The disulphide bonds of insulin

Ryle, Anthony; Sanger, F.; Smith, Leslie F.; Kitai, Ruth

doi:10.1042/bj0600541

Cited by 881 publications

(273 citation statements)

References 16 publications

Supporting

Mentioning

258

Contrasting

Unclassified

Order By: Relevance

“…Finally, an important difference between the diagonal electrophoretic technique described here and those elegantly reviewed by Brown and Hartley [4] should be pointed out. Unlike previous methods, the protein here is reversibly modified before enzymatic (or chemical) digestion and electrophoresis.…”

Section: Discussionmentioning

confidence: 87%

The Determination of the Order of Lysine‐containing

Perham

Jones

1967

European Journal of Biochemistry

View full text Add to dashboard Cite

1.A new diagonal electrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the &-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2.The technique has been successfully tested with insulin. 3. When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-TyrThr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The three basic amino acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4. A common ancestral gene for porcine pepsin and bov&e (calf) rennin is suggested by the close homology between the C-terminal sequences of the two proteins.

show abstract

Section: Discussionmentioning

confidence: 87%

The Determination of the Order of Lysine‐containing

Perham

Jones

1967

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…The A-chain also contains an intra-chain disulfide bond between residues A6-A11. 4,5 In the presence of zinc, the monomers assemble into hexamers where each of the two central zinc ions is coordinated by three HisB10 residues. In the hexameric form the HI molecule exists in two distinct conformations called the T-state and the R-state deviating in the length of the central a-helix in the B-chain.…”

Section: Introductionmentioning

confidence: 99%

Insulin analog with additional disulfide bond has increased stability and preserved activity

et al. 2013

View full text Add to dashboard Cite

Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogs have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Ca-Ca distances, solvent exposure, and side-chain orientation in human insulin (HI) structure. This insulin analog had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared with HI. Addition of the disulfide bond also resulted in a 34.6°C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the Bchain thought to be directly involved in fibril formation was not modified. Importantly, this analog was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analog from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analog featuring a fourth disulfide bond with increased structural stability and retained function.

show abstract

“…High voltage electrophoresis of peptide mixtures was carried out on Whatman No. 3 MM paper in cooled tanks [22] a t 60 V/cm. Thc electrophoresis buffers were pyridine -acetic acid a t pH 6.5 and 3.5, 8 O/, acetic acid-2 O/, formic acid a t pH I .9 and ammonium carbonate (1 O/,) a t pH 8.9 (cf.…”

Section: Methodsmentioning

confidence: 99%

Horse Liver Alcohol Dehydrogenase

Jörnvall

Harris

1970

European Journal of Biochemistry

View full text Add to dashboard Cite

The ethanol-active isoenzyme of alcohol dehydrogenase from horse liver has been carboxymethylated with iodo[2-14C]acetate in 6 M guanidine hydrochloride. A study of the tryptic digest of the carboxymethylated enzyme has shown that it contains 14 unique sequences with [2-l4C]-carboxymethylcysteine, two with tryptophan, one .with C-terminal phenylalanine and one with a blocked N-terminal residue which represents the N-terminal sequence in the protein chain. This number of unique residues found in peptides is in each case half the number previously found by direct amino acid analysis (based on a molecular weight of 80000) of the intact enzyme. These results support the view that the native isoenzyme is a dimer consisting of two similar and probably identical polypeptide chains each containing about 370 amino acid residues. Failure to demonstrate the presence of more than one type of chain by chromatography of the carboxymethylated enzyme on DEAE-cellulose provides additional support for the dimer hypothesis. An alternative proposal [I31 that the enzyme is composed of two pairs of dissimilar chains each with a molecular weight of 20000 appears to be incompatible with thcse results.Alcohol dehydrogenase from horse liver is known to exist in the form of isoenzymes which possess different substrate specificities [l-31, and enzyme preparations consisting either of the purified ethanolactive component, or of mixtures of isoenzymes in which this form has been the predominant component, have been extensively studied. This isoenzyme has a molecular weight of about 80000 [4,5] and in its most active form contains 28 free sulphhydry1 (-SH) groups [6]. Inhibition of enzyme activity by iodoacetic acid has been shown to be due to a specific reaction with the SH groups of two cysteine residues, which, moreover, occur in the same sequence in the primary structure of the enzyme protein. At the same time it was established that the number of peptides produced by tryptic digestion was about half the number that would have been expected from its content of lysine and arginine residues. On the basis of these results it was suggested [7,8] that the active enzyme with a molecular weight of 80000 could consist of two similar and possibly identical polypeptide chains and, although no free N-terminal residues could be detected [9,5], the presence in the protein of two moles of C-terminal phenylalanine [ 5 ] , Unusual Abbreviations. CM-, carboxymethyl ; dansyl, dimethylaminonaphthalene 5-sulphonyl chloride.

show abstract

The disulphide bonds of insulin

Abstract: In order to deduce the unique structure of ox insulin it is necessary to know its molecular weight. In previous papers a value of 12 000 was assumedc since physical measurements suggested that this was the weight of the smallest unit that existed in

Cited by 881 publications

References 16 publications

The Determination of the Order of Lysine‐containing

The Determination of the Order of Lysine‐containing

Insulin analog with additional disulfide bond has increased stability and preserved activity

Horse Liver Alcohol Dehydrogenase

Contact Info

Product

Resources

About