Characterization of proinsulin-like growth factor-II E-region immunoreactivity in serum and other biological fluids

Insulin-like growth factor-II (IGF-II) purified from human serum consists of 67 amino acids. However, mRNA sequence predicts a long carboxy-terminal extension (E-region), which on translation would yield a bigger IGF-II prohormone than the peptide isolated from serum. A peptide containing the predicted IGF-II prohormone sequence extending from Asp69 to Tyr84 (E-II) was synthesized by the solid state method and used to generate a polyclonal antiserum. Using this E-II antiserum, a specific RIA for IGF-II prohormone forms was developed. [125I]E-II was used as tracer, and synthetic E-II was used for the standard curve. The antiserum was highly sensitive and specific for E-II. It also recognized pro-IGF-II-E21, a 9.8-kilodalton (kDa) synthetic peptide which consisted of IGF-II plus the first 21-amino acid sequence of the IGF-II E-peptide region. Human biological fluids assayed included serum and amniotic, seminal, and cerebral spinal fluids. Sera from patients with chronic renal failure (CRF) and nonislet cell tumor hypoglycemia (NICTH) had the highest levels of E-II immunoreactivity. Amniotic and seminal fluids and acromegalic plasma had intermediate levels. E-II levels in normal, cord, and pregnancy sera were low, but measurable. To further characterize the molecular forms of the apparent E-II immunoreactivity, sera pooled from five normal subjects, five CRF patients, and one patient with NICTH were chromatographed separately over a Sephadex G-50 column in formic acid. With all samples, there was a major peak of E-II immunoreactivity at about 15 kDa, consistent with the predicted size of the IGF-II prohormone. There was a second smaller peak at about 10 kDa in NICTH serum. With CRF serum, there was a prominent peak at 3 kDa, which probably consisted of breakdown products of the IGF-II E-peptide region. The 15-kDa peak was highest in NICTH serum. With all three samples, IGF-I eluted in a single peak at about 7 kDa and was low in NICTH serum. There were two peaks of IGF-II. The first coincided with the peak of E-II at 15 kDa, while the second comigrated with IGF-I. By Western immunoblot analysis, the E-II antiserum detected pro-IGF-II-E21 as a band at 10 kDa, but it did not recognize 7.5-kDa recombinant IGF-II, which did not have the E-peptide region.(ABSTRACT TRUNCATED AT 400 WORDS)