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Biomedical subjects

U Hellman

Publications and source records attributed to U Hellman.

At least 109 records · Page 6Linked to original sources
PubMed1993

Yeast TKL1 gene encodes a transketolase that is required for efficient glycolysis and biosynthesis of aromatic amino acids.

A gene encoding transketolase, TKL1, was cloned from the budding yeast Saccharomyces cerevisiae using oligonucleotide primers derived from protein sequence data. The TKL1 sequence predicts a 74-kDa polypeptide which is related to other transketolases. A sequence comparison revealed that the transketolases can be subdivided into three evolutionary branches. We also found that the transketolases are related to another vitamin B1-dependent enzyme: the E1 subunit of pyruvate dehydrogenase from Escherichia coli. Gene disruption and overexpression experiments were used to investigate the function of transketolase in yeast. We found that growth on fermentable carbon sources, but not on gluconeogenic carbon sources, is reduced in cells disrupted for TKL1. This suggests that the glycolytic efficiency is impaired. Growth on fermentable carbon sources is also reduced in cells that overexpress TKL1. Finally, we found that cells disrupted for TKL1 are unable to grow in the absence of aromatic amino acids. This is most likely due to the fact that transketolase is required for the synthesis of erythrose-4-P, a precursor of the aromatic amino acids.

Amino Acid Sequence↗
PubMed1993

The retinal pigment epithelial membrane receptor for plasma retinol-binding protein. Isolation and cDNA cloning of the 63-kDa protein.

Retinol, a metabolic precursor of retinal and retinoic acid, is transported in plasma by the plasma retinol-binding protein (RBP). The cellular uptake of retinol from RBP is believed to involve a specific membrane receptor for RBP. In retinal pigment epithelium the RBP receptor appears to be an oligomeric protein complex, and we have previously identified a 63-kDa membrane protein as part of this receptor. The 63-kDa protein (p63) has now been isolated, and we have cloned the corresponding cDNA. In a data base search no sequences similar to p63 were identified. Hydropathy analyses of the 533 amino acids deduced from the cDNA sequence did not indicate an N-terminal signal sequence or obvious transmembrane regions. In vitro translation of synthetic mRNA encoding p63, in the presence of heterologous microsomes, verified that p63 does not become cotranslationally membrane-inserted. Transcripts for p63 are abundantly expressed in retinal pigment epithelium with no detectable expression in several other tissues. Southern blotting analysis of bovine and human genomic DNA revealed several hybridizing fragments suggesting a complex organization of the corresponding genes.

Amino Acid Sequence↗
PubMed1993

Mapping of the antibody- and receptor-binding domains of granulocyte colony-stimulating factor using an optical biosensor. Comparison with enzyme-linked immunosorbent assay competition studies.

An automated optical biosensor instrument for measuring molecular interactions (Pharmacia BIAcore) has been used to characterise the epitopes recognised by 15 monoclonal antibodies raised against recombinant human granulocyte colony-stimulating factor (G-CSF). The BIAcore combines an autosampler and integrated microfluidic cartridge for the introduction and transportation of samples to the sensor chip surface, with surface plasmon resonance to detect binding events. A rabbit anti-mouse Fc antibody, coupled to the sensor surface in situ using conventional protein chemistry techniques, was used to capture an anti-G-CSF monoclonal antibody. G-CSF was bound to this antibody by injection over the sensor surface. Multi-site binding experiments were then performed in which other anti-G-CSF monoclonal antibodies were injected sequentially over the surface, and their ability to bind to the G-CSF in a multimolecular complex monitored in real time. Results obtained using the biosensor have been compared with data obtained by cross competition studies using biotinylated antibodies or antibody binding studies using chemically or enzymatically derived G-CSF peptide fragments or synthetic peptides. The results of these studies are in excellent agreement with the data from the BIAcore, although modification of the antibody or G-CSF occasionally altered the epitope affinity.

Animals↗
PubMed1993

Molecular cloning and characterization of ficolin, a multimeric protein with fibrinogen- and collagen-like domains.

We have previously identified and purified transforming growth factor-beta 1 (TGF-beta 1)-binding proteins from porcine uterus membranes (Ichijo, H., Rönnstrand, L., Miyagawa, K., Ohashi, H., Heldin, C.-H., and Miyazono, K. (1991) J. Biol. Chem. 266, 22459-22464). One of these TGF-beta 1-binding proteins, with a molecular weight of 40,000, was purified to homogeneity and subjected to amino acid sequence analysis. The amino acid sequences obtained were used to isolate two closely related cDNA clones from a porcine uterus cDNA library. The deduced amino acid sequences revealed that both cDNAs encoded proteins that were mainly composed of fibrinogen-like and collagen-like domains. Therefore, they were denoted ficolin-alpha and ficolin-beta. Expression of ficolin-alpha and -beta cDNA in mammalian cells revealed that ficolin forms dimers, trimers, and several higher order of oligomers, whose molecular weights fit well with those of the purified TGF-beta 1-binding proteins from porcine uterus. Moreover, immunoblotting analysis using a peptide anti-serum against ficolin indicated that the TGF-beta 1-binding proteins identified in porcine uterus are ficolin-alpha, -beta, and their oligomers or closely related molecules. However, recombinant ficolin-alpha and -beta did not bind TGF-beta 1, despite the similarities in molecular weights and immunoreactivity with the material from the natural source. It is possible that a specific posttranslational modification of ficolin or interaction with another component is needed for TGF-beta 1 binding. Analysis by Northern blotting revealed that the expression of ficolin-alpha mRNA is relatively restricted and most abundant in placenta and lung. On the other hand, ficolin-beta was mainly expressed in skeletal muscle. The in vivo functions of ficolin will be discussed.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of microsomal acyl-CoA thioesterase. A member of the rat liver microsomal carboxylesterase multi-gene family.

We have isolated and characterized an acyl-CoA thioesterase from rat liver microsomes. The enzyme consists mainly of a monomer of 59 kDa. However, the final preparation was found to contain minor amounts of a trimeric form of the protein. The enzyme was purified more than 85-fold from isolated microsomes and used for NH2-terminal sequence analysis and for analysis of peptides isolated after proteolytic digestion. The NH2-terminal sequence was unique but highly conserved compared to those of other carboxylesterases. Internal sequence data, covering almost 20% of the protein, showed high similarity to the deduced amino acid sequences from a cDNA encoding a carboxylesterase synthesized in the liver and subsequently secreted to the blood [Alexson, S. E. H., Finlay, T. H., Hellman, U., Diczfalusy, U. & Eggertsen, G., unpublished results] and nonspecific rat liver microsomal carboxylesterase with isoelectric point of 6.1 [Robbi, M., Beaufay, H. & Octave, J.-N. (1990) Biochem. J. 269, 451-458], thus confirming earlier suggestions that this enzyme is a member of the microsomal carboxylesterase multigene family. The peptide sequences contained two of the four conserved cysteic acid residues found in other carboxylesterases. Amino acid analysis indicated that the protein contains five cysteine residues in contrast to most other described carboxylesterases which contain four highly conserved cysteins. The purified protein was used for immunization and the antiserum was used to detect the protein as well as its trimeric form, which is a minor component, in isolated rat liver microsomes. The antiserum recognized proteins of similar sizes in microsomes and 100,000 x g supernatant prepared from hamster brown adipose tissue, a tissue known to contain very high activity of carboxylesterase, and to recognize carboxylesterases isolated from porcine and rabbit liver.

Amino Acid Sequence↗
PubMed1993

A potassium channel toxin from the secretion of the sea anemone Bunodosoma granulifera. Isolation, amino acid sequence and biological activity.

A peptide toxin affecting potassium channels was isolated from the sea anemone Bunodosoma granulifera. It facilitates acetylcholine release at avian neuromuscular junctions, competes with dendrotoxin I, a probe for voltage-dependent potassium channels, for binding to synaptosomal membranes of rat brain with a Ki of 0.7 nM and suppresses K+ currents in rat dorsal root ganglion neurones in culture. It represents a new structural type of potassium channel toxin with the sequence V1RCDWFKETA10CRHAKSLGNC20RTSQKYRANC30AKTLQCC37 (M(r) 4275, three disulfides).

Amino Acid Sequence↗
PubMed1993

Ca2+ binding of latent transforming growth factor-beta 1 binding protein.

Latent transforming growth factor-beta 1 binding protein (LTBP) is a constituent of the latent high molecular weight complex of TGF-beta 1 in human platelets. In the present communication, we show that LTBP binds Ca2+ in its free form as well as in the latent TGF-beta 1 complex. The binding of Ca2+ induces a structural change which protects the molecule against proteolysis and changes its elution position when analyzed by anion exchange chromatography. The in vitro activation of TGF-beta 1 is not influenced by the presence of Ca2+. The possible significance of Ca2+ binding of LTBP is discussed.

Calcium↗
PubMed1993

Unambiguous identification of histone H1 in Trypanosoma cruzi.

The existence of histone H1 has been questioned in Trypanosomatids. We report here the presence of a histone H1 in the chromatin of Trypanosoma cruzi. This protein was purified by narrow-bore reversed phase HPLC and its amino acid composition analyzed and compared with histones H1 from other species. Furthermore, the purified chromosomal protein was digested with proteases and the amino acid sequences of the resulting peptides were analyzed by the automated Edman degradation. The sequences obtained were found to present a high degree of homology when compared to the carboxy terminal domain of other known histones H1.

Amino Acid Sequence↗
PubMed1993

Fragments of chromogranin A are present in the urine of patients with carcinoid tumours: development of a specific radioimmunoassay for chromogranin A and its fragments.

Chromogranin A is a well-known protein constituent in granules of neuroendocrine cells. It is also known that plasma levels of chromogranin A increase considerably in patients with neuroendocrine tumours and thus chromogranin A is used as a marker for these tumours. In the present study, we have shown that fragments of chromogranin A are excreted into the urine in some patients with carcinoid tumours. The chromogranin A molecule appeared in the urine N-terminally cleaved at amino acid positions 116 and 210, which are previously reported cleavage sites of the molecule. The fragments identified were mainly of about 35 kDa in size. The unprocessed chromogranin A molecule was not excreted in the urine. Five out of 40 patients excreting the fragments had slight tubular dysfunction in the kidneys. We also showed that these renally excreted split products of chromogranin A were immunogenic and could be used for production of antibodies against chromogranin A. These antibodies were used both for immunocytochemistry and for the development of a specific and sensitive radioimmunoassay for chromogranin A and its fragments. Measurements of plasma chromogranin A by radioimmunoassay appeared to be a better marker for tumour growth than were measurements of chromogranin A in the urine.

Adult↗
PubMed1993

Presence of histone H2B in Trypanosoma cruzi chromatin.

The organization of chromatin in protists presents some characteristic features. In Trypanosoma cruzi, no condensation of chromatin into chromosomes is observed during cell division. A systematic characterization of histones should provide information on this peculiar behaviour. Histone H2B from this parasite was characterized by selective dissociation from chromatin in 0.8 M NaCl, by its elution pattern in narrow-bore reversed phase high performance liquid chromatography, by polyacrylamide gel electrophoresis and by partial sequencing of its amino terminal domain. This chromosomal protein differs from histone H2B of other species. The first 12 amino acids are missing which explains its lower molecular weight when compared to human histone H2B. Correspondingly, the amino terminal domain of T. cruzi histone H2B is 25-30% shorter than other histones H2B. Moreover, three out of four acetylation sites present in human histone H2B are missing in T. cruzi histone H2B. The differences in size and in acceptor sites for acetylation of T. cruzi histone H2B when compared to human histone H2B may represent a functional feature to consider for the understanding of the chromatin cycle of condensation in this parasite.

Amino Acid Sequence↗
PubMed1992

On the post-translational modifications at the C-terminal domain of the major cysteine proteinase (cruzipain) from Trypanosoma cruzi.

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi, has a 130 amino acid-long C-terminal domain, which, although microheterogeneous in SDS-PAGE, has a single N-terminal amino acid sequence. Most of the Thr residues present at the beginning of this sequence are modified; the nature of this modification is still unknown, but O-glycosylation and phosphorylation seem both to be absent. The only potential site for N-glycosylation (Asn 254) is glycosylated in vivo. Most of the eight Cys residues are involved in disulfide bridges. The results are consistent with cruzipain being made of two well-defined domains, a catalytic one with high homology to cathepsin L, and a C-terminal domain, linked to the former by a 'hinge' corresponding to the Pro- and Thr-rich region at its N-terminus.

Amino Acid Sequence↗
PubMed1992

Assignment of interchain disulfide bonds in platelet-derived growth factor (PDGF) and evidence for agonist activity of monomeric PDGF.

Platelet-derived growth factor (PDGF) is a dimeric factor stabilized by disulfide bonds. Using an approach involving partial reduction of PDGF, we have identified the 2nd and 4th cysteine residues in the PDGF chains as the cysteine residues forming interchain disulfide bonds. Analysis of PDGF mutants in which the 2nd and 4th cysteine residues were mutated to serine residues revealed that the disulfide bonds are arranged in a cross-wise manner, with the 2nd cysteine residue in one chain being linked to the 4th cysteine residue in the other. A PDGF B-chain mutant, in which both the 2nd and 4th cysteine residues were substituted with serine residues, migrated as a monomer in sodium dodecyl sulfate gel electrophoresis and retained receptor binding activity. When analyzed in receptor dimerization and autophosphorylation assays, this mutant showed agonistic activity. Thus, structural information has been obtained that will allow the large scale production of properly folded monomeric PDGF, as well as design of specific PDGF heterodimers.

Amino Acid Sequence↗
PubMed1992

Identification of two C-terminal autophosphorylation sites in the PDGF beta-receptor: involvement in the interaction with phospholipase C-gamma.

Two novel sites of autophosphorylation were localized to the C-terminal tail of the PDGF beta-receptor. To evaluate the importance of these phosphorylation sites, receptor mutants in which Tyr1009, Tyr1021 or both were replaced with phenylalanine residues, were expressed in porcine aortic endothelial (PAE) cells. These mutants were similar to the wild type receptor with regard to protein tyrosine kinase activity and ability to induce mitogenicity in response to PDGF-BB. However, both the Y1009F and Y1021F mutants showed a decreased ability to mediate association with and the tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) compared to the wild type PDGF beta-receptor; in the case of the Y1009F/Y1021F double mutant, no association or phosphorylation of PLC-gamma could be detected. These data show that tyrosine phosphorylation of PLC-gamma is dependent on autophosphorylation of the PDGF beta-receptor at Tyr1009 and Tyr1021.

Amino Acid Sequence↗
PubMed1992

Extremely divergent histone H4 sequence from Trypanosoma cruzi: evolutionary implications.

Trypanosoma cruzi presents six histones electrophoretically resolved in three gel systems. Indirect evidence shows that one of these histones, named e, corresponds to H4 in other species. We present evidence that histone e is H4 by sequencing its amino terminal end. The amino terminal of T. cruzi histone H4, unlike that of other H4s examined thus far is not blocked. Moreover, this protein presents two variants. This partial amino acid sequence of T. cruzi histone H4 differs greatly from homologous sequences of human, yeast, or Tetrahymena. Since the conservatism of the core histones (H2A, H2B, H3, and H4) is clearly illustrated by comparative sequence analyses, the data shown here demonstrates that T. cruzi histone H4 is the most divergent reported. Quantitative analysis of the data suggests that the rate of substitutions in the histone H4 amino terminal sequence varies among different lineages. We postulate a slow-down in the evolutionary rate of histone H4 amino terminal domain in the metazoa branch related perhaps to the appearance of a novel function for this domain.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of a hemoglobin-derived opioid peptide from the human pituitary gland.

An opioid nonapeptide was isolated from fresh frozen human pituitaries. Its primary structure (Leu-Val-Tyr-Pro-Trp-Thr-Gln-Arg) was identical to fragment 32-40 of the beta-, delta-, gamma- and epsilon-chains of human hemoglobin. A larger peptide of about 4.5 kDa, which generated a fragment containing the nonapeptide on trypsin digestion, showed an amino acid composition similar to fragment 1-41 of the beta-chain of human hemoglobin. The nonapeptide interacted with mu-opioid receptors in rat brain homogenates using [3H]-(D-Ala2, MePhe3, Gly-ol5)-enkephalin and with sigma-receptors using (+)-[3H]-3-(3-hydroxyphenyl)-N-1-(propyl)piperidine, respectively. The affinities for mu-opioid receptors were in the same range as those observed for the structurally related beta-casomorphins. However, the isolated peptide showed markedly higher affinity at sigma-binding sites when compared to the beta-casomorphins or other opioid peptides. The opioid potency of this peptide as determined in the guinea-pig ileum myenteric plexus muscle preparation, was significant but less than that observed for the beta-casomorphins.

Amino Acid Sequence↗
PubMed1991

Differential degradation of a recombinant albumin-binding receptor in Escherichia coli.

The degradation in Escherichia coli of the recombinant serum-albumin-binding receptor derived from streptococcal protein G was investigated using a dual-affinity fusion approach. The proteolytic degradation of the receptor was characterized when fused to human proinsulin and human secretin. Several cleavages occurred at sequences not normally regarded as proteolytically sensitive, such as the dipeptide sequences Ile-Gly, Val-Ser and Ser-Ala. Depending on the fusion partner, large differences in the degradation of the albumin-binding domain were observed. Thus, susceptibility to proteolysis of a recombinant protein can be affected by a neighbouring domain.

Amino Acid Sequence↗
PubMed1991

Identification of three amino acids in the platelet-derived growth factor (PDGF) B-chain that are important for binding to the PDGF beta-receptor.

Platelet-derived growth factor (PDGF) is a disulfide-linked dimeric protein composed of two homologous polypeptide chains denoted A and B. Two types of PDGF receptors, alpha and beta, have been characterized. Whereas PDGF-AA binds only to PDGF alpha-receptors, PDGF-BB binds to both receptor types with high affinity. To map the regions of the PDGF B-chain that confer its ability to bind with high affinity to the PDGF beta-receptor, we expressed PDGF A/B-chain chimeras in COS cells and analyzed them with regard to PDGF alpha- and beta-receptor binding. A systematic analysis revealed that replacement of Asn-115, Arg-154, and Ile-158 of the PDGF B-chain with the corresponding A-chain amino acids led to a dramatic decrease in the affinity for the beta-receptor. Conversely, introduction of B-chain amino acids into the A-chain in the region spanning from Asn-115 to Ile-158 yielded a product with high affinity for the beta-receptor. These data thus indicate that Asn-115, Arg-154, and Ile-158 are likely to be part of the active site of the PDGF B-chain.

Amino Acid Sequence↗