Biomedical subjects

J D Hulmes

Publications and source records attributed to J D Hulmes.

At least 37 records · Page 2Linked to original sources

PubMed1991

Selective cleavage of polypeptides with trifluoroacetic acid: applications for microsequencing.

Cleavage of small polypeptides (less than 30 amino acid residues) by trifluoroacetic acid (TFA) under a variety of reaction conditions including time, temperature, TFA phase, and sample supports has been examined by N-terminal sequencing. Treatment with gas-phase TFA at room temperature will cleave polypeptide chains preferentially at the N-terminal side of serine and threonine residues. When liquid-phase TFA is used, additional cleavage at the C-terminal side of aspartic acid was detected. These procedures are applicable for directly treating samples immobilized on sequencer supports (glass fiber filters or polyvinylidene difluoride membranes) to verify the presence of a polypeptide with a blocked N-terminus as well as to obtain internal sequence data at subnanomole levels.

Adenosine Triphosphatases↗

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

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

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

Purification of an inhibitor of erythroid progenitor cell cycling and antagonist to interleukin 3 from mouse marrow cell supernatants and its identification as cytosolic superoxide dismutase.

We have isolated a protein from media conditioned by a murine marrow-derived cell line (PB6) and from mouse marrow supernatants that antagonizes interleukin 3-dependent proliferation of cells in culture and reversibly inhibits DNA synthesis of erythroid progenitor cells (BFU-E) in vitro. This protein, p16 (monomer Mr = 16 kD on SDS-PAGE), was purified to homogeneity and amino acid sequencing of a polypeptide fragment yielded a sequence identical to that of murine cytosolic Cu,Zn-containing superoxide dismutase (SOD). The identification of p16 as SOD was confirmed by the detection of SOD enzymatic activity in pure p16 fractions, and when a commercial human erythrocytic SOD preparation was tested it showed the same cell inhibitory activities as p16. These observations show that superoxide dismutase is able to affect the cycling and growth factor responses of hematopoietic cells, activities that have not previously been associated with this enzyme.

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

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↗

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

cDNA sequence encoding the 16-kDa proteolipid of chromaffin granules implies gene duplication in the evolution of H+-ATPases.

Vacuolar H+-ATPases function in generating protonmotive force across the membranes of organelles connected with the vacuolar system of eukaryotic cells. This family of H+-ATPases is distinct from the two other families of H+-ATPases, the plasma membrane-type and the eubacterial-type. One of the subunits of the vacuolar H+-ATPase binds N,N'-dicyclohexylcarbodiimide (DCCD) and has been implicated in the proton-conducting activity of these enzymes. We have cloned and sequenced the gene encoding the DCCD-binding protein (proteolipid) of the H+-ATPase of bovine chromaffin granules. The gene encodes a highly hydrophobic protein of 15,849 Da. Hydropathy plots revealed four transmembrane segments, one of which contains a glutamic residue that is the likely candidate for the DCCD binding site. Sequence homology with the vacuolar proteolipid and with the proteolipids of eubacterial-type H+-ATPases was detected. The proteolipids from Escherichia coli, spinach chloroplasts, and yeast mitochondria matched better to the NH2-terminal part of the vacuolar protein. The proteolipids of bovine mitochondria and Neurospora mitochondria matched better to the COOH-terminal end of the vacuolar proteolipid. These findings suggest that the proteolipids of the vacuolar H+-ATPases were evolved in parallel with the eubacterial proteolipid, from a common ancestral gene that underwent gene duplication.

Amino Acid Sequence↗

PubMed1988

Aspartic acid-484 of nascent placental alkaline phosphatase condenses with a phosphatidylinositol glycan to become the carboxyl terminus of the mature enzyme.

A carboxyl-terminal chymotryptic peptide from mature human placental alkaline phosphatase was purified by HPLC and monitored by a specific RIA. Sequencing and amino acid assay showed that the carboxyl terminus of the peptide was aspartic acid, representing residue 484 of the proenzyme as deduced from the corresponding cDNA. Further analysis of the peptide showed it to be a peptidoglycan containing one residue of ethanolamine, one residue of glucosamine, and two residues of neutral hexose. The inositol glycan is apparently linked to the alpha carboxyl group of the aspartic acid through the ethanolamine. Location of the inositol glycan on Asp-484 of the proenzyme indicates that a 29-residue peptide is cleaved from the nascent protein during the post-translational condensation with the phosphatidylinositol-glycan.

Alkaline Phosphatase↗

PubMed1988

Primary structure of equine pituitary prolactin.

Equine prolactin was determined to be a single chain protein of 199 amino acid containing two tryptophan and six cysteine residues, as found in other mammalian prolactins. The primary sequence of equine prolactin was obtained by automated Edman analyses of S-carboxymethylated protein and proteolytic fragments of modified protein. Of the known prolactin sequences, equine prolactin shows closest homology with porcine (93%) and fin whale (87-91%) prolactins. Genetic mutations have produced changes in 17 of 199 residues of equine prolactin relative to its putative ancestral precursor. Since equine growth hormone has undergone alterations in 4 of 191 residues relative to this putative precursor protein, these results support the theory that prolactins are evolving at a faster rate than growth hormones. Consistent with the previously determined circular dichroic spectrum of equine prolactin, 60% of the protein is predicted to form alpha helices.

Amino Acid Sequence↗

PubMed1987

Cholecystokinin octapeptides purified from chinchilla and chicken brains.

Cholecystokinin octapeptides (CCK8's) have been purified from methanol extracts of 30 chinchilla and 50 chicken brains containing 9.3 nmol and 8.5 nmol of the peptides respectively. Immunoreactive CCK was concentrated on a DEAE trisacryl column and purification effected by two successive HPLC steps. The peptides were each shown to have a sulfated tyrosine. The sequences of the two peptides are compared with the corresponding CCK8's of pig and guinea pig (GP). Chinchilla & GP: D Y V G W M D F; Chicken & pig: D Y M G W M D F. Since chinchilla insulin resembles other mammalian insulins more than does GP insulin, it is of particular interest that the CCK8's of these two species are identical and raises the question as to whether other brain-gut peptides of the chinchilla, which is a New World mammal as is the GP, would resemble those of the GP or the corresponding peptides of Old World mammals.

Amino Acid Sequence↗

PubMed1986

Guinea pig 33-amino acid gastrin.

Only two 34 amino acid gastrin precursors have previously been purified and sequenced, those of pig and of human. The larger molecular form generally accounts for only about 5% of antral gastrin in most species. This report describes the purification of "big gastrin" from guinea pig (GP) antra. Two hundred grams of antra were defatted with acetone and the acetone cakes were extracted with 0.1M NH4HCO3. The extract was concentrated by adsorption onto and batch elution from QA-52 anion exchange cellulose. Fractionation on a mu Bondapak C18 cartridge resolved 3.6 nmol of the larger peptide from 61 nmol of immunoreactive gastrin in the original extract. Two additional HPLC steps brought the peptide to final purity. GP big gastrin is a 33 amino acid peptide with the following sequence: less than ELGPQVPAHLRTDLSKKQGPWAEEEAAYGWMDF# The GP peptide is different from pig G34 in 6 of the 17 NH2-terminal amino acids as well as in the previously reported deletion of a glutamic acid in the COOH-terminus.

Amino Acid Sequence↗

PubMed1986

Isolation, sequence, and developmental profile of a brain-specific polypeptide, PEP-19.

By comparing the HPLC profiles of cerebellar extracts from adult and neonatal rats, a developmentally regulated polypeptide, termed PEP-19, was identified. The concentration of PEP-19 rose from 0.1 nmol/g of cerebellum at birth to 2 nmol/g at 20 days postpartum. The polypeptide could also be detected at lower levels in olfactory bulbs of adult rats but was absent in cerebral cortex, brain stem, and all non-neural tissues examined. HPLC-purified PEP-19 contained 61 amino acids and had a molecular size of 7.6 kDa. The native polypeptide is blocked at its amino terminus but was sequenced following proteolytic and chemical fragmentation. The primary amino acid sequence was determined to be: X (S-E) R Q S A G A T N G K D K T S G D N D G Q K K V Q E E F D I D M D A P E T E R A A V A I Q S Q F R K F Q K K K A G S Q S. PEP-19 has a unique sequence, but shares some homology with several calcium binding proteins including the beta chain of S100 and intestinal calcium binding protein. This polypeptide is the primary translation product of cerebellar poly(A)+ mRNA.

Age Factors↗

PubMed1986

Partial sequencing of human adult, human fetal, and bovine intestinal alkaline phosphatases: comparison with the human placental and liver isozymes.

Purification, molecular weights, amino acid compositions, and partial sequencing of intestinal alkaline phosphatases (EC 3.1.3.1) from human adult, human fetal, and bovine sources is reported. Additional sequence information is presented for the bovine liver isozyme. Comparisons are made of the partial primary structures of intestinal alkaline phosphatases with those of the isozymes from liver and placenta. Homologies among these isozymes provide structural data to corroborate some concepts of the etiology of these isozymes and refute others.

Alkaline Phosphatase↗