Biomedical subjects
Publications and source records attributed to M W Hunkapiller.
A 4,000-dalton dynorphin was isolated from porcine pituitary. It has 32 amino acids (Mr = 3,986), with the previously described heptadecapeptide (now called dynorphin A) at its amino terminus and a related tridecapeptide, dynorphin B, at its carboxyl terminus. The two peptides are separated by the "processing signal" Lys-Arg.
The amino-terminal amino acid sequences of the four major peptides (Mr 41,000, 50,000, 55,000, and 62,000) present in purified preparations of Electrophorus electricus nicotinic acetylcholine receptor (AcChoR) have been determined for 24 cycles by automated sequence analysis procedures yielding four unique polypeptide sequences. The sequences showed a high degree of similarity, having identical residues in a number of positions ranging between 37% and 50% for specific pairs of subunits. Comparison of the sequences obtained with those of the subunits of similar molecular weight from Torpedo californica AcChoR revealed an even higher degree of homology (from 46% to 71%) for these two highly diverged species. Simultaneous sequence analysis of the amino termini present in native, purified Electrophorus AcChoR showed that these four related sequences were the only ones present and that they occur in a ratio of 2:1:1:1, with the smallest subunit ("alpha 1") being present in two copies. Genealogical analysis suggests that the subunits of both Torpedo and Electrophorus AcChoRs derive from a common ancestral gene, the divergence having occurred early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as the highly conserved structure of the AcChoR complex along animal evolution, suggest that each of the subunits evolved to perform discrete crucial roles in the physiological function of the AcChoR.
Explore the source record for details and available documents.
The amino-terminal sequence of the Tn3 transposase protein was determined to be Pro-Val-Asp-Phe-Leu-Thr-Thr-Glu-Gln-Val-Glu-Ser.... This was determined both from an active transposase protein purified from a transposase overproducing mutant strain and from a hybrid transposase-beta-galactosidase fusion protein. The amino acid sequence corresponded to the DNA sequence of the transposase gene beginning at an ATG initiation codon, as previously predicted from the analysis of transposase-beta-galactosidase gene fusions.
A new miniaturized protein and peptide sequenator has been constructed which uses gas phase reagents at the coupling and cleavage steps of the Edman degradation. The sample is embedded in a matrix of Polybrene dried onto a porous glass fiber disc located in a small cartridge-style reaction cell. The protein or peptide, though not covalently attached to the support, is essentially immobile throughout the degradative cycle, since only relatively apolar, liquid phase solvents pass through the cell. This instrument can give useful sequence data on as little as 5 pmol or protein, can perform extended sequence runs (greater than 30 residues) on subnanomole quantities of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and can sequence hydrophobic peptides to completion. The sequenator is characterized by a high repetitive yield during the degradation, low reagent consumption, low maintenance requirements, and a degradative cycle time of only 50 min using a complete double cleavage program.
Thirteen of the first fifteen amino acids from the NH2-terminus of the primary sequence of human cytochrome c oxidase subunit I and eleven of the first twelve amino acids of subunit II have been identified by microsequencing procedures. These sequences have been compared with the recently determined 5'-end proximal sequences of the HeLa cell mitochondrial mRNAs and unambiguously aligned with two of them. This alignment has allowed the identification of the putative mRNA for subunit I, and has shown that the initiator codon for this subunit is only three nucleotides away from the 5'-end of its mRNA; furthermore, the results have substantiated the idea that the translation of human cytochrome c oxidase subunit II starts directly at the 5'-end of its putative mRNA, as had been previously inferred on the basis of the sequence homology of human mitochondrial DNA with the primary sequences of the bovine subunit.
Gap junctions, strongly implicated as channels for direct cell-to-cell communication, have been isolated from rat liver in high yield and purity. These gap junction fractions contain few morphologically recognizable contaminants, but NaDodSO4/polyacrylamide gel electrophoresis reveals a number of polypeptides. With the exception of a nonjunctional component of Mr 38,000 and some poorly soluble material, including collagen, all the polypeptides have very similar or identical two-dimensional peptide maps and arise from proteolytic cleavage of the COOH-terminus or aggregation of a Mr 28,000 protein. We report the sequence of the NH2-terminal 52 amino acids of this protein. The polypeptide (Mr approximately equal to 10,000) characteristic of trypsin-treated gap junction preparations is shown to be two distinct polypeptides, both derived from the Mr 28,000 protein.
Radioactive human fibroblast-derived beta interferon has been synthesized in vivo after induction of human diploid fibroblast cells with poly I:C. The radioactive protein is not synthesized and secreted by uninduced cells. It reacts with antibody made against homogeneous human beta interferon and has the same N-terminal amino acid sequence as human beta interferon.
The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.
Explore the source record for details and available documents.
The purification of human fibroblast interferon has been simplified to a two-step procedure consisting of affinity chromatography on Blue Sepharose and sodium dodecyl sulfate polyacrlamide gel electrophoresis. A preliminary amino acid composition and the sequence of the 13 amino-terminal residues of homogeneous interferon prepared by this method is reported.
Homogeneous human lymphoblastoid interferon with an apparent molecular size of 18,500 daltons was characterized by its amino acid composition. Analysis of the amino terminal sequence by Edman degradation indicates that the sequence is unique.
Mouse interferons of three size classes (A, 35,000 to 40,000 daltons; B, 26,000 to 33,000 daltons; and C, 20,000 daltons) were purified from Ehrlich ascites tumor cells infected with Newcastle disease virus. The sequences of the first 24 amino acids (No. 17 has not been identified) of interferons A and B are identical. The sequence of the first 20 amino acids of interferon C differs from that of A and B in 18 positions. There is partial homology in amino terminal sequence between mouse interferons A (or B) and a human fibroblast interferon and between mouse interferon C and a human lymphoblastoid interferon.
Two neuroactive peptides, A and B, have been isolated from the atrial gland in the reproductive tract of Aplysia. Each of the two peptides is able to induce egg-laying behavior in recipient animals. In vitro recordings from the abdominal ganglion show that both peptides also trigger longlasting discharges in the bag cell neurons at concentrations around 0.1 muM. The peptides were purified by a combination of ammonium sulfate precipitation, agarose gel filtration, and cation exchange chromatography. Each peptide has 34 amino acid residues. Microsequencing together with carboxypeptidase Y degradation and analysis of tryptic peptides revealed the following sequence for peptide A: H-Ala-Val-Lys-Leu-Ser-Ser-Asp-Gly-Asn-Tyr-Pro-Phe-Asp-Leu-Ser-Lys-Glu-Asp-Gly -Ala-Gln-Pro-Tyr-Phe-Met-Thr-Pro-Arg-Leu-Arg-Phe-Tyr-Pro-Ile. Peptide B differs from A in only four positions. The first nine residues of B are: Ala-Val-Lys-Ser-Ser-Ser-Tyr-Glu-Lys-, whereas residues 10-34 of B are identical to those of A. The calculated M(r) of A is 3924 and that of B is 4032. The pI of peptide A as determined by isoelectric focusing in polyacrylamide gels is 7.9-8.1 and that of peptide B is 9.0-9.2. It is estimated that each atrial gland contains at least 150 mug of peptide A and 50 mug of B. Neither peptide resembles the egg-laying hormone isolated from bag cell neurons. It is postulated that the atrial gland peptides are released during copulation, and then by interacting with neuronal receptors in the head ganglia and pleuroabdominal connectives they cause the bag cells to afterdischarge, thereby releasing egg-laying hormone.
An octacosapeptide that we named pro-somatostatin has been isolated from acid extracts of porcine hypothalami and found to have the amino acid sequence Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys. This octacosapeptide possesses high somatotropin (growth hormone) and prolactin release-inhibiting activity in vitro. It also crossreacts strongly with antisera generated against the somatostatin tetradecapeptide. This octacosapeptide is most likely a precursor (pro-hormone) of somatostatin in the hypothalamus. The existence of still larger molecular size precursors of somatostatin was also observed.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.