摘要:

Human ceruloplasmin from fresh serum has been purified by chromatography on hydroxyapatite and Con A‐Sepharose. Quantitative immunoelectrophoretic analysis of fresh serum, stored serum and fractions from the different purification steps for human ceruloplasmin has been carried out. A combination of the latter, advanced technique with amino acid analysis, molecular weight determination by size chromatography, urea treatment, staining for oxidase activity and enzymatic proteolysis, has revealed that: 1) human ceruloplasmin is a heterogeneous mixture of two glycoproteins (x) differing only in their carbohydrate content and 2) the protein part contains at least one very labile peptide bond which upon enzymatic hydrolysis gives rise to peptides with molecular weights of 93,000 (y) and 24,000 (z) dalton, respectively. The two glycoproteins are immunochemically identical. The y peptide is immunochemically partially identical, and the z peptide immunochemically non‐identical, with the parent molecule. The y and z peptides are non‐identical. On the basis of these observations a simplified two‐dimensional model of human ceruloplasmin is p

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1975.tb02409.x

出版商:Blackwell Publishing Ltd    

年代:1975

数据来源: WILEY

摘要:

Fluorescamine was shown to be an excellent terminating agent for blocking unreacted amino groups during solid phase peptide synthesis. A comparison of the termination efficiency of fluorescamine versus that of acetylation revealed that the former method gave superior products as assessed by peptide analysis, dansyl‐amino end group determination and biological assay. In addition, fluorescamine terminated fragments were converted to non‐fluorescent spirolactones during the deprotection stage. These spirolactones were stable to subsequent solid phase reaction conditions and were readily removed from the target pept

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1975.tb02410.x

出版商:Blackwell Publishing Ltd    

年代:1975

数据来源: WILEY

摘要:

An insoluble preparation of rat liver cathepsin D was obtained by coupling the enzyme to Enzacryl Polyacetal (EPA‐cathepsin) and to CNBr‐activated Sepharose 4B. EPA‐cathepsin was active toward the synthetic hexapeptides (Gly‐Phe‐Leu)2and did not split hemoglobin. The optimum pH of splitting was displaced upward by 1.5 units to pH 5.0. The enzyme exhibited maximum activity at 60°C. No appreciable loss of activity was seen on storage of the enzyme for 4 months or after repeated use of the preparations. Coupling of rat liver cathepsin D to activated Sepharose gave preparations active towards both protein and synthetic substrates. The preparations were totally inactive in acid media and exhibited maximum activity at pH 7.0, that is, under physiological conditions. Optimum temperature was 65°. The specific activity of the preparations (pH 7.0, 65°) was 60–110% that of the free enzyme in acid media. Proteolytic activity of the Sepharose‐coupled cathepsin D was not inhibited by pepstatin, whereas that of the free enzyme was fully inhibited by this reagent. A sarcoma cathepsin, similar in some of its properties to the rat liver enzyme, was also coupled to CNBr‐activated Sepharose 4B. The preparation split protein substrates at pH 7.0 and possessed enhanced thermostability. The enzymes fixed on Sepharose showed i

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1975.tb02411.x

出版商:Blackwell Publishing Ltd    

年代:1975

数据来源: WILEY

摘要:

Galline, a protamine from rooster sperm nuclei, has already been separated into its components, G‐I‐G‐VIII. The amino acid composition of the homogeneously purified fraction G‐I is determined decisively to be Arg11, Ser2, Gly3, Val1and Tyr2, and the molecular weight is 2,908 as hydrochloride. The complete amino acid sequence was deduced as follows from the results of analyses of the tryptic and chymotryptic peptides: H‐Ser‐Gly‐Gly‐Val‐Arg‐Arg‐Arg‐Arg‐Tyr‐Gly‐Ser‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Arg‐Tyr‐OH.Another purified fraction, G‐V, has the amino acid composition, Arg24, Thr1, Ser8, Gly3, Ala2, Pro2and Tyr2as described previously, and the molecular weight 6,274 as hydrochloride. The complete amino acid sequence was established by combining analytical results of tryptic, chymotryptic and thermolysin peptides as well as those of carboxypeptidase and leucine aminopeptidase digestion as follows: H‐Ala‐Arg‐Tyr‐Arg‐Ser‐Gly‐Arg‐Ser‐Arg‐Ser‐Arg‐Arg‐Thr‐Arg‐Arg‐Arg‐Arg‐Ser‐Pro‐Arg

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1975.tb02412.x

出版商:Blackwell Publishing Ltd    

年代:1975

数据来源: WILEY

摘要:

A method of resolving CD spectra in α‐helix, β‐structure and random coil conformations is described. The residue ellipticities for α‐helix and β‐structure given by Greenfield&Fasman or by Chen, Yang&Martinez are used together with CD spectra from at least two similar peptides to determine, by an iterative least‐squares method, the number of amino acids in the three reference conformations as well as a set of residue ellipticities characteristic of the random coils of the family of peptides in question, but not necessarily of other peptides. The fits between computed and experimental spectra improve significantly and systematic deviations disappear by allowing the random coil coefficients to vary from one family of proteins to another, a liberty justified by the different types of random coils that have been encountered.The method of analysis showed that 5 M urea did not change the conformations of C‐peptides of proinsulin from ox, pig and duck, all being mainly in the random coil conformation and all having 3–4 amino acids in β‐structure. Bovine insulin and proinsulin showed a transfer of amino acids from α‐helix to β‐structure with increasing concentrations of urea, the latter at a higher concentration, indicating a stabilizing effect of the connecting peptide. The numbers of amino acids found in the α‐helical conformation in insulin and proinsulin were equal and in agreement with the X‐ray crystallographic data for insulin when the Greenfield&Fasman coefficients for α‐helix and β‐structure were employed, whereas the Chen, Yang&Martinez coefficients yielded too few amino acids in α‐helix in proinsulin. Both sets of coefficients estimate more

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1975.tb02413.x

出版商:Blackwell Publishing Ltd    

年代:1975

数据来源: WILEY

摘要:

In order to investigate the possible role of the Trp residue in ACTH as a charge‐transfer donor in the activation of ACTH receptors, two ACTH analogues (β‐corticotrophins (1–24) with L‐Ser1and D‐Ser1, respectively) containing pentamethylphenylalanine instead of Trp have been synthesized. In these syntheses a new, alkaline‐labile, aminoprotecting group, the methylsulphonylethyloxycarbonyl group, was employed. The association constants of ACTH (1–24) and [Pmp9]‐ACTH (1–24)* with the watersoluble acceptor paraquat,

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1975.tb02414.x

出版商:Blackwell Publishing Ltd    

年代:1975

数据来源: WILEY

摘要:

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10%) and subsequently submitted to gel‐filtration. The large polypeptide subfractions were citraconylated and resubmitted to different gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C‐terminal position because one methionyl residue is situated at the C‐terminal position in the native protein. The polypeptide with 132 residues possessed N‐acetylated residue at N‐terminal position; therefore this polypeptide is located at the N‐terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present result

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1975.tb02415.x

出版商:Blackwell Publishing Ltd    

年代:1975

数据来源: WILEY

摘要:

Affinity chromatography has been used to purify adenosine deaminase from various sources: calf spleen, calf intestinal mucosa, chicken duodena and human erythrocytes. For this purpose a specific inhibitor, 9‐(p‐aminobenzyl) adenine, was synthesized and covalently joined to agarose. Adenosine deaminase is selectively retained by such an inhibitor‐resin when highly impure solutions are chromatographed through it. After elution from the resin with guanylurea, a competitive inhibitor, the enzyme is homogeneous and can be recovered in yields of 80% or more and the same number of multiple forms of the enzyme is present in the purified preparation and in the crude ex

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1975.tb02416.x

出版商:Blackwell Publishing Ltd    

年代:1975

数据来源: WILEY

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1975.tb02417.x

出版商:Blackwell Publishing Ltd    

年代:1975

数据来源: WILEY

ISSN:0367-8377    

DOI:10.1111/j.1399-3011.1975.tb02418.x

出版商:Blackwell Publishing Ltd    

年代:1975

数据来源: WILEY