Biomedical subjects

H S Lu

Publications and source records attributed to H S Lu.

At least 73 records · Page 4Linked to original sources

PubMed1990

Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver--conditioned medium.

We have identified a novel growth factor, stem cell factor (SCF), for primitive hematopoietic progenitors based on its activity on bone marrow cells derived from mice treated with 5-fluorouracil. The protein was isolated from the medium conditioned by Buffalo rat liver cells. It is heavily glycosylated, with both N-linked and O-linked carbohydrate. Amino acid sequence following removal of N-terminal pyroglutamate is presented. The protein has potent synergistic activities in semisolid bone marrow cultures in conjunction with colony-stimulating factors. It is also a growth factor for mast cells. In two companion papers, we present the sequences of partial SCF cDNAs, identify SCF as a c-kit ligand, and map the SCF gene to the Sl locus of the mouse.

Amino Acid Sequence↗

PubMed1990

Primary structure and functional expression of rat and human stem cell factor DNAs.

Partial cDNA and genomic clones of rat stem cell factor (SCF) have been isolated. Using probes based on the rat sequence, partial and full-length cDNA and genomic clones of human SCF have been isolated. Based on the primary structure of the 164 amino acid protein purified from BRL-3A cells, truncated forms of the rat and human proteins have been expressed in E. coli and mammalian cells and have been shown to possess biological activity. SCF is able to augment the proliferation of both myeloid and lymphoid hematopoietic progenitors in bone marrow cultures. SCF exhibits potent synergistic activities in conjunction with colony-stimulating factors, resulting in increased colony numbers and colony size.

Amino Acid Sequence↗

PubMed1990

Tissue kallikrein-binding protein is a serpin. I. Purification, characterization, and distribution in normotensive and spontaneously hypertensive rats.

Kallikrein-binding protein was purified to apparent homogeneity from rat serum by Affi-Gel Blue, DEAE-Sepharose CL-6B, Sephacryl S-200 chromatography, and preparative gel electrophoresis or high performance liquid chromatography. The purified protein migrates as a single band of 60 kDa in a sodium dodecyl sulfate-polyacrylamide gel under reducing conditions. It is an acidic protein with isoelectric points ranging from 4.2 to 4.6. The amino terminus of the binding protein is an Asp residue as determined by sequence analysis. It forms a 92-kDa sodium dodecyl sulfatestable complex with kallikrein with a t1/2 of 18 min. Western blot and radioimmunoassay showed a distribution of the kallikrein-binding protein in serum, urine, and various tissues with a 5-10-fold lower amount in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). A full length cDNA clone encoding the kallikrein-binding protein was isolated from a rat liver cDNA library by immunoscreening and the translated amino acid sequence matches the amino-terminal 29-amino acid sequence of the binding protein. The cDNA sequence shares 68.8% identity with human alpha 1-antichymotrypsin and is identical to that of a rat hepatic protein. Dot blot analysis shows that kallikrein-binding protein is expressed at high levels in the liver and at low levels in the lung, salivary gland, and kidney. Its mRNA level in the liver decreases by 2-fold after acute phase inflammation and is higher in male than in female rats. Genomic Southern blot analyses reveal restriction fragment length polymorphisms between SHR and WKY rats in the binding protein locus. The results indicate that rat kallikrein-binding protein belongs to the serpin superfamily and its level is significantly reduced in the spontaneously hypertensive rats.

Amino Acid Sequence↗

PubMed1990

Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon.

Recombinant DNA-derived consensus alpha interferon was expressed in Escherichia coli and purified. Isoelectric focusing of this purified protein indicated the presence of three isoelectric subforms of pI 6.1, 6.0, and 5.7. These three subforms were preparatively separated by isoelectric focusing using Immobiline polyacrylamide gel and did not exhibit apparent differences in biological activity and tertiary structure. The pI 5.7 subform could also be separated from the pI 6.1 and 6.0 subforms by reverse-phase HPLC. Automated N-terminal amino acid sequence analysis of the pI 6.1 and 6.0 subforms yielded sequences corresponding to the methionyl and des-methionyl forms of the protein, respectively. Sequence analysis of the pI 5.7 subform indicated that its N terminus is blocked. To further determine the structure of the blocking moiety in the pI 5.7 subform, a blocked N-terminal tryptic peptide was isolated from HPLC peptide mapping of the S-carboxymethylated derivative. Results obtained from mass spectroscopic and amino acid analyses of this peptide suggest that it is blocked with an acetyl group at the N-terminal cysteine residue.

Amino Acid Sequence↗

PubMed1990

Revised amino acid sequences of the three major phospholipases A2 from Bungarus fasciatus (banded krait) venom.

The structures of three cardiotoxin-like proteins obtained from the venom of Bungarus fasciatus (banded krait) were elucidated previously (Lu and Lo, 1980, 1981). Since their molecular sizes are similar to that of phospholipase A2 and since they show weak phospholipase A2 activities (Chang et al., 1983), a further study of their primary structures was carried out. Fractions Va, Vb-2 and VI, corresponding to the former V-2, V-3 and VI were determined to be typical phospholipases A2. Among 118 amino acid residues, they all have in common a Pro29 between Gly28 and Gly30, the latter two residues being implicated in Ca2+ binding together with Tyr26 and Asp47.

Amino Acid Sequence↗

PubMed1989

Purification and characterization of two related but distinct metalloproteinase inhibitors secreted by bovine aortic endothelial cells.

Two metalloproteinase inhibitors were purified from serum-free medium conditioned by bovine aortic endothelial cells. One of these inhibitors, with a molecular weight of 30,000-34,000 (reduced) is identified as tissue inhibitor of metalloproteinases; the second inhibitor has a molecular weight of 27,500 (reduced) and 20,400 (unreduced), is not recognized by an antiserum against bovine tissue inhibitor of metalloproteinases, appears unglycosylated, and has 51% identity with tissue inhibitor of metalloproteinases by NH2-terminal amino acid sequence analysis. This inhibitor has antiproteinase activities similar to those of tissue inhibitor of metalloproteinases, with inhibition of classical collagenase, type IV collagenase, and gelatinases but not trypsin, plasmin, or bacterial collagenase. Other properties shared with tissue inhibitor of metalloproteinases include trypsin sensitivity, acid and heat resistance, and inactivation by reduction-alkylation. The presence of these inhibitors in endothelial cells suggests that they may play important roles in protecting the integrity of the vascular basement membrane.

Amino Acid Sequence↗

PubMed1989

Disulfide structures of human interleukin-6 are similar to those of human granulocyte colony stimulating factor.

The amino acid sequences of human interleukin-6 and granulocyte colony stimulating factor are approximately 30% homologous in the N-terminal region. The relative positions of four half-cystines in human interleukin-6 (IL-6) match four of the five in human granulocyte colony stimulating factor. Labeling experiments of recombinant interleukin-6 with tritiated iodoacetate confirmed that the molecule forms two intramolecular disulfide bonds and contains no detectable level of free sulfhydryls. By isolation and characterization of tryptic and subtilytic peptides obtained from different proteolytic digestions, the disulfide bonds of the IL-6 molecule were assigned to Cys44-Cys50 and Cys73-Cys83. The two disulfide bridges form two small loops which are separated by 22 amino acids. These structures are similar to those of recombinant granulocyte colony stimulating factor.

Amino Acid Sequence↗

PubMed1989

Disulfide and secondary structures of recombinant human granulocyte colony stimulating factor.

Molecular characteristics and secondary structures of recombinant methionyl human granulocyte colony stimulating factor produced by genetically engineered Escherichia coli are described. Limited radiolabeling of the protein with tritiated iodoacetate and determination of the labeled residue revealed that this recombinant protein contains only one free cysteine at position 17 which is not essential for activity. The free cysteine is inaccessible to modification unless the molecule is unfolded under denaturing conditions. The molecule forms two disulfide bridges which were assigned as Cys(36)-Cys(42) and Cys(64)-Cys(74) based on the results of isolation and characterization of disulfide-containing peptides obtained from a subtilisin digest of the intact protein. CD analyses and secondary structure prediction suggest that the molecule is abundant in alpha-helical structures.

Amino Acid Sequence↗

PubMed1989

Human urinary kallikrein. Complete amino acid sequence and sites of glycosylation.

Human glandular kallikrein was purified from urine and subjected to detailed structural characterization. The protein was carboxymethylated with iodoacetic acid and digested with TPCK-trypsin, Staphylococcal aureus V-8 protease and endo LysC peptidase. The resulting peptide fragments were separated by reverse-phase HPLC using C-4 columns and acetonitrile-trifluoroacetic acid gradient elution. The complete amino acid sequence of the carboxymethylated derivative was elucidated by sequence analysis and alignment of peptides derived from different proteolytic cleavages. A procedure using in situ CNBr cleavage of a large endo LysC peptidase-derived peptide followed by direct sequencing was carried out to provide overlap for two glycosylation sites at residues 78 and 84. Three Asn-linked glycosylation sites were confirmed by the direct sequence analysis of the isolated glycopeptides. However, the third glycosylation at Asn-144 occurs only in 60% of kallikrein molecules. Reverse-phase HPLC effectively separates two species of HUK which correspond to molecules glycosylated and non-glycosylated at Asn-144, respectively. The human urinary kallikrein contains 238 amino acid residues with Ile and Ser as N- and C-terminal amino acids, respectively. The primary structure is completely identical to that deduced from a human genomic DNA sequence (F.K. Lin et al., manuscript in preparation) and is different in one amino acid (Lys-162 vs. Glu-162) from that deduced from pancreatic or kidney cDNA sequence.

Amino Acid Sequence↗

PubMed1989

[Synovial osteochondromatosis: several valuable clinical observations in 12 cases].

In the treatment of twelve cases of synovial osteochondromatosis, the loose bodies were studied clinically, pathologically and roentgenologically. Several notable clinical features were observed: (1) the mass of a big loose body is splintery and liable to break up into small pieces. (2) Microscopically, the loose bodies are usually built by piling of multiple small cartilaginous nodules, surrounded by fibrous tissues. (3) Radiologically, calcification and ossification in the loose bodies or intrasynovial nodules neither is related with the diameter, nor is always in the central position. These findings are of value to the diagnosis of the mentioned disease, especially phase 3, rather than relying on the criterion of the number of loose bodies.

Adolescent↗

PubMed1988

Structural characterization of recombinant consensus interferon-alpha.

Recombinant consensus interferon-alpha is derived from genetically modified Escherichia coli containing a synthetic gene constructed from a consensus of interferon sequences. The purified and biologically active protein has been subjected to detailed structural characterization including sequence determination and peptide isolation and identification. The homogeneous consensus interferon-alpha preparation contains two chromatographically indistinguishable homologous polypeptides with one containing an extra methionyl residue at the amino terminus. The delineated amino acid sequence of the protein is identical to that expected from the coding sequence of the gene. Correct oxidation of the molecule has been confirmed with two intramolecular disulfide linkages observed at Cys(1)-Cys(99) and Cys(29)-Cys(139).

Amino Acid Sequence↗

PubMed1988

Control of misincorporation of de novo synthesized norleucine into recombinant interleukin-2 in E. coli.

Interleukin-2 produced from a recombinant E. coli was found to contain as much as 19% norleucine in place of methionine in a minimal medium fermentation. Medium supplementation experiments and use of a leucine-requiring mutant host strain indicated the origin of norleucine to be de novo biosynthesis by reactions involving the enzymes of the leucine biosynthetic pathway. The misincorporation was highly suppressed by addition of either L-leucine or L-methionine to the fermentation and completely suppressed by adding both amino acids.

Aminocaproates↗

PubMed1988

Identification of unusual replacement of methionine by norleucine in recombinant interleukin-2 produced by E. coli.

Moderate amounts of norleucine incorporation into recombinant interleukin-2 (IL-2) produced in E. coli have been detected. Incorporation of norleucine occurs both at the amino terminal and internal methionines as confirmed by the isolation of norleucine-containing tryptic peptides which eluted later than the respective methionine-containing peptides by reverse-phase HPLC. The occurrence of norleucine in intact protein and modified peptides was determined by amino acid analysis and amino acid sequencing including Edman degradation and fast atom bombardment mass spectrometry. In the subsequent paper, we determined that norleucine incorporation is caused by the endogenous synthesis of norleucine in E. coli.

Amino Acid Sequence↗

PubMed1988

Narrow-bore high-performance liquid chromatography of phenylthiocarbamyl amino acids and carboxypeptidase P digestion for protein C-terminal sequence analysis.

Carboxypeptidase P digestion followed by narrow-bore high-performance liquid chromatography of phenylthiocarbamyl amino acids is employed for polypeptide C-terminal end group and sequence determination. Carboxypeptidase P digestion of polypeptides provides specific cleavage of protein C-terminal amino acids. The digestion offers the advantage that it can be carried out in either 10 mM sodium acetate or water at pH 4.0 in the presence of an enzyme activator, Brij-35. The narrow-bore high-performance liquid chromatography of all 20 phenylthiocarbamyl-amino acids has provided quantitative analysis at low picomole levels. This efficient and sensitive procedure is particularly useful for examining in vivo excision of protein C-termini and for verifying the integrity of various protein products produced by recombinant DNA techniques.

Amino Acid Sequence↗

PubMed1986

Use of narrow-bore high-performance liquid chromatography for microanalysis of protein structure.

We report here systematic approaches to microanalysis of protein structures using narrow-bone high-performance liquid chromatography of protein, peptide and derivatized [phenylthiocarbamyl (PTC-) and phenylthiohydantoinyl (PTH-)] amino acids. The utilization of columns with small diameters (2 mm or less) has improved resolution and sensitivity and enabled protein structure analysis at the low pmol level. Preparative isolation of proteins and peptides of pmol quantity is achieved and separation and identification of PTC- and PTH-amino acids can be routinized at the low pmol to subpmol level. The use of diode-array detection enables simultaneous multiple-wavelength monitoring and spectral retreat, which greatly enhances flexibility and usefulness of the present methods. In combination with protein microsequencing techniques, these narrow-bore high-performance liquid chromatographic procedures can facilitate structural analysis of minutely available proteins of biochemical and physiological significance.

Amino Acid Sequence↗

PubMed1986

Protein secretion from Saccharomyces cerevisiae directed by the prepro-alpha-factor leader region.

The Saccharomyces cerevisiae secretory process was studied by evaluating secretion efficiency, processing efficiency, and the efficiency of protein folding for hybrid proteins containing the yeast prepro-alpha-factor leader region. Secretion of three proteins, beta-endorphin, calcitonin, and a consensus alpha-interferon (IFN-Con1), were compared in terms of secretion efficiency into the culture medium, beta-Endorphin and calcitonin, both small proteins, were found to be efficiently secreted from logarithmically grown cells. In contrast, the larger IFN-Con1 accumulated in the periplasmic space and cell wall. The glycosylated, unprocessed prepro-alpha-factor/IFN-Con1 fusion protein was also found to be secreted into the culture medium. The presence of (Glu-Ala) dipeptides in the alpha-factor spacer peptide increased the efficiency of cleavage at Lys-Arg in the prepro-alpha-factor/IFN-Con1 protein fusion. Purified secreted IFN-Con1 was structurally characterized to determine the effect of passage through the yeast secretory pathway on the fidelity and efficiency of protein folding. The disulfide structure of the secreted protein was found to be identical with that reported for the native human alpha-interferons.

Amino Acid Sequence↗

PubMed1984

Solid-phase protein and peptide sequencing using either 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate or phenylisothiocyanate.

Automated solid-phase sequencing using 4-N,N-dimethylaminoazobenzene 4'-isothiocyanate (DABITC) double coupling or regular phenylisothiocyanate (PITC) degradation procedures have been investigated. Employing sensitive high-performance liquid chromatography for the identification of amino acid thiohydantoin derivatives (PTH and DABTH), both methods were capable of sequencing immobilized peptides or proteins at the subnanomole levels. In the sequencing program using DABITC, alternate methanol and dichloroethane washes and automated conversion using methanolic HCl containing dithiothreitol were introduced to obtain clean thiazolinones and to ensure high recovery yields of the thiohydantoins. Using regular PITC degradation with a 59-min program, the background peaks of the side products could be reduced to enhance HPLC identification. Peptides or proteins attached to the glass beads or resins via the carboxyl terminii or epsilon-amino groups of lysyl residues could be readily sequenced up to 30 identifiable degradation cycles, where the sequencing is generally terminated due to the increased background components.

Amino Acid Sequence↗

PubMed1984

Primary structure of human triosephosphate isomerase.

Human placental triosephosphate isomerase was isolated by an improved procedure and recovered with the highest specific activity ever reported. Employing this purification procedure, sufficient amounts of the enzyme were obtained for detailed primary structural studies. For sequences analysis, the enzyme was reduced and carboxymethylated and subjected to tryptic and chymotryptic digestions. The peptide mixtures were separated by high-performance liquid chromatography using octyl or alkylphenyl reverse-phase columns and trifluoroacetic acid/acetonitrile gradient elution systems. Sequence analyses of the intact enzyme, tryptic, chymotryptic, and cyanogen bromide peptides were accomplished using high-sensitivity solid-phase sequencing procedures with either 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate or phenylisothiocyanate. The primary structure of human triosephosphate isomerase is constructed from the alignment of the tryptic peptides with the analysis of the overlapping chymotryptic peptides. The enzyme is a dimeric molecule consisting of two identical polypeptide chains with 248 amino acid residues and a calculated subunit molecular mass of 26,750 daltons. A comparison of the amino acid sequences from the human placental enzyme and from other species such as rabbit, chicken, and coelacanth muscles showed relatively high sequence homology, indicating that the evolution of the enzyme is very conservative. The amino acids of the active-site pocket and the subunit-subunit contact sites exhibit few changes.

Amino Acid Sequence↗