Secreted mouse prolactin (PRL) and stored ovine PRL. I. Biochemical characterization, isolation, and purification of their electrophoretic isoforms.
The biochemical nature of the electroisomers of secreted mouse PRL and stored ovine PRL were compared. When examined on alkaline polyacrylamide gels, they exhibited electrophoretic heterogeneity. The electrophoretic isomers had the same molecular size by examination of Ferguson plots and therefore differed only in net negative charge at alkaline pH. There was no apparent charge heterogeneity in the PRL preparations when they were electrophoresed at a pH below the pKa of the side-chain carboxyl groups of aspartic acid and glutamic acid; however, they exhibited size heterogeneity owing to aggregation. The slowest migrating electroisomers (from alkaline gels) converted spontaneously into faster migrating forms at 37 C in either acid (pH 4.0) or alkaline (pH 8.0) environments. The rate constant (determined at 37 C) for the transformation of the native PRL into faster migrating isoforms was 10.76 X 10(6) sec-1 at pH 8.0 and 0.50 X 10(6) sec-1 at pH 4.0. When secreted mouse PRL was incubated in alkaline (pH 10.0) conditions at 25 C, ammonia was released as the conversion reaction occurred. These results indicate that the electroisomers separated at alkaline pH are charge isoforms differing only in the number of free carboxyl groups of glutamic and/or aspartic acid residues. The des-amido isoforms were fractionated and purified. Polyacrylamide gel electrophoresis at alkaline pH was used to separate the charge isomers from each other. Each isoform was then excised from the separating matrix and recovered via electrophoretic elution. The homogeneity of each isoform was monitored by polyacrylamide gel electrophoresis.
D000641:Ammonia / Q000378:metabolism; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D051379:Mice; D011388:Prolactin / Q000031:analogs & derivatives / Q000302:isolation & purification* / Q000378:metabolism; D012756:Sheep
D000641:Ammonia; D011388:Prolactin