Proteins isolated from regenerating sciatic nerves of rats form aggregates following posttranslational amino acid modification.
Soluble proteins of regenerating sciatic nerves of rats can be posttranslationally, covalently modified by a variety of radioactive amino acids. The present study shows that once modified by a mixture of 15 amino acids, many of those proteins form aggregates that are unable to pass through a 0.45-micron filter and pellet following 20,000g centrifugation (suggesting a size of greater than 2 x 10(6) Da). Aggregation of proteins also occurs following modification by Arg or Lys alone, but does not occur following protein modification in nonregenerating nerves or in brain. Aggregates are not disrupted by treatment with 100 mM beta mercaptoethanol or by exposure to 1.0 M NaCl, but aggregates are solubilized by treatment with urea and by boiling in 1.5% SDS. Amino acid analysis of proteins modified by a mixture of [3H]amino acids shows a similar proportion of posttranslationally incorporated Ser, Pro, Val, Ala, Leu, Phe, Lys, and Arg in the soluble and pelletable fractions. Two-dimensional PAGE profiles of soluble and pelletable modified proteins show that the modified proteins in both fractions are in similar pI and molecular weight ranges, except that the soluble modified proteins include a high-molecular-weight component that is absent in the pelleted modified proteins. Kinetic studies show that while half-maximal levels of protein modification occur within 30 seconds of incubation, the appearance of the pelletable modified protein fraction is delayed significantly. These results indicate that amino acid modification of soluble proteins in regenerating sciatic nerves of rats results in physical changes in those proteins so that they form high-molecular-weight aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)
Ingoglia
N A
NA
Department of Physiology, UMDNJ-New Jersey Medical School, Newark 07103.
Chakroborty
G
G
Yu
M
M
Luo
D
D
Sturman
J A
JA
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008297:Male; D009416:Nerve Regeneration; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012584:Sciatic Nerve / Q000502:physiology*; D014316:Tritium
D000596:Amino Acids; D009419:Nerve Tissue Proteins; D012346:RNA, Transfer, Amino Acyl; D014316:Tritium