Role of Methionine in the Initiation of the Biosynthesis of Bovine Proinsulin

1973 ◽  
Vol 51 (6) ◽  
pp. 783-788 ◽  
Author(s):  
C. C. Yip ◽  
C. C. Liew
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Defined Medium ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Edman Degradation ◽  
Tissue Slices ◽  
Fetal Bovine ◽  
Bovine Pancreas

Slices of fetal bovine pancreas were used to study the initiation of proinsulin biosynthesis. The pancreatic slices were incubated with radioactive methionine, phenylalanine, or leucine, in a defined medium. The incorporation of amino acid into peptides in the tissue slices was measured for 2–3 h. Two types of radioactive peptides, "free" and "blocked," were identified by ion-exchange chromatography. Most of the radioactive "blocked" peptides labelled with [3H]phenylalanine and [35S]methionine were hydrolyzed by proteases, except for about 20% of those labelled with [35S]methionine, which also showed higher resistance to acid hydrolysis.Microsomes were isolated from the tissue slices after incubation and were extracted with acid alcohol. The radioactive proteins in the extract were reacted with a solid immunosorbant against insulin. Analysis of the immunoadsorbed radioactive peptides by Edman degradation showed the presence of both methionine and phenylalanine as the N-termini. It was concluded that methionine was an initiating amino acid in the biosynthesis of bovine proinsulin.

The Primary Structures of the Core Antenna Polypeptides from Rhodopseudomonas marina

1989 ◽  
Vol 44 (5-6) ◽  
pp. 407-414 ◽  
Author(s):  
René A. Brunisholz ◽  
I. Bissig ◽  
R. Wagner-Huber ◽  
G. Frank ◽  
F. Suter ◽  
...  
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Edman Degradation ◽  
Amino Acid Residues ◽  
Photosynthetic Membrane ◽  
Photosynthetic Membranes ◽  
Antenna Complex ◽  
Primary Structures

Abstract The antenna complex B 880 of Rp. marina has been isolated by applying ion-exchange chromatography on Whatman DE -52 resin and sucrose density centrifugation of LDAO-solubilized photosynthetic membranes. The antenna polypeptides B 880-α and B 880-β were pre­pared by organic solvent extraction of extensively dialyzed and freeze-dried B 880 antenna complex material or photosynthetic membranes. Gel filtration on Sephadex LH-60 and ion-exchange chromatography on Whatman DE -32 resin in the presence of organic solvents and an additional step on a C-8 reversed phase column yielded pure α-and β-apoproteins. Their complete primary structures have been elucidated using automated Edman degradation and carboxypeptidase diges­tion. According to quantitative Edman degradation the ratio of B 880-α and B 880-β has been determined as 1:1 in the isolated antenna complex as well as in the photosynthetic membrane. B 880-α of Rp. marina, presumably N -form ylated, consists of 52 amino acid residues and is 75, 56, 52 and 44% homologous to the corresponding core antenna polypeptides of Rs. rubrum, Rp. viridis, Rb. capsulatus and Rb. sphaeroides. In contrast, B 880-β (56 amino acid residues) is less homologous to the corresponding core β-antenna polypeptides of the same strains (57. 51. 41 and 42%). It shows an extended N-terminal domain as compared to the B 880-a polypeptide. Apart from the typical structural features of bacterial membrane-bound antenna polypeptides (three domain structure, His-residue in the hydrophobic stretch) the antenna polypeptides of Rp. marina are structurally related to polypeptides of core antenna complexes with strong near infra­red circular dichroism signals.

scholarly journals Confirmation of the 1–20 amino acid sequence of human adrenocorticotrophin

1973 ◽  
Vol 133 (1) ◽  
pp. 11-13 ◽  
Author(s):  
H. P. J. Bennett ◽  
P. J. Lowry ◽  
C. McMartin
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Sequence ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Countercurrent Distribution ◽  
Edman Degradation ◽  
Sequence Determination ◽  
Carboxypeptidase B ◽  
Aminopeptidase M

The tryptic fragments of natural human adrenocorticotrophin, were separated by countercurrent distribution and a correction in positions 25, 26, 27 and 30 was made by Riniker et al. (1972) in a study of the fragment containing residues 22–39. We have purified the remaining tryptic fragments, namely residues 1–8, 9–15, 16–21 and 17–21, by using ion-exchange chromatography on CM-cellulose and have carried out sequence determination by using the subtractive Edman degradation procedure and digestion with aminopeptidase M and carboxypeptidase B. These results have confirmed the proposed sequence for human adrenocorticotrophin in regions 6–7, 10–14 and 17–20, which had previously been arrived at only by analogy with the invariant sequence found in the three other mammalian adrenocorticotrophin species that had been investigated.

Quantitative Chromatographic Studies on Urinary Amino Acid Excretion

1968 ◽  
Vol 14 (1) ◽  
pp. 12-21 ◽  
Author(s):  
Richard P Geer ◽  
Richard K Hantman ◽  
Cyrus V Swett
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Acid Excretion ◽  
Amino Acid Nitrogen ◽  
Urinary Amino

Abstract Amino acid excretions of 82 individuals were quantitatively determined by ion-exchange chromatography. The results are expressed as µmoles amino acid per day, divided by milligrams α-amino acid nitrogen per day. This index is independent of age and provides a more useful method of representation than those presently employed in the literature.

Effect of Deproteinating Agents (Picric Acid and Sulfosalicylic Acid) on Analysis for Free Amino Acids in Swine Blood and Tissue

1969 ◽  
Vol 52 (5) ◽  
pp. 981-984 ◽  
Author(s):  
J E Knipfel ◽  
D A Christensen ◽  
B D Owen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino ◽  
Biological Fluids ◽  
Picric Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sulfosalicylic Acid ◽  
Coefficients Of Variation

Abstract Amino acid analyses were performed on samples of blood, liver tissue, loin muscle, and ham muscle by ion exchange chromatography after deproteination of the samples with picric acid or sulfosalicylic acid (SSA). Resolution of threonine and serine from the ion exchange column was poor when SSA was used as the deproteinating agent. Twelve of sixteen amino acids were higher (P < 0.05) in serum deproteinated with picric acid as compared to concentrations determined after SSA deproteination. Amino acid values for ham muscle tended to be higher after deproteination with picric acid; however, with liver and loin muscle samples, the values were somewhat higher after SSA deproteination. In both serum and tissue analyses, coefficients of variation were lower for niGSt amino acids when picric acid was utilized as the deproteinating agent. The latter observation, in particular, suggests that picric acid is preferable to SSA as a deproteinating agent before amino acid analyses of biological fluids. Standardization of methods of deproteination is needed to allow meaningful comparisons of data.

Isolation of a Crosslinked Peptide from Hitman Fibrin and Development of a Radioimmunoassay

1979 ◽  
Author(s):  
R. Canfield ◽  
B. Lahiri ◽  
R. D’Alisa ◽  
V. Butler ◽  
H. Nossel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Edman Degradation ◽  
Cnbr Cleavage ◽  
Normal Hemostasis

Factor XIIIa introduces up to six crossllnklng bonds per molecule of fibrin; the bonds between the γ chains on adjacent fibrin molecules form most rapidly. Since cross linking is essential for normal hemostasis and is likely to be important in tests to detect thrombosis, we have attempted to develop a radioimmunoassay that exhibits specificity for the γ chain crosslinks. The immunogen consisted of a 54 amino acid, crosslinked peptide, isolated from purified human γ-γ chains following CNBr cleavage, gel filtration on Sephadex G-50 and ion-exchange chromatography on SP-Sephadex. Amino acid analysis and Edman degradation through step 24 confirmed the sequence of Chen and Doolittle (Biochemistry 10: i486, 1971), and the two degradation steps that failed to liberate the expected PTH-amino acids matched the reported location of the Gin-Lys crosslinks. Antisera were obtained against this immunogen coupled either to bovine thyroglobulin or bovine serum albumin. All antisera elicited bound immunogen that was covalently coupled to ribonuclease radiolabeled with 125I as a tracer. The unlabeled γ-γ, crosslinked peptide effectively inhibited binding (0.03-0.08 picomoles for 50% inhibition), while with some antisera up to 500 times more of the 27 amino acid γ monomer peptide was required for the same degree of inhibition. Fibrinogen and fragment D also were poor Inhibitors. The results Indicate that it is possible by radioimmunoassay to distinguish the COOH-termlnal region of the γ-γ dlmer from that of uncrosslinked molecules.

scholarly journals Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1968 ◽  
Vol 106 (2) ◽  
pp. 531-541 ◽  
Author(s):  
P T Grant ◽  
K. B. M. Reid
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Bovine Insulin ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Performic Acid ◽  
Amino Acid Residues ◽  
Islet Tissue ◽  
A Chain

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11·5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.

scholarly journals Studies on the protein-bound chondroitin sulphate of bovine cortical bone

1968 ◽  
Vol 107 (1) ◽  
pp. 41-49 ◽  
Author(s):  
G. M. Herring
Keyword(s):  
Ion Exchange ◽  
Cortical Bone ◽  
Chondroitin Sulphate ◽  
Bone Sialoprotein ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Mild Conditions ◽  
The Third ◽  
Apparent Homogeneity

A fraction containing chondroitin sulphate, isolated from bovine cortical bone under mild conditions, was separated by ion-exchange chromatography into three fractions with apparent homogeneity on electrophoresis and ultracentrifugation. Two of these appeared to consist of chondroitin sulphate bound to a glycoprotein ‘core’ that had similarities to the bone sialoprotein described previously. The differences in composition of the two fractions were considered to be due to variation in the number or lengths of the polysaccharide chains. The presence of xylose and the alkali-lability of the bond between protein and polysaccharide suggested the presence of a xylosylserine linkage. The third fraction had the properties of a relatively pure chondroitin sulphate which contained a small amount of peptide. These fractions differed considerably from the protein–polysaccharide complexes of epiphysial and other cartilages, and their relevance to the possible role of glycosaminoglycans is discussed.

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