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Biomedical subjects

Y C Pan

Publications and source records attributed to Y C Pan.

At least 91 records · Page 5Linked to original sources
PubMed1986

Rapid enzymatic degradation of growth hormone-releasing hormone by plasma in vitro and in vivo to a biologically inactive product cleaved at the NH2 terminus.

The effect of plasma on degradation of human growth hormone-releasing hormone (GRH) was examined in vitro and in vivo using high performance liquid chromatography (HPLC), radioimmunoassay (RIA), and bioassay. When GRH(1-44)-NH2 was incubated with human plasma, the t1/2 of total GRH immunoreactivity was 63 min (RIA). However, HPLC revealed a more rapid disappearance (t1/2, 17 min) of GRH(1-44)-NH2 that was associated with the appearance of a less hydrophobic but relatively stable peptide that was fully immunoreactive. Sequence analysis indicated its structure to be GRH(3-44)-NH2. Identity was also confirmed by co-elution of purified and synthetic peptides on HPLC. Biologic activity of GRH(3-44)-NH2 was less than 10(-3) that of GRH(1-44)-NH2. After intravenous injection of GRH(1-44)-NH2 in normal subjects, a plasma immunoreactive peak with HPLC retention comparable to GRH(3-44)-NH2 was detected within 1 min and the t1/2 of GRH(1-44)-NH2 (HPLC) was 6.8 min. The results provide evidence for GRH inactivation by a plasma dipeptidylaminopeptidase that could limit its effect on the pituitary.

Adult↗
PubMed1986

Guinea pig glucagon differs from other mammalian glucagons.

Mammalian glucagon is thought to be highly conserved. Glucagons from pig, cow, human, rat, and hamster have identical amino acid sequences, whereas the amino acid contents of rabbit and camel glucagons are consistent with this 29-amino acid sequence. It had earlier been reported that guinea pig (GP) glucagon contains 40 amino acids. In the current study, glucagon was purified from two GP pancreata by a series of three HPLC steps after acid-alcohol extraction and acetone precipitation. GP glucagon is a 29-amino acid peptide that differs from other mammalian glucagons by substitution of Gln for Asp in position 21, Leu for Val in position 23, Lys for Gln in position 24, Leu for Met in position 27, and Val for Thr in position 29. In view of the marked changes in the COOH-terminal of GP glucagon, receptor binding studies were performed using both rat and GP liver membranes. Labeled synthetic porcine glucagon has similar binding in the two systems and its binding is inhibited to a similar degree by synthetic porcine glucagon, whereas GP glucagon is 10-fold less potent at inhibiting binding in both systems. This suggests that glucagon receptor binding sites in the GP are evolutionarily more conserved than is GP glucagon.

Amino Acid Sequence↗
PubMed1985

Guinea pig "little" gastrin is a hexadecapeptide.

Gastrin heptadecapeptides (gastrins I and II which differ in the presence of sulfate on the tyrosine of the latter) have been purified and sequenced from several mammalian species including pig, dog, cat, sheep, cow, human and, more recently, rat. This report describes the purification of "little" gastrin from guinea pig (GP) antra. GP antra were defatted with acetone and the acetone cakes were extracted with 0.1M NH4HCO3. The extract was concentrated by adsorption to DE53 anion exchange cellulose and the peak eluates were fractionated on a Sephadex G50F column. The peptides were brought to final purity by 3 successive HPLC steps. The GP sequence compared to other species is shown: (formula: see text) Thus GP "little" gastrins I and II are hexadecapeptides due to a deletion of a glutamic acid in the region 6-9 from the N-terminus.

Amino Acid Sequence↗
PubMed1985

Purification of dog VIP from a single animal.

VIP, a potent vasodilator peptide, is reported to be identical in pig, cow, human and rat but to differ in four amino acids in chicken. This report describes the purification of dog VIP from the small intestine of a single animal. The purification method is based on tissue extraction with a sequence of organic solvents. The extracted VIP is concentrated onto cation-exchange cellulose and brought to purity by three HPLC steps. A 30% final yield of pure VIP was obtained from the original extract. Dog VIP was found to have the following sequence: His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala -Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn. Thus the amino acid sequence of dog VIP is identical with all the mammalian VIP's which have been reported. This suggests that a high degree of conservation throughout the molecule may be required for VIP bioactivity.

Amino Acid Sequence↗
PubMed1985

Guinea pig has a unique mammalian VIP.

Mammalian vasoactive intestinal peptide (VIP) has been reported to be identical in four species. This report describes the extraction of guinea pig (GP) intestinal VIP, its purification and sequence. Frozen intestines were extracted in five volumes of methanol and the methanol cakes reextracted with acid. VIP in the acid extract was concentrated onto ion-exchange cellulose and was brought to final purity through a series of HPLC steps. GP VIP differs from other mammalian VIP's by four amino acid substitutions: (sequence in text) This is further evidence that the GP gastroenteropancreatic axis has a unique evolutionary separation from other mammals.

Amino Acid Sequence↗
PubMed1985

Purification and partial sequencing of bovine liver alkaline phosphatase.

Bovine liver alkaline phosphatase has been purified to homogeneity by procedures that include reverse-phase HPLC. The pure enzyme has an apparent Mr of 160,000 and is composed of what appears to be two identical monomers of Mr 82,000. About 80% of the material yielded the amino-terminal sequence Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-?-Tyr-?-Arg-Asp-Gln-Ala-Gln. The minor component was extended at the amino terminus by two residues that have not yet been identified, i.e., ?-?-Leu-Val-Pro-Glu-Lys-Glu-Lys-Asp-Pro-?-Tyr-?-Arg-Asp-Gln-Ala-Gln.

Alkaline Phosphatase↗
PubMed1985

Isolation and characterization of a cDNA and a pseudogene for mouse lactate dehydrogenase-A isozyme.

A mouse lactate dehydrogenase-A cDNA was isolated and it was shown to contain the 393bp of the protein-coding sequence and 488bp of the 3' untranslated region. The amino acid sequence deduced from its open reading frame provided independent evidence for the sequence of residues 201-331 of mouse LDH-A subunit (muscle). This cDNA clone was used as a probe to isolate a mouse genomic clone containing a truncated, processed LDH-A pseudogene. This pseudogene showed 81.6% homology at 713 positions compared with the LDH-A cDNA sequence. The divergence of this pseudogene was estimated to have occurred 39 million years ago.

Animals↗
PubMed1984

Use of high-performance liquid chromatography for preparing samples for microsequencing.

A method has been developed for the microsequencing of protein at subnanomole levels. The protein is carboxymethylated and freed of salts and reagents by reversed-phase chromatography prior to automated Edman degradation on a gas-phase sequencer. The carboxymethylated protein can also be fragmented chemically or enzymatically for further sequence analysis. The analytical techniques used to monitor the progress of the reactions all have picomole level sensitivity.

Amino Acid Sequence↗
PubMed1984

Characterization of small amounts of peptides and proteins.

The use of microchemical methods for the characterization of both natural and recombinant proteins of biomedical importance is discussed. The methods include gel electrophoresis, high-performance liquid chromatography, protein fragmentation, amino acid analysis and automated Edman degradation. Each procedure is applicable at the picomole level.

Journal Article↗
PubMed1984

A species of human alpha interferon that lacks the ability to boost human natural killer activity.

Most species of recombinant leukocyte interferons (IFN-alpha A, -alpha B, -alpha C, -alpha D, -alpha F, -alpha I, and -alpha K) were capable of boosting human natural killer (NK) activity after a 2-hr treatment of cells at a concentration of 1-80 units/ml. In contrast, recombinant human IFN-alpha J was found to be incapable of augmenting NK activity after exposure of cells for 2 hr to concentrations as high as 10,000 units/ml. This inability of IFN-alpha J to boost NK activity was not complete because, after exposure of cells to a high concentration of IFN-alpha J (10,000 units/ml) for 18 hr, boosting of cytolysis was observed. IFN-alpha J appeared to interact with receptors for IFN on NK cells since it was found to interfere with the boosting of NK activity by other species of IFN-alpha. In contrast to its deficient ability to augment NK activity, IFN-alpha J has potent antiviral and antiproliferative activities. Such extensive dissociation of these biological activities has not been observed previously with any other natural or recombinant IFN species. Thus, this IFN species may be useful for evaluating the relative importance of various biological activities on the therapeutic effects of IFN, for understanding structure-function relationships, and for determining the biochemical pathways related to the various biological effects of IFN.

Amino Acid Sequence↗
PubMed1984

Purification to homogeneity and partial characterization of interleukin 2 from a human T-cell leukemia.

A method utilizing reversed-phase high-performance liquid chromatography has been developed for the purification to homogeneity of interleukin 2 (IL-2) isolated from a human T-cell leukemia. A final purification of 500,000-fold was obtained with a specific activity of pure IL-2 of 10(9) units/mg. The amino acid analysis of natural IL-2 is strikingly similar to the composition deduced from sequence analysis of a cDNA coding for human IL-2. Protein sequence analysis of CNBr-derived peptides yields data consistent with the sequence proposed from cloned cDNA. The availability of homogeneous IL-2 will allow accurate biological studies of its activity free from the contamination of the numerous lymphokine species that are known to be co-produced with IL-2 during the induction procedure.

Amino Acid Sequence↗
PubMed1983

Purification and properties of a type beta transforming growth factor from bovine kidney.

Type beta transforming growth factor (TGF-beta) has been purified 200 000-fold from bovine kidneys. This peptide is characterized by its ability to induce anchorage-dependent normal rat kidney cells to grow in soft agar in the presence of epidermal growth factor (EGF); TGF-beta is not mitogenic for cells grown in monolayer culture. Purified TGF-beta does not compete with EGF for binding to membrane receptors. The concentration of TGF-beta required to elicit a half-maximal response for formation of colonies greater than 3100 micron2 in the soft agar assay is 2-3 pM (55 pg/mL) when assayed in the presence of 0.8 nM EGF (5 ng/mL). The four-step purification procedure which includes chromatography of acid--ethanol tissue extracts on polyacrylamide sizing gels, cation exchange, and two steps of high-pressure liquid chromatography results in a 10% overall yield of colony-forming activity with a recovery of 3-4 micrograms/kg. Amino acid analysis of purified TGF-beta shows 16 half-cystine residues per mole. Analysis of the purified polypeptide by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels indicates that TGF-beta is composed of two closely related polypeptide chains cross-linked by disulfide bonds. In the absence of beta-mercaptoethanol, the colony-forming activity is associated with a single silver-staining band of molecular weight 25 000; in the presence of beta-mercaptoethanol, the TGF-beta is converted to an inactive species that migrates as a single band of molecular weight 12 500-13 000. Sequence analysis indicates that at least the first 15 N-terminal amino acids of the two TGF-beta subunits are identical.

Amino Acid Sequence↗
PubMed1983

Molecular features and immunological properties of lactate dehydrogenase C4 isozymes from mouse and rat testes.

With the use of a computer graphics system, spacefilling models of mouse and rat testis lactate dehydrogenase C4 (LDH-C4) isozymes were constructed from amino acid sequence and published x-ray diffraction data. Thirty-two residues that differ between the mouse and rat LDH-C4 sequences are also displayed on the monomeric and tetrameric lactate dehydrogenase molecules. Immunological properties of mouse and rat LDH-C4 isozymes are compared and related to these 32 differences. Possible relationships between the structure, especially the amino acid sequence, and the unique enzymatic properties of LDH-C4 isozymes are also discussed.

Amino Acid Sequence↗
PubMed1983

Amino acid sequence studies on lactate dehydrogenase C4 isozymes from mouse and rat testes.

Carboxymethylated sperm-specific lactate dehydrogenase isozyme C4 (LDH-C4) proteins from mouse and rat testes were cleaved with cyanogen bromide and trypsin. Proteins were also citraconylated and digested with trypsin. In the case of mouse LDH-C4 isozyme, all 7 CNBr and 11 limited tryptic (arginine) peptides were isolated and sequenced. Some of the CNBr peptides were further fragmented with trypsin and chymotrypsin and their compositions and/or sequences characterized. Also, 34 of the 36 expected tryptic peptides were purified, and their compositions and sequences determined. Amino acid sequences of these peptides purified from mouse LDH-C4 were overlapped into a complete covalent structure of the 330 residues. For rat LDH-C4, 5 of 6 expected CNBr peptides, 5 of 8 expected arginine peptides, and 28 of the 34 expected tryptic peptides were isolated, and their compositions and sequences were determined. Some of the CNBr and arginine peptides were further fragmented with chymotrypsin, thermolysin, or V8 protease, and their compositions and/or sequences characterized. The amino acid sequence of 85% of the 330 residues from rat LDH-C subunit has been unambiguously determined, and the sequences of the remaining regions were tentatively aligned on the basis of peptide compositions and sequence homologies with the other known lactate dehydrogenase sequences, including mouse LDH-C. A comparison of the proposed rat LDH-C sequence with the complete covalent structure of mouse LDH-C indicates that 27 differences are located in the established rat LDH-C sequence of 280 residues and that 5 additional differences are in the tentative sequence of the remaining 50 amino acids.

Amino Acid Sequence↗
PubMed1983

Evolutionary relationships of vertebrate lactate dehydrogenase isozymes A4 (muscle), B4 (heart), and C4 (testis).

The amino acid sequence variation among seven lactate dehydrogenase isozymes from dogfish muscle, chicken muscle and heart, pig muscle and heart, and mouse and rat testes were compared with respect to the whole lactate dehydrogenase polypeptide chain as well as their four functional domains. The coenzyme-binding domain is more conserved than the substrate-binding domain. The sequence of the loop and helix alpha D region of testicular LDH-C4 isozymes is very different from those of somatic LDH-A4 and LDH-B4 isozymes, while the NH2-terminal arm is extremely variable. The most parsimonious phylogenetic tree among these seven vertebrate lactate dehydrogenase sequences clearly indicates that the LDH-A4 and LDH-B4 isozymes are more closely related to each other than either to the LDH-C4 isozymes.

Amino Acid Sequence↗
PubMed1983

Studies of amino-acid sequence in dihydrofolate reductase from a human methotrexate-resistant cell line KB/6b. Structural and kinetic comparison with mouse L1210 enzyme.

The partial amino acid sequence of dihydrofolate reductase (DHFR, EC 1.5.1.3) from human KB/6b cells has been determined by using 3.5 mg of protein. Peptides covering the entire polypeptide chain were recovered from preparative peptide maps generated by the combination of paper chromatography and electrophoresis at pH 4.4 Peptide maps from mouse L1210 DHFR were also generated for comparison. Amino acid sequence of 75% of the 186 amino acid residues in the polypeptide chain of human KB/6b DHFR was obtained from Edman degradations and the remaining sequence was deduced from the amino acid compositions, from electrophoretic mobilities of related peptides and from the sequence homologies with other known mammalian DHFR sequences. A comparison of the proposed human DHFR sequence with the previously known sequences of mouse enzyme [Stone, et al. (1979) J. Biol. Chem. 245, 480-488] indicates that 18 differences are located in the established sequence of 139 residues and that 5 additional differences are in the tentative sequence of the remaining 47 amino acids. Kinetic properties of human KB/6b and mouse L1210 DHFR, which were determined in parallel experiments, are also compared. The possible structural-functional relationships between human and mouse DHFR are discussed.

Amino Acid Sequence↗
PubMed1983

Pig brain contains cholecystokinin octapeptide and several cholecystokinin desoctapeptides.

A sequential method employing methanol extraction of the COOH-terminal fragment of cholecystokinin (CCK) from pig brain followed by HCl extraction of the more basic CCK peptides was used as the first step in purification of these peptides. Recovery was monitored with two different assays, one directed to the COOH terminus of CCK and the other to the NH2 terminus. The amino acid content and sequence were determined for each of five peptides after purification. The only peptide containing COOH-terminal immunoreactivity was CCK-octapeptide (CCK8). The other four peptides did not contain CCK8 and had lost one or two additional amino acids, perhaps as a consequence of the action of carboxypeptidases. These peptides were shown to be CCK33-desnonapeptide, CCK39-desnonapeptide and -desdecapeptide, and a large molecular weight precursor, CCK58-desnonapeptide, containing 19 amino acids (Ala-Val-Gln-Lys-Val-Asp-Gly-Glu-Ser-Arg-Ala-His-Leu-Gly-Ala-Leu-Leu-Ala-Arg) NH2-terminal to CCK39. The three NH2-terminal fragments of CCK58, CCK39, and CCK33 were about equally prominent. The brain, unlike the gut, appears to cleave CCK8 rapidly from a precursor peptide but to process the NH2-terminal portions of the molecule more slowly and incompletely.

Amino Acid Sequence↗