scholarly journals Precursors in the biosynthesis of vasopressin and oxytocin in the rat. Characteristics of all the components in high-performance liquid chromatography

1982 ◽  
Vol 208 (2) ◽  
pp. 339-349 ◽  
Author(s):  
R W Swann ◽  
C B Gonzalez ◽  
S D Birkett ◽  
B T Pickering
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Supraoptic Nucleus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Wistar Rat ◽  
Common Precursor ◽  
Rat Pituitary ◽  
Pituitary Glands

A reverse-phase high performance liquid-chromatography (h.p.l.c.) protocol has been developed, whereby all the major known posterior-pituitary components that are derived from the processing of pro-oxytocin and pro-vasopressin can be separated one from another. Thus, in a single chromatographic step, it has been possible to separate vasopressin (VP), oxytocin (OT), oxytocin-neurophysin (rOT-Np), vasopressin-neurophysin (rVP-Np) and vasopressin-glycopeptide (rVP-GP) from acid extracts of the neurointermediate lobes of rat pituitary glands. All these peptides except rVP-GP were labelled in the neural lobe by 24h after a hypothalamic injection of [35S]cysteine, whereas all except VP were labelled by 24h after a similar injection of [3H]leucine. Three major labelled proteins were isolated from 20 min [35S]cysteine-injected rats when extracts of the supraoptic nucleus were subjected to Sephadex G-75 chromatography, h.p.l.c. and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Immunoprecipitation with antisera raised against rat neurophysins, VP and OT revealed 21000- and 19000-mol.wt. common precursors to VP and rVP-Np and a 15000-mol.wt. common precursor to OT and rOT-Np. Some immunoreactive rVP-Np could occasionally be detected in the Vo of Sephadex G-75 chromatograms of Wistar rat supraoptic-nucleus extracts, but no evidence of [35S]neurophysin in this fraction was obtained from h.p.l.c. fingerprinting of its S-carboxymethylated tryptic digests. Radioimmunoassay for rVP-Np and rOT-Np revealed that about 70-80% of the total recovered immunoreactive neurophysin (IR-Np) in the supraoptic nucleus eluted from Sephadex G-75 and h.p.l.c. in the positions of rVP-Np and rOT-Np. Evidence is presented for an approx. 20000-mol.wt. rOT-Np in both Wistar and Brattleboro rats and for an approx. 20000-mol.wt. component in the Brattleboro rat that is recognized by vasopressin-neurophysin antisera.

A glyco-phosphoprotein in human milk

1987 ◽  
Vol 54 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Norihiro Azuma ◽  
Kunio Yamauchi
Keyword(s):  
Molecular Weight ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Gel Electrophoresis ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Reversed Phase ◽  
Ultracentrifugal Analysis

SummaryA highly glycosylated phosphoprotein (HGPP) was isolated from a human casein fraction by reversed-phase high-performance liquid chromatography. This component contained carbohydrates to ∼ 38·2% (w/w) and phosphorus to ∼ 1·6% (w/w). The molecular weight of this HGPP as estimated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis ∼ 41000. Ultracentrifugal analysis revealed that the sedimentation coefficient of the HGPP was 2·6S in a 10 mM-imidazole-HCl buffer at pH 7·0 and 27 °C, but this component interacted with human κ-casein and formed a complex with s = 10·4S.

Purification of goats' milk casein by reversed-phase high-performance liquid chromatography and identification of αs1-casein

1987 ◽  
Vol 54 (3) ◽  
pp. 361-367 ◽  
Author(s):  
Jan Mikkelsen ◽  
Peter Højrup ◽  
Jens Knudsen
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Reversed Phase ◽  
Terminal Sequence ◽  
Phase Gradient ◽  
Milk Casein

SummaryGoats’ milk caseins were separated into four components in a single run using reversed-phase gradient high-performance liquid chromatography. The purity of the isolated components was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis, amino acid analysis and determination of the N-terminal residue. By a comparison with previously published results for goats’ milk caseins the four peaks were identified as κ-, αs1-, αs2- and β-casein. In order to confirm the existence of αsl-casein in goats’ milk, this component was sequenced for 44 steps, revealing a sequence homologous to bovine αsl-casein and almost identical to the N-terminal sequence previously published by Boulanger et al. (1984).

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