The primary structure of the calcium-transporting adenosine triphosphatase of rabbit skeletal sarcoplasmic reticulum. Soluble tryptic peptides from the succinylated carboxymethylated protein.

The isolation and the determination of the amino-acid sequences of the soluble tryptic peptides, derived by cleavage at arginine residues, of the succinylated (3-carboxypropionylated) S-carboxymethylated adenosine triphosphatase protein of rabbit skeletal sarcoplasmic reticulum are described. Treatment of the protein with succinic anhydride gave a derivative that was readily digested with trypsin, yielding two distinct sets of peptides. One set comprises large, relatively hydrophobic, peptides that are highly aggregated (or insoluble) in aqueous solution and that have been identified, by several criteria, with the portion of the protein embedded in the lipid bilayer in the sarcoplasmic reticulum. The second set, which is described here, comprises peptides that have properties typical of those derived from soluble globular proteins and that constitute that part of the protein external to the lipid bilayer. The sequences of these soluble tryptic peptides contain 586 unique residues. Details of the isolation of the peptides and the determination of the sequences are contained in Supplementary Publication SUP 50102 (88 pages) which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

The primary structure of the calcium ion-transporting adenosine triphosphatase protein of rabbit skeletal sarcoplasmic reticulum. Peptides derived from digestion with cyanogen bromide, and the sequences of three long extramembranous segments.

The isolation and characterization of the soluble peptides from the CNBr digest of the calcium ion-transporting adenosine triphosphatase protein of rabbit skeletal sarcoplasmic reticulum are described. The 562 unique residues of the protein were placed in sequences. The remaining part of the protein (about 500 residues) yielded long hydrophobic sequences that contained all but one of the tryptophan residues of the protein and that were probably derived largely from the intramembranous parts of the protein. Three long stretches of primary structure, constituting half of the protein, have been reconstructed from the information presented here together with the sequences found in peptides from other digests of the protein. The secondary structures of these sequences have been predicted. A model for the primary structure of the protein is presented and the implications discussed. Details of the isolation of peptides are contained in Supplementary Publication, SUP 50105 (29 pages), which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.     

Comparison of the primary structures of ribonuclease U2 isoforms.

The primary structures of the two isoforms of ribonuclease U2, RNAases U2-A and U2-B, were analysed and compared with each other. Among the chymotryptic peptides obtained from the reduced and S-carboxymethylated enzymes, only peptides C-3 were different from each other in terms of chromatographic behaviour on reverse-phase h.p.l.c. On the basis of chemical analyses of these peptides, it was shown that RNAase U2-B had an isopeptide bond in which Asp-32 was linked to Gly-33 through the beta-carboxy group in its side chain instead of the alpha-carboxy group. Deamidation of Asn-32 in RNAase U2-A led to the formation of this unusual linkage. The previously reported sequence of RNAase U2 [Sato & Uchida (1975) Biochem. J. 145, 353-360] was corrected by changing amino acid residues at eight different positions and by inserting an asparagine residue at position 32. The numbering of the positions of amino acid residues located downstream of Asn-32 was therefore shifted by 1. Accordingly, RNAase U2-A was shown to be composed of 114 amino acid residues.     

Primary structures of cysteine-containing peptides from the calcium ion-transporting adenosine triphosphatase of rabbit sarcoplasmic reticulum.

A preliminary investigation of the primary structure of the Ca(2+-transporting ATPase (adenosine triphosphatase) protein of rabbit skeletal-muscle sarcoplasmic reticulum is reported. The preparation of derivatives of delipidated protein in a form suitable for sequence analysis is described. Tryptic peptides containing S-carboxymethylcysteine residues were isolated from the reduced carboxymethylated protein, and their sequences were partially determined. The results are consistent with mol.wt. about 105000 for the polypeptide, and the absence of extended repeated lengths of sequence. The distribution of tryptophan and cysteine residues between large, aggregated peptides and soluble tryptic peptides shows that these residues are concentrated in different regions of the primary structure. This observation agrees with other evidence that these residues are, on the whole, widely separated in the native protein. The details of the procedures used to isolate the peptides, and the evidence for the determination of their sequences, are given Supplementary Publication SUP 50085 (30 pages), which has been deposited at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem.J. (1978) 169, 5.     

Studies on adenosine triphosphate transphosphorylases. Amino acid sequence of rabbit muscle ATP-AMP transphosphorylase

The total amino acid sequence of rabbit muscle adenylate kinase has been determined, and the single polypeptide chain of 194 amino acid residues starts with N-acetylmethionine and ends with leucyllysine at its carboxyl terminus, in agreement with the earlier data on its amino acid composition [Mahowald, T. A., Noltmann, E. A., & Kuby, S. A. (1962) J. Biol. Chem. 237, 1138-1145] and its carboxyl-terminus sequence [Olson, O. E., & Kuby, S. A. (1964) J. Biol. Chem. 239, 460-467]. Elucidation of the primary structure was based on tryptic and chymotryptic cleavages of the performic acid oxidized protein, cyanogen bromide cleavages of the 14C-labeled S-carboxymethylated protein at its five methionine sites (followed by maleylation of peptide fragments), and tryptic cleavages at its 12 arginine sites of the maleylated 14C-labeled S-carboxymethylated protein. Calf muscle myokinase, whose sequence has also been established, differs primarily from the rabbit muscle myokinase's sequence in the following: His-30 is replaced by Gln-30; Lys-56 is replaced by Met-56; Ala-84 and Asp 85 are replaced by Val-84 and Asn-85. A comparison of the four muscle-type adenylate kinases, whose covalent structures have now been determined, viz., rabbit, calf, porcine, and human [for the latter two sequences see Heil, A., Müller, G., Noda, L., Pinder, T., Schirmer, H., Schirmer, I., & Von Zabern, I. (1974) Eur. J. Biochem. 43, 131-144, and Von Zabern, I., Wittmann-Liebold, B., Untucht-Grau, R., Schirmer, R. H., & Pai, E. F. (1976) Eur. J. Biochem. 68, 281-290], demonstrates an extraordinary degree of homology.(ABSTRACT TRUNCATED AT 250 WORDS)     

The preparation and properties of a group of proteins from the high-sulphur fraction of wool

A method is reported for the preparation of a group of three proteins from the S-carboxymethylated high-sulphur fraction of wool. These proteins have been partially characterized by their tryptic peptides. All have similar structural features and show an interesting homology within the group and some similarities of sequence with a different group of wool high-sulphur proteins. The evidence for the sequence of some of the peptides is given in a supplementary paper that has been deposited as Supplementary Publication 50008 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.     

L-lactate 2-monooxygenase from Mycobacterium smegmatis. Cloning, nucleotide sequence, and primary structure homology within an enzyme family

L-Lactate 2-monooxygenase catalyzes the oxidation of L-lactate to acetate and carbon dioxide. The catalytic mechanism has been extensively investigated but very little is known about which amino acid residues may play a role in catalysis. As a first step toward this goal, the gene for this protein from Mycobacterium smegmatis has been cloned and sequenced. Peptide sequencing data for L-lactate 2-monooxygenase was used to construct three sets of fully redundant tetradecamer oligonucleotide probes, which were hybridized to restriction-digested M. smegmatis DNA. An approximately 3-kilobase pair PstI fragment hybridized with two of the probes. This region was subsequently isolated and cloned into Escherichia coli. From this size-fractionated gene bank, a 3.1-kilobase pair genomic DNA fragment was isolated by colony hybridization to two of the oligonucleotide probes. The complete gene for L-lactate 2-monooxygenase was contained on this fragment as shown by DNA sequencing of the whole insert. The DNA sequence codes for a mature protein that is 393 amino acids in length with a subunit molecular weight of 43,072 (including the FMN). The protein sequence shows impressive homology with the primary structures of two mechanistically related proteins, yeast flavocytochrome b2 (Lederer, F., Cortial, S., Becam, A.-M., Haumont, P.-Y., and Perez, L. (1985) Eur. J. Biochem. 152, 419-428; Guiard, B. (1985) EMBO J. 4, 3265-3272) and spinach glycolate oxidase (Volkita, M., and Somerville, C. R. (1987) J. Biol. Chem. 262, 15825-15828; Cederlund, E., Lindqvist, Y., Soderlund, G., Br?ndén, C.-I., and Jornvall, H. (1988) Eur. J. Biochem. 173, 523-530). For each residue proposed from the crystal structure of glycolate oxidase to be involved in catalysis (Lindqvist, Y., and Br?ndén, C.-I. (1989) J. Biol. Chem. 264, 3624-3628), an identical residue was found in a homologous position in lactate oxidase. Furthermore, most of these residues occur in regions whose sequences are highly conserved between lactate oxidase, flavocytochrome b2, and glycolate oxidase.     

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptides from digestion with a staphylococcal proteinase.

The extracellular proteinase of Staphylococcus aureus strain V8 was used to digest the NADP-specific glutamate dehydrogenase of Neurospora crassa. Of 35 non-overlapping peptides expected from the glutamate content of the polypeptide chain, 29 were isolated and substantially sequenced. The sequences obtained were valuable in providing overlaps for the alignment of about two-thirds of the sequences found in tryptic peptides [Wootton, J. C., Taylor, J, G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 739-748]. The blocked N-terminal peptide of the protein was isolated. This peptide was sequenced by mass spectrometry, and found to have N-terminal N-acetylserine by Howard R. Morris and Anne Dell, whose results are presented as an Appendix to the main paper. The staphylococcal proteinase showed very high specificity for glutamyl bonds in the NH4HCO3 buffer used. Partial splits of two aspartyl bonds, both Asp-Ile, were probably attributable to the proteinase. No cleavage of glutaminyl or S-carboxymethylcysteinyl bonds was found. Additional experimental detail has been deposited as Supplementary Publication SUP 50053 (5 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K, from whom copies may be obtained under the terms given in Biochem. J. (1975) 1458 5.     

The amino acid sequence of rabbit J chain in secretory immunoglobulin A.

The primary structure of rabbit J chain, which occurs covalently bound to secretory IgA, was determined. J chain was isolated in its S-carboxymethylated form, in one step, by SDS/PAGE followed by electro-elution; 5 nmol of protein (approx. 75 micrograms), in all, was necessary for the determination of the complete sequence by the 'shot-gun' microsquencing technique; with the use of several site-specific endoproteinases, the various digests of S-carboxymethylated J chain were separated by micro-bore reverse-phase h.p.l.c. and the partial N-terminal sequences of all peptides were analysed. From the sequence alignment, gaps were filled by further extensive sequencing of the relevant overlapping fragments isolated from selected digests. Rabbit J chain comprises 136 amino acid residues, out of which eight are conserved cysteine residues, and is more closely similar to the human sequence (73.5% identify) than to the mouse sequence (68% identity). There is one unique glycosylation site at asparagine-48.     

Formation of a covalent disulfide-linked antithrombin-albumin complex by an antithrombin variant, antithrombin "Northwick Park"

Antithrombin is a major proteinase inhibitor of the blood coagulation system. Its inherited deficiency or abnormality is often associated with thromboembolism. Antithrombin "Northwick Park," a functionally inactive variant antithrombin, has recently been shown by us (Lane, D.A., Flynn, A., Ireland, H., Erdjument, H., Samson, D., Howarth, D., and Thompson, E. (1987) Br. J. Haematol. 65,451-456) to be present in plasma, in part, as a high Mr (approximately 120,000) component which has a characteristic electrophoretic mobility in agarose gels in the absence of denaturing agents. In this communication, we present evidence that this Mr approximately 120,000 variant component is comprised of an antithrombin-albumin covalent disulfide-linked complex. This proposal is supported by results of: (a) fast atom bombardment mass spectrometry of the isolated reduced, S-carboxymethylated, trypsin-digested Mr approximately 120,000 complex; (b) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this complex and its reduced and S-carboxymethylated constituents; (c) immunoblotting of these polyacrylamide gels with antisera specific for antithrombin and albumin; (d) NH2-terminal sequence analysis of one of the isolated, S-carboxymethylated proteins that comprise the Mr approximately 120,000 complex; and (e) fast atom bombardment mass spectrometry of its tryptic peptides.     

The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with pepsin and trypsin

Peptides obtained by tryptic digestion of carboxymethylated and maleylated aspartate aminotransferase and of the aminoethylated enzyme were isolated and the complete amino acid sequences of most of them were determined. Digestion of the carboxymethylated protein with pepsin produced a complex mixture of peptides that allowed some overlapping of the tryptic peptides (Fig. 4); in addition, peptides were obtained that had not been found in either of the tryptic digests. From these studies about 400 amino acid residues were identified. Experimental details and confirmatory data for the results presented here are given in a supplementary paper that has been deposited as Supplementary Publication 50011 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1972) 126, 5.     

Isolation and structure elucidation of a novel 5-kDa peptide from neurohaemal lobes of the corpora cardiaca of Locusta migratoria (Insecta, Orthoptera)

Two predominant peptides have been isolated from neurohaemal lobes of corpora cardiaca of 8000 adults of Locusta migratoria. Both peptides have been unambiguously characterized by automated peptide microsequencing and liquid secondary-ion mass spectrometry as a 50-residue peptide (5K peptide) and a 48-residue isologue (5K' peptide). Computer search of sequence data banks did not reveal any significant similarity with other identified proteins. The 5K peptides are remarkably rich in alanine residues (25%) and contain a stretch of five consecutive alanines. This structure suggests that these molecules could correspond to spacer peptides. This assumption is corroborated in the accompanying paper [Lagueux et al. (1990) Eur. J. Biochem. 187, 249-254] on the molecular cloning of the precursor protein which attributes to the 5K peptides a role analogous to that of the C peptides of insulins.     

Antithrombin-III Denver, a reactive site variant

Antithrombin-III Denver is a mutant protein which differs from the normal in being defective in serine protease binding (Sambrano, J. E., Jacobson, L. J., Reeve, E. B., Manco-Johnson, M. J., and Hathaway, W. E. (1986) J. Clin. Invest. 77, 887-893). It was isolated from the blood of an individual heterozygous for the abnormal gene by: affinity separation on heparin-Sepharose to obtain an antithrombin fraction, and gel filtration of the species present following complexing of the antithrombin fraction with a small excess of thrombin. The reduced, S-carboxymethylated protein formed a mixture of soluble tryptic peptides which was fractionated on Vydac C18. A single, unique peptide not present in a parallel experiment with normal antithrombin-III was isolated. This peptide was identified by sequence analysis and synthesis to correspond to residues 394-399 in the known sequence of the inhibitor, with leucine replacing reactive site P'1 residue Ser394. Although chromatograms of the tryptic peptides from the normal and mutant proteins were otherwise indistinguishable, the existence of additional residue replacements is not excluded. Measurements of the rate of thrombin binding by the mutant protein with p-aminobenzamidine as a fluorescent indicator showed that the second-order rate constant is reduced drastically. Meaningful measurements with the mutant protein could only be made in the presence of heparin and revealed a reduction of about 4000-fold in the rate constant.     

Complete amino acid sequence of copper-zinc superoxide dismutase from Drosophila melanogaster

The complete amino acid sequence of the Drosophila melanogaster Cu,Zn superoxide dismutase subunit has been determined by automated Edman degradation. Sequence analyses were performed on the intact S-carboxymethylated protein, two fragments derived from CNBr cleavage, and three peptides recovered from mouse submaxillary protease digestion of the reduced and S-carboxymethylated enzyme. The peptides were aligned by characterizing peptides yielded by trypsin and Staphylococcus aureus V8 protease. All the peptides studied were purified exclusively by reverse-phase columns of HPLC and were analyzed with an improved liquid-phase sequencer. A molecular weight of 15,750 (subunit) was calculated from the 151 residues sequenced. The amino acid sequence of the Drosophila superoxide dismutase subunit is compared with that of four other eucaryotes: man, horse, cow, and yeast. Comparison of the five primary structures reveals very different rates of evolution at different times. Copper-zinc superoxide dismutase appears to be a very erratic evolutionary clock. Val-Val-Lys-Ala- Val-Cys-Val-Ile-Asn-Gly-Asp-Ala-Lys-Gly-Thr-Val-Phe-Phe-Glu-Gln- Glu-Ser-Ser-Gly-Thr-Pro-Val-Lys-Val-Ser-Gly-Glu-Val-Cys-Gly-Leu- Ala-Lys-Gly-Leu-His-Gly-Phe-His-Val-His-Glu-Phe-Gly-Asp-Asn-Thr- Asn-Gly-Cys-Met-Ser-Ser-Gly-Pro-His-Phe-Asn-Pro-Tyr-Gly-Lys-Glu- His-Gly-Ala-Pro-Val-Asp-Glu-Asn-Arg-His-Leu-Gly-Asp-Leu-Gly-Asn- Ile-Glu-Ala-Thr-Gly-Asp-Cys-Pro-Thr-Lys-Val-Asn-Ile-Thr-Asp-Ser- Lys-Ile-Thr-Leu-Phe-Gly-Ala-Asp-Ser-Ile-Ile-Gly-Arg-Thr-Val-Val-Val- His-Ala-Asp-Ala-Asp-Asp-Leu-Gly-Gln-Gly-Gly-His-Glu-Leu-Ser-Lys- Ser-Thr-Gly-Asn-Ala-Gly-Ala-Arg-Ile-Gly-Cys-Gly-Val-Ile-Gly-Ile- Ala-Lys.     

The primary structure of component 8c-1, a subunit protein of intermediate filaments in wool keratin. Relationships with proteins from other intermediate filaments.

Component 8c-1, one of four highly homologous component-8 subunit proteins present in the microfibrils of wool, was isolated as its S-carboxymethyl derivative and its amino acid sequence was determined. Large peptides were isolated after cleaving the protein chemically or enzymically and the sequence of each was determined with an automatic Sequenator. The peptides were ordered by sequence overlaps and, in some instances, by homology with known sequences from other component-8 subunits. The C-terminal residues were identified by three procedures. Full details of the various procedures used have been deposited as Supplementary Publication SUP 50133 (4 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5. The result showed that the protein comprises 412 residues and has an Mr, including the N-terminal acetyl group, of 48,300. The sequence of residues 98-200 of component 8c-1 was found to correspond to the partial or complete sequences of four homologous type I helical segments previously isolated from helical fragments recovered from chymotryptic digests of microfibrillar proteins of wool [Crewther & Dowling (1971) Appl. Polym. Symp. 18, 1-20; Crewther, Gough, Inglis & McKern (1978) Text. Res. J. 48, 160-162; Gough, Inglis & Crewther (1978) Biochem. J. 173, 385]. Considered in relation to amino acid sequences of other intermediate-filament proteins, the sequence is in accord with the view that keratin filament proteins are of two types [Hanukoglu & Fuchs (1983) Cell (Cambridge, Mass.) 33, 915-924]. Filament proteins from non-keratinous tissues, such as desmin, vimentin, neurofilament proteins and the glial fibrillary acidic protein, which form monocomponent filaments, constitute a third type. It is suggested that as a whole the proteins from intermediate filaments be classed as filamentins, the three types at present identified forming subgroups of this class. The significant homologies between types I, II and III occur almost exclusively in segments of the chain that have been identified as having a coiled-coil structure together with the relatively short sections connecting these segments. The non-coiled-coil segments at the C- and N-termini show no significant homology between types, nor is homology in these segments apparent in all members of one type. Component 8c-1 does not show homology in its terminal segments with the known sequence of any other filamentin.(ABSTRACT TRUNCATED AT 400 WORDS)     

The amino acid sequence of a cereal Bowman-Birk type trypsin inhibitor from seeds of Jobs' tears (Coix lachryma-jobi L.)

The major trypsin inhibitor from seeds of Jobs' tears (Coix lachryma-jobi) was purified by heat treatment, fractional precipitation with (NH4)2SO4, ion-exchange chromatography on DEAE-Sepharose, gel-filtration on Sephadex G-75 and preparative reverse-phase HPLC. The complete amino acid sequence was determined by analysis of peptides derived from the reduced and S-carboxymethylated protein by digestion with trypsin, chymotrypsin and the S. aureus V8 protease. The polypeptide contained 64 amino acids with a high content of cysteine. The sequence exhibited strong homology with a number of Bowman-Birk inhibitors from legume seeds and similar proteins recently isolated from wheat and rice.     

The amino acid sequence of Neurospora NADP-specific glutamate dehydrogenase. Peptic and chymotryptic peptides and the complete sequence.

Peptic and chymotryptic peptides were isolated form the NADP-specific glutamate dehydrogenase of Neurospora crassa and substantially sequenced. Out of 452 residues in the polypeptide chain, 265 were recovered in the peptic and 427 in the chymotryptic peptides. Together with the tryptic peptides [Wootton, J. C., Taylor, J. G., Jackson, A. A., Chambers, G. K. & Fincham, J. R. S. (1975) Biochem. J. 149, 749-755], these establish the complete sequence of the chain, including the acid and amide assignments, except for seven places where overlaps are inadequate. These remaining alignments are deduced from information on the CNBr fragments obtained in another laboratory [Blumenthal, K. M., Moon, K. & Smith, E. L. (1975), J. Biol. Chem. 250, 3644-3654]. Further information has been deposited as Supplementary Publication SUP 50054 (17 pages) with the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained under the terms given in Biochem. J. (1975) 145, 5.     

Tryptic digestion of the alpha subunit of human choriogonadotropin

In order to further characterize chemical, physicochemical, and immunochemical properties, as well as structure-function relationships, of the common alpha subunit of human glycoprotein hormones, a tryptic core was prepared from the alpha subunit of human choriogonadotropin. The core was purified in greater than 80% yield using gel permeation and anion-exchange chromatography, and, following reduction and S-carboxymethylation, the constituent peptides were purified by gel permeation and high performance liquid chromatography. The disulfide-bridged peptides comprising the alpha core were identified as residues 1-35 and residues 52-91 by amino acid composition and amino acid carboxyl sequence analyses of the reduced, S-carboxymethylated peptides. The alpha tryptic core contained both N-asparagine carbohydrate moieties, but was devoid of residues 36-51 and the carboxyl-terminal serine at position 92. The small peptides cleaved from residues 36-51, a known potential O-glycosylation region of the alpha subunit, were purified and identified. The tryptic core retained full immunopotency relative to the intact subunit in the binding to polyclonal and monoclonal antibodies directed against the alpha subunit. The region consisting of residues 36-51 is not part of the epitope recognized by these antibodies. With antisera generated to the reduced, S-carboxymethylated subunit, peptide 1-35, but not 52-91, was immunoreactive. This finding is consistent with the known dominant antigenicity of the amino-terminal region in the reduced, S-carboxymethylated molecule. The core exhibited no appreciable interaction with the complementary beta subunit, and, not surprisingly, was unable to compete with intact hormone binding in a radioreceptor assay using rat testicular homogenates. Circular dichroic spectroscopy was used to probe gross features of tertiary structure (240-300 nm) and secondary structure (190-240 nm). The tryptic core and each of the two constituent peptides exhibited spectra above 240 nm that resembled that of the reduced, S-carboxymethylated subunit more than that of the native material, thus suggesting a significant loss of tertiary structure in the core and isolated peptides. This finding is unexpected in consideration of the full retention of immunopotency by the alpha core although consistent with failure of the core to combine with intact complementary beta subunit. The intact subunit as well as the isolated constituent peptides exhibit little if any helicity in aqueous solution. Interestingly, the reduced, S-carboxymethylated chain and peptide 52-91 displayed helicity in 80% trifluoroethanol, a helicogenic solvent.     

Rooster comb hyaluronate-protein, a non-covalently linked complex.

Hyaluronate from rooster comb was isolated by ion-exchange chromatography on DEAE-cellulose from tissue extracts and papain digests. The preparations were labelled with [14C]acetic anhydride and subjected to CsCl-density-gradient centrifugation in 4 M-guanidinium chloride in the presence and absence of 4% ZwittergentTM 3-12. A radioactive protein fraction was separated from the hyaluronate when the zwitterionic detergent was also present. The protein could also be separated from the glycosaminoglycan by chromatography on Sepharose CL-6B eluted with the same solvent mixture. The protein fraction contained three protein bands of Mr 15,000-17,000 as assessed by polyacrylamide-gel electrophoresis in 0.1% SDS, and seemed to lack lysozyme activity. No evidence of other protein or amino acid(s) covalently linked with the hyaluronate was obtained. The hyaluronate-protein complex may be re-formed upon mixing the components, the extent of its formation depending on the conditions used. The results show that, as in chondrosarcoma [Mason, d'Arville, Kimura & Hascall (1982) Biochem. J. 207, 445-457] and teratocarcinoma cells [Prehm (1983) Biochem. J. 211, 191-198] the rooster comb hyaluronate also is not linked covalently to a core protein.     

Cloning and sequence analysis of a rat liver cDNA encoding acyl-peptide hydrolase

Acyl-peptide hydrolase catalyzes the removal of an N alpha-acetylated amino acid residue from an N alpha-acetylated peptide. Two overlapping degenerate oligonucleotide probes based on the sequence of a CNBr tryptic peptide, derived from purified rat acyl-peptide hydrolase, were synthesized and used to screen a rat liver lambda gt11 cDNA library. A 2.5-kilobase cDNA was cloned and sequenced. This clone contained 2364 base pairs of rat acyl-peptide hydrolase sequence but lacked a translational initiation codon. Using a 220-base pair probe derived from near the 5'-end of this almost full-length cDNA to rescreen the library, full-length clones were isolated, which contained an in-frame ATG codon at nucleotides 6-8 and encoded the NH2-terminal sequence, Met-Glu-Arg-Gln.... The DNA sequence encoded a protein of 732 amino acid residues, 40% of which were confirmed by protein sequence data from 19 CNBr or CNBr tryptic peptides. The isolated enzyme is NH2-terminally blocked (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), and based on the NH2-terminal protein sequence deduced from the DNA sequence and the sequence of the most NH2-terminal CNBr peptide, it is likely that the NH2-terminal residue is an acetylated methionine residue, since such residues are frequently juxtaposed to glutamyl residues (Persson, B., Flinta, C., von Heijne, G., and Jornvall, H. (1985) Eur. J. Biochem. 152, 523-527). The RNA blot analysis revealed a single message of 2.7 kilobases in various rat tissues examined. Although this enzyme is known to be inhibited by diisopropyl fluorophosphate and acetylalanine chloromethyl ketone (Kobayashi, K., and Smith, J. A. (1987) J. Biol. Chem. 262, 11435-11445), no strong similarity in protein sequence has been found with other serine proteases. This result suggests that acyl-peptide hydrolase may be a unique serine protease.