D R Marshak

The extension of neurites by chicken embryo cerebral cortical neurons can be measured quantitatively at low cell density in serum-free, defined medium. An acidic, heat-stable protein fraction from bovine brain has been shown to have neurite extension activity in this assay. We report the use of reversed-phase HPLC to purify a neurite extension factor from this fraction to apparent homogeneity. The protein was characterized by NaDodSO4/PAGE. In the presence of reducing agents, the protein migrated as a single band, with an apparent molecular weight of 6500. In the absence of reducing agents, the protein showed bands at apparent molecular weights of 6500, 21,000-22,000, 30,000, and 40,000. Reduction and S-carboxymethylation of the protein abolished all biological activity and resulted in a shift of the apparent molecular weight to 11,000. The amino acid composition of the purified neurite-extension factor was nearly identical to that of bovine brain S100 beta. The amino acid sequences of peptides derived from trypsin or cyanogen bromide digests of the protein were identical to those found in S100 beta and accounted for 71 of 91 amino acids in the protein. However, three peptides obtained from cyanogen bromide digestion of the nonreduced protein appeared to be disulfide-linked dimers. Our results indicate that a biological activity, neurite extension, which is critical for the development of the nervous system, is associated with a disulfide form of S100 beta.

The human anti-oncoprotein p53 is shown to be a substrate of cdc2. The primary site of phosphorylation is serine-315. Serine-315 is phosphorylated by both p60-cdc2 and cyclin B-cdc2 enzymes. The phosphorylation of p53 is cell cycle-dependent. The abundance of p53 also oscillates during the cell cycle. The protein is largely absent from cells that have just completed division but accumulates in cells during G1 phase. Phosphorylation by cdc2 might regulate the antiproliferative activity of p53.

p53 is a sequence-specific DNA-binding oligomeric protein that can activate transcription from promoters bearing p53-binding sites. Whereas the activation region of p53 has been identified within the amino terminus, the location of the specific DNA-binding domain has not been reported. Thermolysin treatment of p53 protein generates a stable protease-resistant fragment that binds with marked specificity to p53 DNA-binding sites. Amino-terminal sequencing of the fragment located the thermolysin cleavage site to residue 91. Because the fragment does not contain the cdc2 phosphorylation site at Ser-315, we conclude that the the site-specific DNA-binding domain of p53 spans the central region of the protein. The vast majority of the mutations in oncogenically derived p53 proteins are located within this central portion of the molecule. Such mutant p53 proteins exhibit defective sequence-specific DNA-binding. Although thermolysin digestion of mutant p53 proteins generates proteolytic patterns that differ from wild-type protein, one mutant tested, His-273, generates a resistant fragment that migrates with a similar electrophoretic mobility to the wild-type protease-resistant fragment. Interestingly, although intact mutant His-273 protein binds to DNA at 20 degrees C, the thermolysin-resistant mutant fragment does not. In addition, the central protease-resistant, site-specific binding region of wild-type p53 does not demonstrate nonspecific DNA-binding. Thus, although sequences outside of the central region of p53 contribute to both nonspecific DNA-binding and oligomerization, they are not required for sequence-specific DNA-binding.

The primary structure of gonadotropin-releasing hormone (GnRH) isolated from whole brains of lamprey is pGlu-His-Tyr-Ser-Leu-Glu-Trp-Lys-Pro-Gly-NH2. This unique decapeptide was isolated and purified from brain extracts by reverse-phase high performance liquid chromatography. The structure of the peptide was established from chymotryptic fragments that were identified by protein sequence analysis and fast atom bombardment mass spectrometry. The peptide reacts with an antiserum raised against mammalian GnRH and is structurally identified as a member of the GnRH family by the amino and carboxyl termini of pGlu1-His2 and Pro9-Gly10NH2, the conservation of Ser4 in the internal segment of the molecule and its length of 10 amino acids. For the first time, amino acid substitutions are found in positions 3 and 6, critical for biological potency and conformation, respectively. Additionally, a second form of GnRH (lamprey II GnRH), representing about 10% of the total GnRH immunoreactive material in the brain, was isolated; its amino acid composition differs by 3 residues from lamprey I GnRH. Synthetic lamprey I GnRH elevates plasma estradiol in adult female lampreys.