure of H2-proteinase, a non-hemorrhagic metalloproteinase, isolated from the venom of the habu snake, Trimeresurus flavoviridis

The complete amino acid sequence of and the locations of disulfide bridges in H2-proteinase, a major non-hemorrhagic proteinase isolated from the venom of the habu Trimeresurus flavoviridis, have been determined and compared with those of HR2a, one of the hemorrhagic metalloproteinases in this venom. The strategy involved consisted of structural analysis of peptides in digests with cyanogen bromide, lysyl endopeptidase, trypsin, Staphylococcus aureus V8 protease and thermolysin. Peptides were purified by gel filtration followed by reversed-phase HPLC. H2-proteinase is a non-glycosylated single chain polypeptide consisting of 201 amino acids with an amino-terminal pyroglutamic acid, a calculated molecular weight of 22,991 and a net charge of +14 at neutral pH. There was no evidence of heterogeneity of the sequence. H2-proteinase has a typical zinc-chelating sequence and its overall sequence identity with HR2a is 73.6%. The 3 disulfide bridges in H2-proteinase link Cys-117 to Cys-196, Cys-158 to Cys-180, and Cys-160 to Cys-163, in the same manner as in the case of HR2a. In striking contrast to HR2a, it contains en extra free cysteine residue at position 94 which becomes reactive to a sulfhydryl reagent in the presence of a denaturant.     

Primary structure of a hemorrhagic metalloproteinase, HT-2, isolated from the venom of Crotalus ruber ruber

Crotalidae and Viperidae snake venoms contains several kinds of metalloproteinases which cause localized hemorrhage by direct action on blood vessel walls. We report here the entire amino acid sequence and the disulfide bridge locations of HT-2, one of the hemorrhagic toxins isolated from the venom of Crotalus ruber ruber (red rattlesnake). The non-reduced protein was first cleaved at methionine residues to provide a set of 8 fragments, which covered the entire sequence of HT-2. The disulfide bridge locations of HT-2 were also determined by using these primary fragments. The unambiguous sequence for the whole protein was then established by conventional methods using lysyl endopeptidase and thermolysin digests. HT-2 consisted of 202 amino acid residues with two disulfide bridges, which were assigned to Cys-117-Cys-197 and Cys-157-Cys-164. HT-2 had a typical zinc-chelating sequence His-Glu-X-X-His (residues 142-146) found in thermolysin, and its overall sequence showed, respectively, 50, 52, and 53% identities to those of HR2a, H2-proteinase, and the metalloproteinase domain of HR1B. However, the disulfide bridge locations of HT-2 were different from those in the other metalloproteinases. The primary structure of HT-2 was more closely related to that of Ht-d from Crotalus atrox recently determined (81% identity). From the structural comparison with five metalloproteinases so far elucidated, six conservative amino acid residues, which may possibly be related to the induction of the hemorrhagic activity, were suggested to be present in these toxins.     

The position of the disulfide bonds in human plasma alpha 2 HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma alpha 2 HS-glycoprotein were determined. alpha 2 HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)--Cys-14 (A-chain), Cys-71--Cys-82, Cys-96--Cys-114, Cys-128--Cys-131, Cys-190--Cys-201 and Cys-212--Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of alpha 2 HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the S--S bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two S--S bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second S--S loops and the end of domains A and B, and the positions of the ordered structures.     

Amino acid sequence of the sex steroid binding protein of human blood plasma

The amino acid sequence of the sex steroid binding protein (SBP) from human plasma has been determined. The SBP subunit consists of a 373-residue polypeptide chain containing two disulfide bonds and three oligosaccharide chains. The sequence was solved primarily by analysis of peptides derived by cleavage at either lysyl or methionyl residues. In our preparations, approximately half of the protein molecules have the amino-terminal sequence Arg-Pro-Val-Leu-Pro; the other half lack Arg-Pro and begin with the valine. Preparations of Hammond et al. [Hammond, G. L., Robinson, P. A., Sugino, H., Ward, D. N., & Finne, J. (1986) J. Steroid Biochem. 24, 815] have an additional leucine at the amino terminus, making a total of 373 residues in the chain. Oligosaccharide chains are placed at Thr-7 and at Asn residues 351 and 367. The two disulfide bonds connect Cys-164 to Cys-188 and Cys-333 to Cys-361. The reported heterogeneity of preparations of the molecule may result in part from the amino-terminal microheterogeneity, in part from variations in the oligosaccharide moieties, and possibly in part from rearrangements involving cyclic imide formation in two Asn-Gly sequences. Certain hydrophobic segments are suggested as possible components of the steroid-binding sites. The protein shows no homology either with the cDNA-derived sequences of the estrogen and glucocorticoid receptors found by others to be homologous with each other or with any other protein sequence in the 1986 data base.     

Determination of disulfide array and subunit structure of taste-modifying protein, miraculin

The taste-modifying protein, miraculin (Theerasilp, S. et al. (1989) J. Biol. Chem. 264, 6655-6659) has seven cysteine residues in a molecule composed of 191 amino acid residues. The formation of three intrachain disulfide bridges at Cys-47-Cys-92, Cys-148-Cys-159 and Cys-152-Cys-155 and one interchain disulfide bridge at Cys-138 was determined by amino acid sequencing and composition analysis of cystine-containing peptides isolated by HPLC. The presence of an interchain disulfide bridge was also supported by the fact that the cystine peptide containing Cys-138 showed a negative color test for the free sulfhydryl group and a positive test after reduction with dithiothreitol. The molecular mass of non-reduced miraculin (43 kDa) in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was nearly twice the calculated molecular mass based on the amino acid sequence and the carbohydrate content of reduced miraculin (25 kDa). The molecular mass of native miraculin determined by low-angle laser light scattering was 90 kDa. Application of a crude extract of miraculin to a Sephadex G-75 column indicated that the taste-modifying activity appears at 52 kDa. It was concluded that native miraculin in pure form is a tetramer of the 25 kDa-peptide and native miraculin in crude state or denatured, non-reduced miraculin in pure form is a dimer of the peptide. Both tetramer miraculin and native dimer miraculin in crude state had the taste-modifying activity.     

Amino acid sequence of a unique protease from the crayfish Astacus fluviatilis

The amino acid sequence of a protease from the crayfish Astacus fluviatilis has been determined from overlapping sets of peptides derived largely by cleavage at Met, Lys, or Arg residues. The protein comprises 200 amino acid residues in a single polypeptide chain, corresponding to a molecular mass of 22,614 daltons. Two disulfide bonds link Cys-42 to Cys-198 and Cys-64 to Cys-84. The sequence of this invertebrate protease appears to be unique since it has no homologous relationship to any of the known protein sequences.     

Reinvestigation of the disulfide bridge arrangement in human pro-opiomelanocortin N-terminal segment (hNT 1-76)

The cystine bridge structure of the amino-terminal fragment of human pro-opiomelanocortin has been reinvestigated. Highly purified amino-terminal fragment 1-76 was rapidly isolated from human pituitaries using only reverse-phase liquid chromatography (RP-HPLC). This peptide was then subjected to trypsin and V8-protease digestion and the products separated by RP-HPLC and subjected to amino acid and microsequence analysis. The results show that disulfide bridges link Cys-2 to Cys-24 and Cys-8 to Cys-20. Amino acid analysis and amino sugar determination confirm (i) the previously proposed sequence and (ii) the suggestion of the presence of two glycosylation sites in this molecule. These are most probably located at Thr-45 (O-glycosylation) and at Asn-65 (N-glycosylation).     

Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme

Determination of the nucleotide sequence of a cDNA for batroxobin, a thrombin-like enzyme from Bothrops atrox, moojeni venom, allowed elucidation of the complete amino acid sequence of batroxobin for the first time for a thrombin-like snake venom enzyme. The molecular weight of batroxobin is 25,503 (231 amino acids). The amino acid sequence of batroxobin exhibits significant homology with those of mammalian serine proteases (trypsin, pancreatic kallikrein, and thrombin), indicating that batroxobin is a member of the serine protease family. Based on this homology and enzymatic and chemical studies, the catalytic residues and disulfide bridges of batroxobin were deduced to be as follows: catalytic residues, His41, Asp86, and Ser178; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys230, Cys118-Cys184, Cys150-Cys163, and Cys174-Cys199. The amino-terminal amino acid residue of batroxobin, valine, is preceded by 24 amino acids. This may indicate that the amino-terminal hydrophobic peptide (18 amino acids) is a prepeptide and that the hydrophilic peptide (6 amino acids), preceded by the putative prepeptide, is a propeptide.     

The amino acid sequence of the sex steroid-binding protein of rabbit serum

The amino acid sequence of the sex steroid-binding protein (SBP or SHBG) of rabbit serum, specific for binding testosterone and 5 alpha-dihydrotestosterone, was determined using a complementary combination of mass spectrometric and Edman degradation techniques. The monomeric unit of the homodimeric protein is a single chain glycopeptide of 367 amino acid residues, with N-linked oligosaccharide side chains at Asn-345 and Asn-361 and disulfide bonds connecting Cys-158 to Cys-182 and Cys-327 to Cys-355. The polypeptide molecular weight of the monomer calculated from the sequence is 39,769. The molecular weight of the homodimer including 9% carbohydrate is 87,404. The sequence contains a relatively hydrophobic segment between Trp-241 and Leu-282, which includes many leucine residues in an alternating pattern. An amino acid sequence repeat is also located within that segment. Both of these patterns are present in human SBP and in the androgen-binding protein of rat epididymis. The sequence data indicate that the previously reported microheterogeneity of rabbit SBP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis reflects variants generated by differential glycosylation of the monomer rather than different gene products. Seventy-nine percent of the amino acids of rabbit SBP are identical to those of human SBP; rabbit SBP thus joins human SBP and rat androgen-binding protein in one gene family that is distinct from the steroid hormone receptor superfamily. It appears that the problem of binding sex steroid hormones has been solved independently in two different gene families that contain completely different steroid-binding domains. Since the nonhomologous steroid-binding domains of both families of proteins recognize essentially the same steroid structure, it will be interesting to determine the structural basis of the two different protein designs that lead to similar steroid-binding specificity.     

The position of the disulfide bonds in human plasma α2HS-glycoprotein and the repeating double disulfide bonds in the domain structure

The positions of the inter- and intra-chain disulfide bonds of human plasma α₂HS-glycoprotein were determined. α₂HS-glycoprotein was digested with acid proteinase and then with thermolysin. The disulfide bonds containing peptides were separated by reversed-phase HPLC and detected by SBD-F (7-fluorobenzo-2-oxa-1,3-diasole-4-sulfonic acid ammonium salt) method. One inter-disulfide bond containing peptide and five intra-disulfide bond containing peptides (A-chain) were purified and identified as Cys-18 (B-chain)-Cys-14 (A-chain), Cys-71-Cys-82, Cys-96-Cys-114, Cys-128-Cys-131, Cys-190-Cys-201 and Cys-212-Cys-229, respectively. The location of the intra-disulfide bonds revealed that the A-chain of α₂HS-glycoprotein is composed of three domains. Two domains were shown to possess intramolecular homology judging from the total chain length of the domains, size of the loops formed by the SS bonds, the location of two disulfide loops near the C-terminal end of domains A and B, the distance between two SS bonds of each domain, the amino acid sequence homology between these two domains (22.6%), number of amino acid residues between the second SS loops and the end of domains A and B, and the positions of the ordered structures.     

Complete primary structure of prostatropin, a prostate epithelial cell growth factor

Bovine brain prostatropin is a potent and essential mitogen for prostate epithelial cell growth. The major form of prostatropin contains 154 amino acid residues in a single amino terminally blocked chain corresponding to a molecular weight of 17,400. The amino acid sequence of the 150 carboxy-terminal residues of prostatropin was derived by Edman degradation of overlapping peptides primarily generated by cleavage at lysyl and glutamyl residues. Analysis of the amino-terminal tetradecapeptide by fast atom bombardment mass spectrometry identified the blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 12 residues. Prostatropin residues 15-154 contain the sequence of bovine brain polypeptides recently described as acidic fibroblast growth factor and class I heparin-binding growth factor. The sequence of the first 25 residues of prostatropin is acetyl-Ala-(Gly, Glu)-Glu-Thr-Thr-Thr-Phe-Thr-Ala-Leu-Thr-Glu-Lys-Phe-Asn-Leu-Pro-Leu-Gly -Asn-Tyr-Lys-Lys-Pro. Reduced and carboxymethylated prostatropin exhibits mitogenic activity, suggesting that disulfide bonds among cysteine residues 30, 61, and 97 are not functionally essential. These results demonstrate by rigorous structural analysis that the brain-derived polypeptide previously described only as a mesenchymal and neuroectodermal cell mitogen is also an epithelial cell growth factor that may be involved in support of prostate hyperplasia and adenocarcinoma.     

Subtilosin A, a new antibiotic peptide produced by Bacillus subtilis 168: isolation, structural analysis, and biogenesis

Subtilosin A, a new antibiotic produced by Bacillus subtilis 168, was extracted from culture medium with n-butanol and purified to homogeneity by a combination of gel filtration and thin-layer chromatography. The yield was 5.5 mg from a liter of culture. It had bacteriocidal activity against some gram-positive bacteria. Amino acid analysis and mass spectrometry showed that it was a peptide with a molecular weight of 3398.9, consisting of 32 usual amino acid and some non-amino acid residues. Its amino- and carboxyl-termini were blocked. By analysis of the fragments obtained by partial acid hydrolysis, as well as by chymotryptic and thermolysin digestions of reduced and S-carboxymethylated samples and Achromobacter protease I digestion of performic acid-oxidized samples, the amino acid sequence was determined to be as follows: X-Gly-Leu-Gly-Leu-Trp-Gly-Asn-Lys-Gly-Cys-Ala-Thr-Cys-Ser-(sequence; see text) Ile-Gly-Ala-Ala-Cys-Leu-Val-Asp-Gly-Pro-Ile-Pro-Asp-Glx-Ile-Ala-Gly-Ala. The analyses of cross-linking structures revealed that there were linkages between the amino- and carboxyl-termini and between the Cys-19 and the Glx-28 residues through an unknown residue with a residue weight of 163. Consequently, subtilosin A was deduced to be a cyclic peptide antibiotic with a novel cross-linking structure. The production of subtilosin A begins at the end of vegetative growth and finishes before spore formation. Studies on the correlation between the production of subtilosin A and spore formation with decoyinine in the original strain and in asporogenous mutants of B. subtilis 168 suggested that there was no close correlation between the two phenomena. The production of subtilosin A was repressed by inhibitors of protein and RNA synthesis in contrast to that of many other antibiotic peptides, suggesting that it is synthesized by the mechanism of usual protein synthesis.     

The complete amino acid sequence of the high molecular mass hemorrhagic protein HR1B isolated from the venom of Trimeresurus flavoviridis

Hemorrhage is a common occurrence in a victim bitten by crotalid and viperid snakes, and hemorrhagic components in these various venoms have been isolated and characterized. Previously, we have shown that a low molecular weight hemorrhagic protein (HR2a, 202 amino acid residues) isolated from the venom of Trimeresurus flavoviridis is a member of a new subfamily of metalloproteinases. We now report the complete amino acid sequence of a high molecular mass hemorrhagic protein isolated from the same venom. This protein, HR1B, is a mosaic protein composed of 416 residues containing four asparagine-linked oligosaccharide chains. The amino-terminal half (residues 1-203) of HR1B contains a metalloproteinase domain, the sequence of which is 62% identical to that of HR2a and 52% identical to that of hemorrhagic toxin d isolated from Crotalus atrox venom. The most interesting finding is that the middle region (residues 204-300) of HR1B shows a striking similarity to disintegrins, Arg-Gly-Asp-containing platelet aggregation inhibitors, recently found in several viper venoms. Interestingly, however, this region of HR1B does not contain the Arg-Gly-Asp sequence which is known to be a putative binding site in the disintegrins for the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. We also found that the carboxyl-terminal region (residues 213-336) of the middle part of HR1B shows 30% identity to residues 1543-1656 of von Willebrand factor and that the remaining region at the carboxyl-terminal end is unique and has a cysteine-rich sequence. These results suggest that the middle portion of HR1B, which shows structural similarities to the disintegrins and von Willebrand factor, may be important in synergistically stimulating hemorrhagic activity in the NH2-terminal metalloproteinase domain.     

Association of vesicular stomatitis virus glycoprotein with virion membrane: characterization of the lipophilic tail fragment.

The proteolytic enzyme, thermolysin, degraded the external segment of the membrane glycoprotein of intact vesicular stomatitis (VS) virions but left behind a small nonglycosylated fragment, presumably embedded in the virion membrane. Other proteases generated membrane-associated glycoprotein fragments differing somewhat in molecular weight. The thermolysin-resistant, virion-associated fragment, which can be selectively solubilized by either Triton X-100 or chloroform/methanol, has a molecular weight of 5,200. Amino acid analysis of the glycoprotein fragment reveals a preponderance of hydrophobic amino acids (64% of the residues); the amino-terminal amino acid is alanine as determined by dansylation. Cyanogen bromide digestion of the tail fragment generated two peptides, confirming the presence of one methionine residue per thermolysin-resistant glycoprotein fragment. The secondary structure of this glycoprotein tail peptide is maintained by at least one disulfide bridge. Thermolysin treatment is isolated VS viral glycoprotein in the presence of Triton X-100 also generated a hydrophobic peptide fragment which is very similar to the virion-associated glycoprotein fragment. The amino acid terminus of intact glycoprotein was also found to be alanine as was its dansylated Triton-micellar fragment that resisted thermolytic degradation; this finding suggests that the amino-terminal end of the VS viral glycoprotein is embedded in the virion membrane. These results suggest that the VS viral glycoprotein is an amphipathic molecule, the hydrophilic portion of which contains all the carbohydrate and a lipophilic tail segment which forms lipid or detergent micelles, thus rendering it resistant to proteolysis.     

Structure of potato carboxypeptidase inhibitor: disulfide pairing and exposure of aromatic residues.

The determination of the covalent structure of a carboxypeptidase inhibitor from potatoes containing 39 amino acid residues has been completed by analysis of the pairing of the six half-cystine residues. Since the native inhibitor is resistant to fragmentation by proteases, the protein was first subjected to cleavage at aspartic acid residues by exposure to 0.03 N HCl at 110 degrees C for 10h to yield a fragment containing two chains (residues 6-15 and residues 18-39)held together by three disulfide bonds. Digestion with subtilisin and Pronase, respectively, yielded sets of peptides from which, by diagonal electrophoresis and amino acid analysis, the paired cystinyl residues were identified as Cys-8 to Cys-24, Cys-12 to Cys-27, and Cys-18 to Cys-34. Charge-transfer titration of the native inhibitor with N-methylnicotinamide chloride suggests that one of the two tryptophan residues and the single tyrosine residue are exposed to the solvent.     

Disulfide bond formation and secretion of Escherichia coli heat-stable enterotoxin II.

The Escherichia coli heat-stable enterotoxin II (STII) is a typical extracellular toxin consisting of 48 amino acid residues, of which 4 are cysteine. There are two disulfide bonds, one between Cys-10 and Cys-48 and one between Cys-21 and Cys-36. We examined the involvement of DsbA in the formation of the disulfide bonds of STII and the role of each in the secretion of STII. A dsbA mutant was transformed with a plasmid harboring the STII gene, and STII was not detected either in the cells or in the culture supernatant. Reducing the level of STII brought about the dsbA mutation restored by introducing the wild-type dsbA gene into the mutant strain. These results showed that DsbA is involved in forming the disulfide bonds of STII and that STII without these disulfide bonds is degraded during secretion. We substituted these four cysteine residues in vivo by oligonucleotide-directed site-specific mutagenesis. The amino acid sequence of the purified STII (C48S) and pulse-chase studies revealed that two intermolecular disulfide bonds must be formed to be efficiently secreted and that cleavage between amino acid residues 14 and 15 is probably the first step in the proteolytic degradation of STII.     

A new liposomal formulation for antisense oligodeoxynucleotides with small size, high incorporation efficiency and good stability

The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalian cells showed four-fold bands on non-reduced SDS-PAGE, though it was homogeneous under reduced conditions. It contains three cysteine residues, Cys-25, Cys-103, and Cys-184, which may be concerned with this heterogeneity. Therefore, we examined the formation of a disulfide bond in the recombinant Shh-N and identified three kinds of disulfides with a combination of peptide mapping and NH(2)-terminal amino acid sequencing analysis. Among them, one type of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be separated from the other Shh-Ns using reverse phase HPLC and had no activity of alkaline phosphatase induction in C3H10T1/2 cells. This molecule could also be made by denaturation of the purified Shh-N with guanidine-HCl under non-reduced conditions. On the other hand, the reduced Shh-N and the reduced S-methylated Shh-N at cysteine residues showed approximately 10-fold higher activity compared to the originally purified Shh-N. These results suggested that Shh-N was synthesized as an active form whose three cysteine residues did not form disulfide and inactivated finally by forming a disulfide bond between Cys-103 and Cys-184.     

Mitochondrial adenylate kinase (AK2) from bovine heart. The complete primary structure

The sequence analysis of adenylate kinase isoenzyme 2 (AK2) was completed using a gas-phase sequencer constructed in our laboratory. The enzyme contains 238 amino acid residues in the following order: (sequence; see text) The four cysteine residues of AK2 were reinvestigated. Cys-41 and Cys-233 contain free thiols, which can be carboxymethylated in the intact protein without loss of enzymic activity. Chemical and model-building studies suggest that the pair Cys-43/Cys-93 forms a disulfide in native AK2. The relative molecular mass of AK2, as deduced from the sequence, is 26104. Other methods, including titration of -SH groups, sedimentation equilibrium ultracentrifugation and gel filtration yielded Mr values in the range from 26 000 to 31 500, each value depending on the respective method of determination. Bovine heart AK2 contains 44 residues more than the homologous isoenzyme AK1 (myokinase). As all but one single insertions and deletions cancel, the higher Mr of AK2 is due to 9 residues preceding the N terminus of AK1, a stretch of 30 residues in the middle of the molecule and 6 residues at the end. AK2 and AK1 are similar in their active-site geometry. In contrast, AK2 does not possess any of the three antigenic sites of AK1, which is consistent with the lack of immunological cross-reactivity between AK1 and AK2.     

Analysis of Amino Acid Residues Involved in Catalysis of Polyethylene Glycol Dehydrogenase from Sphingopyxis terrae, Using Three-Dimensional Molecular Modeling-Based Kinetic Characterization of Mutants

Polyethylene glycol dehydrogenase (PEGDH) from Sphingopyxis terrae (formerly Sphingomonas terrae) is composed of 535 amino acid residues and one flavin adenine dinucleotide per monomer protein in a homodimeric structure. Its amino acid sequence shows 28.5 to 30.5% identity with glucose oxidases from Aspergillus niger and Penicillium amagasakiense. The ADP-binding site and the signature 1 and 2 consensus sequences of glucose-methanol-choline oxidoreductases are present in PEGDH. Based on three-dimensional molecular modeling and kinetic characterization of wild-type PEGDH and mutant PEGDHs constructed by site-directed mutagenesis, residues potentially involved in catalysis and substrate binding were found in the vicinity of the flavin ring. The catalytically important active sites were assigned to His-467 and Asn-511. One disulfide bridge between Cys-379 and Cys-382 existed in PEGDH and seemed to play roles in both substrate binding and electron mediation. The Cys-297 mutant showed decreased activity, suggesting the residue's importance in both substrate binding and electron mediation, as well as Cys-379 and Cys-382. PEGDH also contains a motif of a ubiquinone-binding site, and coenzyme Q₁₀ was utilized as an electron acceptor. Thus, we propose several important amino acid residues involved in the electron transfer pathway from the substrate to ubiquinone.     

The Ch21 protein, developmentally regulated in chick embryo, belongs to the superfamily of lipophilic molecule carrier proteins

Ch21, a developmentally regulated low molecular weight protein observed in chick embryo skeletal tissues, is expressed "in vitro" by differentiating chondrocytes at a late stage of development. Here we report the complete amino acid sequence of the protein. 86% of the total amino acid sequence was deduced by sequences of 17 high performance liquid chromatography-separated proteolytic fragments and 33 amino acid residues at the amino-terminal end of protein purified from spent culture medium of hypertrophic chondrocytes. Furthermore we isolated by molecular cloning the corresponding cDNA and determined its nucleotide sequence. By combining protein and nucleotide sequence data we determined the primary structure of the entire Ch21. It consists of 158 amino acids and has a molecular mass of 18.065 kDa. Computer-assisted analysis showed that the Ch21 belongs to the superfamily of low molecular weight proteins sharing a basic framework for binding and transport of small hydrophobic molecules.