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Y C Pan

Publications and source records attributed to Y C Pan.

At least 55 records · Page 3Linked to original sources
PubMed1990

Purification and characterization of glycosyl-phosphatidylinositol-specific phospholipase D.

We have developed a simple immunoaffinity chromatography procedure for the purification of a glycosyl-phosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) from bovine serum. The enzyme was initially purified by a procedure consisting of 9% polyethylene glycol precipitation, Q Sepharose anion-exchange chromatography, S-300 gel filtration, wheat germ lectin-Sepharose, hydroxylapatite agarose, zinc chelate matrix, Mono Q-high performance liquid chromatography (HPLC), and Superose 12 (gel filtration) HPLC. Using this purified material as immunogen, we generated a panel of monoclonal antibodies. A low affinity antibody was selected for the purification of catalytically active GPI-PLD from bovine serum by immunoaffinity chromatography, followed by wheat germ lectin-Sepharose and Mono Q-fast protein liquid chromatography. The latter method provides a simple purification procedure with an overall yield of 26%. The purified enzyme has an apparent molecular weight of about 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of about 5.6 by isoelectric focusing gel analysis. On Superose 12 HPLC, the material purified by the latter method elutes as a single peak with an apparent molecular weight of 200,000 as determined by protein standards. The enzyme activity is inhibited by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid or 1,10-phenanthroline. Phosphatidic acid is the only 3H-labeled product when [3H]myristate-labeled variant surface glycoprotein is hydrolyzed by the purified enzyme. Amino terminal sequence analysis of the intact 100-kDa protein reveals no strong homology to that of any other known protein. Twelve tryptic peptides derived from the intact protein have been subjected to amino acid sequence analysis. Two of them share sequence homology with each other and with the metal ion binding domains of members of the integrin family. Based upon these criteria, it appears that the purified enzyme is distinct from other phospholipases with specificity for inositol phospholipids.

Amino Acid Sequence↗
PubMed1990

Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor.

Two distinct receptors for tumor necrosis factor (TNF) of 55 and 75 kd are expressed at low levels by various cells. The 55 kd TNF receptor was purified from HL60 cells, and partial amino acid sequences were determined. Short degenerate sense and antisense oligonucleotide primers encoding the N- and C-terminal ends of a peptide of 22 amino acid residues were used to amplify a 66 bp cDNA fragment from HL60 RNA by reverse transcriptase-polymerase chain reaction. The cDNA fragment as a probe identified several overlapping clones in a human placenta cDNA library. The open reading frame of the cDNA predicts a 455 amino acid TNF receptor protein with leader, extracellular, transmembrane, and intracellular domains. When expressed in COS-1 cells or in a baculovirus system, the cDNA conferred TNF binding properties comparable to the native receptor. Surprisingly, the 55 kd TNF receptor shows a high degree of sequence homology to the NGF receptor extracellular domain.

Amino Acid Sequence↗
PubMed1990

Identification of a human immunodeficiency virus-1 protease cleavage site within the 66,000 Dalton subunit of reverse transcriptase.

The human immunodeficiency virus-1 reverse transcriptase is a heterodimer of related 51 and 66 kDa subunits. The smaller subunit arises by viral protease-catalyzed cleavage of the carboxy-terminal domain of the 66 kDa species. Comparison of the amino acid composition analyses of the isolated 51 kDa and 66 kDa subunits indicates that the carboxyl terminus of 51 kDa is Phe440. This site was confirmed in vitro using purified recombinant protease and a peptide spanning the postulated cleavage area. The sequence surrounding this site does not show significant homology to other protease cleavage sites in the viral gag and pol precursors; thus, this new information may contribute to our understanding of the sequence specificity of the viral protease.

Amino Acid Sequence↗
PubMed1990

The use of proteolysis and direct N-terminal sequence analysis to study human interleukin-2/receptor interaction on solid support.

An immobilized interleukin-2 receptor which is capable of binding interleukin-2 and suitable for direct N-terminal sequence analysis was employed to study interleukin-2/receptor interactions. Sensitive tryptic sites on the immobilized receptor and its interleukin-2 complex were identified by sequence analyses and compared. The results have revealed that the N-terminal region of interleukin-2 is not involved in receptor binding and the peptide segment covering residues 36-39 in the receptor is probably near or involved in the interleukin-2 binding site. The rapidity and simplicity make this solid phase sequence approach a good method for analyzing interleukin-2/receptor interaction and may be suitable for studying other protein-ligand interactions.

Amino Acid Sequence↗
PubMed1990

Two human TNF receptors have similar extracellular, but distinct intracellular, domain sequences.

Tumor necrosis factor (TNF) is a cytokine with a wide range of biological activities in inflammatory and immunologic responses. These activities are mediated by specific cell surface receptors of 55 kDa and 75 kDa apparent molecular masses. A 75-kDa TNF receptor cDNA was isolated using partial amino acid sequence information and the polymerase chain reaction (PCR). When expressed in COS-1 cells, the cDNA transfers specific TNF-binding properties comparable to those of the native receptor. The predicted extracellular region contains four domains with characteristic cysteine residues highly similar to those of the 55-kDa TNF receptor, the nerve growth factor (NGF) receptor, and the CDw40 and OX40 antigens. The consensus sequence of the TNF receptor extracellular domains also has similarity to the cysteine-rich sequence motif LIM. In marked contrast to the extracellular regions, the intracellular domains of the two TNF receptors are entirely unrelated, suggesting different modes of signaling and function.

Amino Acid Sequence↗
PubMed1990

Purification to homogeneity and partial characterization of cytotoxic lymphocyte maturation factor from human B-lymphoblastoid cells.

A cytokine that can synergize with interleukin 2 to activate cytotoxic lymphocytes was purified to homogeneity. The protein, provisionally called cytotoxic lymphocyte maturation factor (CLMF), was isolated from a human B-lymphoblastoid cell line that was induced to secrete lymphokines by culture with phorbol ester and calcium ionophore. The purification method, utilizing classical and high-performance liquid chromatographic techniques, yielded protein with a specific activity of 8.5 x 10(7) units/mg in a T-cell growth factor assay. Analysis of the purified protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated that CLMF is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. Determination of the N-terminal amino acid sequences of the two subunits revealed that both subunits are not related to any previously identified cytokine. Purified CLMF stimulated the proliferation of human phytohemagglutinin-activated lymphoblasts by itself and exerted additive effects when used in combination with suboptimal amounts of interleukin 2. Furthermore, the purified protein was shown to synergize with low concentrations of interleukin 2 in causing the induction of lymphokine-activated killer cells.

Amino Acid Sequence↗
PubMed1990

Putative N-terminal sequence of murine soluble immune response suppressor (SIRS): significant homology with short neurotoxin 1.

Soluble immune response suppressor (SIRS) is a low-molecular-weight protein (approximately 10,000 daltons) produced by mitogen- or interferon-activated T lymphocytes that can block development of humoral or cell-mediated immune responses in vivo or in vitro. As previously reported, murine SIRS is heterogeneous, eluting in two broad peaks on high performance reverse phase chromatography as well as displaying several broad isoelectric point forms. A putative N-terminal 21 amino acid sequence has been obtained for one of the less hydrophobic isoforms, SIRS-alpha 7. The sequence of SIRS-alpha 7 is unique in mammals but shows a remarkable homology to the family of short neurotoxins (short neurotoxin 1) found in sea snake, adder, and cobra species. A degenerate oligonucleotide probe based on the protein sequence was synthesized and was shown to hybridize to SIRS messenger RNA as measured by SIRS synthesis in a rabbit reticulocyte lysate system. A synthetic polypeptide based on the 21-residue sequence was also prepared and coupled to thyroglobulin or keyhole limpet hemocyanin. These were used to prepare rabbit antisera that neutralize SIRS bioactivity and precipitate authentic SIRS.

Amino Acid Sequence↗
PubMed1989

Limited proteolysis of recombinant human soluble interleukin-2 receptor. Identification of an interleukin-2 binding core.

Limited proteolysis of a recombinant, soluble form of the Tac protein, a human interleukin-2 receptor (rIL-2R), was performed using trypsin, Staphylococcus aureus V8 protease and proteinase K to study the structural requirements of interleukin-2 receptor (IL-2R) for interleukin-2 (IL-2) binding. Sensitive proteolytic sites were found to be clustered in the regions of the polypeptide encoded by exons 3, 5, and 6, with a few semi-sensitive sites located within the two homologous domains encoded by exons 2 and 4. A number of nicked and truncated rIL-2R species generated by proteolysis were assayed for IL-2 binding using recombinant IL-2 (rIL-2) affinity gel and then structurally characterized. The results demonstrated that only the species that consist of the regions encoded by exons 2 and 4, joined by five disulfide bonds, are capable of binding IL-2 and that the presence of semi-sensitive cleavage sites within the two homologous domains had no apparent effect on IL-2 binding. These results suggest that the pattern of the sensitive cleavage sites in rIL-2R is closely related to the structural requirements for IL-2 binding. Based on the experimental results, a highly symmetrical core structure of IL-2R with a total of 135 amino acid residues was identified. This is the smallest protein moiety so far known to be capable of binding IL-2.

Amino Acid Sequence↗
PubMed1989

In vitro assembly of 30S and 70S bacterial ribosomes from 16S RNA containing single base substitutions, insertions, and deletions around the decoding site (C1400).

An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes [Krzyzosiak et al. (1987) Biochemistry 26, 2353-2364] was used to make 10 single base changes around C1400, a residue known to be at the decoding site. C1400 was replaced by U, A, or G, five single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400. Another mutant possessed seven additional nucleotides at the 3' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomes. Modified in vitro reconstitution conditions were required to obtain assembly of all of the synthetic ribosomes. Quantitative HPLC analysis of the protein content of each mutant showed that all of the proteins were present. The ability of synthetic 30S to form 70S particles under functional assay conditions was about 75% that of natural 30S and was unchanged by any of the mutations except for the deletion of G1401, which decreased the association activity under the standard conditions to 35-40% of synthetic 30S. That part of the ribosomal P site which interacts with the anticodon loop of tRNA was investigated by near-UV (greater than 300 nm) induced cross-linking of AcVal-tRNA. Cross-linking depended on both 30S subunits and the correct codon. The cross-linking yield of all mutants with a pyrimidine at position 1400 was equal to control isolated 30S, and the first-order rate constants for cross-linking of those mutants tested were like reconstituted natural 30S. The site of cross-linking for mutants with a C or U insertion between C1400 and G1401 was shifted to the inserted residue. Cross-linking to the base 5' to G1401 rather than to the residue 3' to C1399 indicates that G1401 is an important structural determinant of the P site.

Centrifugation, Density Gradient↗
PubMed1989

Molecular cloning of the chicken myelomonocytic growth factor (cMGF) reveals relationship to interleukin 6 and granulocyte colony stimulating factor.

Normal as well as retrovirally transformed avian myeloid precursor cells require the colony stimulating factor cMGF for their survival, proliferation and colony formation in vitro. cMGF has been shown to be a glycoprotein which is active in the picomolar concentration range. Co-expression of kinase type oncogenes in v-myb or v-myc transformed myeloid cells induces cMGF expression and confers factor independence via an autocrine mechanism. Here we describe the molecular cloning of cMGF from a myeloblast cDNA library and show that it is a 201 amino acid residue secretory protein which is modified by signal peptide cleavage and glycosylation during translocation into the lumen of membrane vesicles. A bacterially expressed trpE-cMGF fusion protein induces proliferation of E26 transformed myeloblasts in a cMGF bioassay suggesting that glycosylation is not absolutely necessary for biological activity. Sequence comparison reveals that cMGF is distantly related to G-CSF and IL-6.

Amino Acid Sequence↗
PubMed1989

Purification of homogeneity and amino acid sequence analysis of a receptor protein for interleukin 1.

The interleukin 1 (IL-1) receptor from mouse EL-4 thymoma cells was purified to homogeneity by a method which utilized ligand affinity chromatography and classical chromatographic techniques. After solubilization of the receptor from intact cells with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the IL-1 binding activity was purified greater than 23,000-fold. Analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and ligand blot demonstrated that a single protein of molecular mass of approximately 80 kDa is the IL-1 binding polypeptide. The purified protein bound IL-1 with a dissociation constant of approximately 1.1 X 10(-10) M, which is indistinguishable from the affinity of the cell-bound receptor. The amino acid composition of this protein is strikingly similar to the composition deduced from the sequence of a cDNA coding for an IL-1 receptor from EL-4 cells. Protein sequence analysis of Staphylococcus aureus V-8 protease-derived peptides yields data consistent with the sequence proposed from cloned cDNA. These studies have demonstrated that the high affinity IL-1 receptor on EL-4 cells is the 80-kDa protein.

Amino Acids↗
PubMed1989

The use of fluorescamine as a detection reagent in protein microcharacterization.

With recent advances in protein microchemistry, compatible methods for the preparation and quantitation of proteins and peptides are required. Fluorescamine, a reagent which reacts with primary amino groups has been used successfully to detect amino acids, peptides, and proteins in various micromethods. This article discusses these methods which include (1) amino acid analysis of protein and peptide hydrolysates with postcolumn fluorescamine derivatization; (2) purification and characterization of proteins and peptides by reversed-phase HPLC with postcolumn fluorescamine derivatization; (3) purification of peptides by two-dimensional chromatography and electrophoresis on thin-layer cellulose with fluorescamine staining; and (4) electroblotting of protein bands from SDS-PAGE to glass fiber filters and polyvinylidene difluoride (PVDF) membranes with fluorescamine staining. In addition, this article also compares a postcolumn fluorescamine detection system with a UV detection system in the applications of amino acid analysis and reversed-phase HPLC protein/peptide analysis.

Amino Acid Sequence↗
PubMed1989

A human immunoglobulin G receptor exists in both polypeptide-anchored and phosphatidylinositol-glycan-anchored forms.

Several cDNA clones encoding the human immunoglobulin G receptor CD16 were isolated from human lung or peripheral blood leukocyte cDNA libraries. Nucleotide sequence comparisons revealed that the cDNAs could be divided into two groups. cDNA clones in one group encode a protein that terminates 4 amino acids after the putative transmembrane domain. Clones in the second group encode a protein with an extra 21 amino acids that could comprise a cytoplasmic domain. Direct peptide sequencing was used to determine the N terminus of the mature CD16 receptor protein and supported the existence of the two forms of the receptor. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C resulted in the release of a large percentage of the CD16 molecules from the cell surface. In contrast, treatment of natural killer cells with phosphatidylinositol-specific phospholipase C did not release any CD16 from the cell surface. These data demonstrate that both polypeptide-anchored and phosphatidylinositol-glycan-anchored forms of the CD16 molecule exist and that they are differentially expressed on neutrophils and natural killer cells.

Amino Acid Sequence↗
PubMed1988

Cloning of cDNA encoding a 32-kDa protein. An accessory polypeptide of the H+-ATPase from chromaffin granules.

The purified H+-ATPase from chromaffin granules is composed of several polypeptides, one of which has an apparent molecular weight of 39,000. Immunoblots with the antibody against this protein and various membrane preparations showed that similar or even identical polypeptides may be associated with the H+-ATPases from synaptic vesicle, kidney microsomes, and lysosomes. A cDNA library was constructed from bovine adrenal medulla, and the cDNA encoding the polypeptide was isolated and sequenced. Search in DNA and protein data banks revealed no significant homology to known genes. Hydrophobicity plot revealed no obvious transmembrane segments with the exception of one stretch of hydrophobic and neutral amino acid starting at leucine 16. The cDNA was shown to encode the entire polypeptide by the virtue of an amino acid sequence corresponding to the N terminus of the open reading frame and by subunit and site-specific antibodies. The cDNA was cloned into an expression vector, transcribed by T7 polymerase, and translated by reticulocyte lysate. Even though the cDNA encodes a protein with a molecular weight of 31,495, the translation product comigrated on sodium dodecyl sulfate gels with the subunit of the purified H+-ATPase. In line with several other subunits of vacuolar H+-ATPases, no signal sequence was detected in the translated gene. Northern blots revealed the presence of a single mRNA of about 1.6 kb in bovine adrenal medulla. However, liver, lung, and kidney may contain additional mRNA of about 1.7 kb.

Adrenal Medulla↗
PubMed1988

Molecular cloning and sequencing of the psaD gene encoding subunit II of photosystem I from the cyanobacterium, Synechocystis sp. PCC 6803.

Photosystem I reaction center was isolated from the cyanobacterium, Synechocystis sp. PCC 6803, in a form which contains seven different polypeptide subunits. One of the subunits, with a molecular mass of about 16 kDa, was isolated, and protein sequence information was obtained for the amino terminus and several tryptic peptides. Oligonucleotide probes, corresponding to these sequences, were used to probe a genomic library, and the gene, designated psaD, encoding subunit II was cloned and sequenced. The gene encodes a polypeptide with a mass 15,644 Da, which exhibits a high degree of similarity to subunit II from tomato, as well as amino acid sequences reported from barley photosystem I. In addition to this gene, three large open reading frames were identified. Two remain unidentified, and the third is highly homologous to anthranilate synthase, component 1 from Escherichia coli and Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1988

Structural analysis of recombinant soluble human interleukin-2 receptor. Primary structure, assignment of disulfide bonds and core IL-2 binding structure.

A purified soluble and functional form of recombinant human interleukin-2 receptor, engineered and expressed in Chinese hamster ovary cells, was structurally characterized. The primary sequence of this 224 amino acid recombinant protein which lacks most of the carboxy-terminal transmembrane and cytoplasmic portions of the intact protein was established by sequence analyses. The disulfide bonds were assigned by comparative peptide mapping of the reduced and non-reduced peptide digests. As in the case of natural interleukin-2 receptor they occur between cysteines 3-147, 46-104, 131-163, and 28/30-59/61. Based on assignment of the disulfide bonds, a structural model of the interleukin-2 receptor for interleukin-2 binding is proposed.

Amino Acid Sequence↗
PubMed1988

Recent microsurgical advances in China.

For more than two decades, microsurgery has been developed, practiced, and refined in China. For the most part, this has taken place independent of a parallel process in the Western World. Only until recent years has there been some communication from China regarding this microsurgical experience. The authors believe that there is much yet to be learned from the wealth of Chinese experience in microsurgery, just as Chinese surgeons have gained valuable information from the West. The authors reviewed the microsurgical literature in China from 1983 to 1986, and present here what we regard to be of particular interest to American microsurgeons, including the areas of digital reconstruction, replantation, flap transfer and transposition, and microsurgical techniques and patient management. Some historical background in the development of Chinese microsurgery is also provided.

Adult↗