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U Hellman

Publications and source records attributed to U Hellman.

At least 127 records · Page 7Linked to original sources
PubMed1991

Self-proteolysis of the cysteine proteinase, cruzipain, from Trypanosoma cruzi gives a major fragment corresponding to its carboxy-terminal domain.

The major cysteine proteinase (cruzipain) purified from Trypanosoma cruzi epimastigotes catalyzes its own degradation in the presence of beta-mercaptoethanol, at 56 degrees C and pH 6. The reaction is affected by the same inhibitors which inhibit the azocaseinase activity, and yields a major 25-kDa fragment, which contains carbohydrate, few, if any, aromatic amino acids, and presents a proline-rich N-terminus (GPGPXPEP...), in addition to a number of small peptides, which can be isolated by reversed-phase HPLC, but are lost during electrophoresis. The results, together with recently published evidence of Mottram et al. and Eakin et al., are compatible with a structure for cruzipain consisting of a conventional cysteine proteinase moiety, linked to a long C-terminal extension including the 25-kDa fragment, which would contain a high proportion of the carbohydrate and the proline residues present in the original 60-kDa molecule.

Amino Acid Sequence↗
PubMed1991

The N-terminal amino acid sequence of phenol hydroxylase contains a dinucleotide-binding sequence motif.

The N-terminal sequence of phenol hydroxylase from Trichosporon cutaneum was determined by Edman degradation of the integral protein and of fragments obtained by hydroxylamine cleavage and by digestion with Staphylococcus V8 protease. A continuous sequence of 80 residues from the N terminus was determined: TKYSESYCDV10, LIVGAGPAGL20 MAARVLSEYV30 RQKPDLKVRI40 IDKRSTKVYN50 GQADGLQCRT60 LESLKNLRLA70 DKIXSEXNDM80. A single N-terminal sequence was detected, suggesting two identical subunits in the dimeric enzyme. We suggest the occurrence of an FAD-binding site near the N terminus. The C-terminal sequence is -LSTA, as determined by carboxypeptidase digestion.

Amino Acid Sequence↗
PubMed1990

Peptide mapping on HIV polypeptides expressed in Escherichia coli. Quality control of different batches and identification of tryptic fragments containing residues of aromatic amino acids or cysteine.

Peptide mapping was used for the quality control of different batches of the recombinant HIV proteins p24 core and p24-gp41, expressed in Escherichia coli. These proteins comprise gag and env region polypeptides of the virus and may serve as suitable components in the diagnosis of HIV infections. The proteins were digested with trypsin and the mixtures were subjected to peptide mapping to prove batch equivalence of p24-gp41 and to isolate fragments of the p24-gp41 digest that differ from those of the p24 core digest. The proteins were reduced with dithiothreitol and the cysteine residues were derivatized by addition of 4-vinylpyridine. Peptide mapping was performed by means of reversed-phase high-performance liquid chromatography. Batch equivalence was proved by comparison of the maps. Peaks present in one map but not in the other were considered to be due to sequence differences or variability in digestion.

Amino Acid Sequence↗
PubMed1990

Amino acid sequence from degu islet amyloid-derived insulin shows unique sequence characteristics.

The main protein of enriched and purified amyloid from Octodon degus pancreatic islets was identified as insulin. The material was reduced and alkylated and the A- and the B-chain were separated by reversed phase chromatography and subjected to Edman degradation and amino acid analysis. It was shown that the A-chain contains two additional C-terminal amino acid residues (i.e. a total of 23 residues) and that the B-chain has a deletion in the C-terminal part (i.e. a total of 29 residues). The obtained sequence follows: A-chain: GIVDQCCNNICTFNQLQNYCNVP B-chain: YSSQHLCGSNLVEALYMTCGRSGFYRPHD.

Amino Acid Sequence↗
PubMed1990

TGF-beta 1 binding protein: a component of the large latent complex of TGF-beta 1 with multiple repeat sequences.

TGF-beta occurs in a latent complex of high Mr. We report the cDNA cloning and an initial structural and functional characterization of a component of the large latent TGF-beta 1 complex, denoted TGF-beta 1 binding protein (TGF-beta 1-BP). Most of the sequence of fibroblast TGF-beta 1-BP is made up of cysteine-rich repeats of two different kinds; there are 16 EGF-like repeats and three repeats with a distant resemblance to EGF, but of a distinct type hitherto not found in any other protein. beta-hydroxylated asparagine residues were identified in two of the EGF-like repeats. TGF-beta 1-BP purified from human platelets is considerably smaller than the fibroblast form (125-160 kd vs. 170-190 kd), suggesting that there is alternative splicing of the TGF-beta 1-BP gene or that TGF-beta 1-BP undergoes cell-specific proteolysis. TGF-beta 1-BP was found not to bind and inactive TGF-beta 1; its role in the latent complex is discussed.

Amino Acid Sequence↗
PubMed1990

Amino acid and carbohydrate composition of a lysosomal cysteine proteinase from Trypanosoma cruzi. Absence of phosphorylated mannose residues.

The size of a lysosomal cysteine proteinase from epimastigotes of Trypanosoma cruzi decreased from 60 to 54 kDa upon treatment with endo beta-N-acetylglucosaminidase H. A lower-molecular weight component (30-35 kDa), which usually accompanies the 60-kDa protein also increased its electrophoretic mobility, and seems to consist of a mixture of degradation products of the enzyme, since both the larger and the smaller components had the same N-terminal sequence as the 60-kDa protein. The amino acid composition of the protein moiety and the composition of the oligosaccharide chains have been determined. The oligosaccharide chains are of the high-mannose type, and contain 6, 7, 8 or 9 mannose residues, as shown both by in vivo labeling with [U-14C]glucose, and by labeling the endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharides of the purified enzyme with tritiated sodium borohydride. The oligosaccharide chains did not contain phosphate residues. Further studies with [U-14C]-labeled total glycoproteins of T. cruzi and enzyme assays, suggest that T. cruzi and other trypanosomatids do not target their lysosomal enzymes to the organelle through the mannose-6-phosphate marker pathway.

Amino Acid Sequence↗
PubMed1990

Localization of platelet-derived endothelial cell growth factor in human placenta and purification of an alternatively processed form.

Platelet-derived endothelial cell growth factor (PD-ECGF) was purified to homogeneity from human term placenta, an organ characterized by extensive angiogenesis. N-terminal amino acid sequencing revealed that placental PD-ECGF was proteolytically processed at Thr-6, in contrast to PD-ECGF purified from human platelets, which is processed at Ala-11. The purified factor stimulated porcine aortic endothelial cells as well as two choriocarcinoma cell lines. Immunohistochemical staining revealed that PD-ECGF was present in the connective tissue cells of the placenta. The possibility that PD-ECGF is involved in the development of the placenta is discussed.

Amino Acid Sequence↗
PubMed1990

Uukuniemi virus S RNA segment: ambisense coding strategy, packaging of complementary strands into virions, and homology to members of the genus Phlebovirus.

We determined the complete nucleotide sequence of the small (S) RNA segment of Uukuniemi virus, the prototype of the Uukuvirus genus within the Bunyaviridae family. The RNA, which is 1,720 nucleotides long, contains two nonoverlapping open reading frames. The 5' end of one strand (complementary to the viral strand) encodes the nonstructural protein NSs (273 residues; molecular weight, 32,019), whereas the 5' end of the viral-sense strand encodes the nucleocapsid protein N (254 residues; molecular weight, 28,508). Thus, the S RNA uses an ambisense coding strategy previously described for the S segment of two phleboviruses and the arenaviruses. The localization of the N protein within the S RNA sequence was confirmed by amino-terminal sequence analysis of all five possible cyanogen bromide fragments obtained from purified N protein. Northern (RNA) blot analyses with strand-specific probes showed that the N and NSs proteins are translated from subgenomic mRNAs about 800 and 850 nucleotides long, respectively. These mRNAs are apparently transcribed from full-length S RNAs of opposite polarities. The two mRNA species were also detected in virus-infected cells. Interestingly, highly purified virions contained full-length S RNA copies of both polarities at a ratio of about 10:1. In contrast, virions contained exclusively negative-strand copies of the M RNA segment. The possible significance of these results for viral infection is discussed. The amino acid sequence of the N protein showed 35 and 32% homology (identity) with the N protein of Punta Toro and sandfly fever Sicilian viruses, two members of the Phlebovirus genus. The NSs proteins were much less related (about 15% identity). In addition, the extreme 5' and 3' ends of the S RNA, which are complementary to each other, also showed a high degree of conservation with the two phleboviruses. These results indicate that the uukuviruses and phleboviruses are evolutionarily related and suggest that the two genera could be merged into a single genus within the Bunyaviridae family.

Amino Acid Sequence↗
PubMed1990

A polyclonal antiserum against chromogranin A and B--a new sensitive marker for neuroendocrine tumours.

Chromogranins A, B, and C, proteins that are co-stored and co-released with peptides and amines, have been identified in a variety of neuroendocrine tissues, both normal and neoplastic. We examined the secretion of chromogranin A and chromogranin A + B by hormone-producing tumours in patients with endocrine pancreatic tumours, carcinoid tumours, pheochromocytomas, and small cell lung cancer. The radioimmunoassay determining the plasma concentrations of chromogranin A + B showed a greater sensitivity than that determining chromogranin A alone. All patients with endocrine pancreatic tumours, carcinoids, and pheochromocytomas had increased levels of chromogranin A + B, whereas a small number of the patients (5/18 with endocrine pancreatic tumours and 1/3 with pheochromocytomas) had normal levels of chromogranin A. Also in immunocytochemical stainings, our polyclonal antiserum detecting both chromogranin A and B showed a greater sensitivity than other available antisera against chromogranin A, B and C. We have demonstrated that a polyclonal antiserum against a mixture of chromogranin A and B might be a more sensitive marker than chromogranin A alone for diagnosing neuroendocrine tumours. This is not surprising, since both chromogranins are widely distributed in neuroendocrine cells.

Amino Acids↗
PubMed1989

Further characterization and partial amino acid sequence of a cysteine proteinase from Trypanosoma cruzi.

A cysteine proteinase from epimastigotes of Trypanosoma cruzi, Tul 2 stock, has been purified to homogeneity from cell-free extracts obtained by freezing and thawing, by a procedure involving ammonium sulfate fractionation, DEAE-Sephacel chromatography, and gel filtration on Sephadex G-200; when necessary, further purification was attained by fast protein liquid chromatography on Mono Q and Superose 6 columns. The purified enzyme was strongly inhibited by leupeptin, antipain and chymostatin (I50 values of 0.25, 0.75 and 1 microM, respectively), little inhibited by elastatinal, and unaffected by pepstatin A. The enzyme is a glycoprotein, as shown by binding to ConA-Sepharose and elution with alpha-methyl-D-mannopyranoside and alpha-methyl-D-glucopyranoside. Partial amino acid sequences were obtained from the N-terminal end (32 amino acids) of the carbamidomethylated enzyme, and from a tryptic peptide (14 amino acids) of the pyridylethylated enzyme. Both regions show considerable homology with papain and some cathepsins, such as cathepsin L, thus showing that the enzyme belongs to the cysteine proteinase family.

Amino Acid Sequence↗
PubMed1989

Dual affinity fusion approach and its use to express recombinant human insulin-like growth factor II.

A dual affinity fusion concept has been developed in which the gene encoding the desired product is fused between two flanking heterologous genes encoding IgG- and albumin-binding domains. Using sequential IgG and serum albumin affinity chromatography, a full-length tripartite fusion protein is obtained. This approach was used to recover a full-length fusion product in Escherichia coli containing the human insulin-like growth factor II (IGF-II). Surprisingly, the recombinant IGF-II showed increased stability against proteolytic degradation in E. coli when produced as a dual affinity fusion protein, as compared to an N-terminal fusion protein. After site-specific cleavage of the tripartite fusion protein, IGF-II molecules with immunological and receptor binding activity were obtained without renaturation steps. The results demonstrate that proteins can fold into biologically active structures, even if provided with large flanking heterologous protein domains. The concept was further used to characterize the specific degradation of recombinant IGF-II in this heterologous host.

Amino Acid Sequence↗
PubMed1989

Chromogranins--new sensitive markers for neuroendocrine tumors.

Chromogranins A, B and C, proteins that are costored and coreleased with peptides and amines, have been identified in a variety of endocrine and nervous tissues, both normal and neoplastic. We examined the secretion of chromogranin A and chromogranin A + B by hormone-producing tumors in patients with endocrine pancreatic tumors (EPT), carcinoid tumors, pheochromocytomas and small cell lung cancer (SCLC). Radioimmunoassay (RIA) of the plasma/serum concentrations of chromogranin A + B showed a greater sensitivity than RIA of chromogranin A alone. All patients with EPT, carcinoids and pheochromocytomas had increased levels of chromogranin A + B, whereas a small number of the patients (5/18 with EPT and 1/3 with pheochromocytomas) had normal levels of chromogranin A. Also in immunocytochemical stainings, our polyclonal antiserum detecting both chromogranin A and B showed a greater sensitivity than other available antisera against chromogranin A, B and C.

Adenoma, Islet Cell↗
PubMed1989

Expression of three recombinant homodimeric isoforms of PDGF in Saccharomyces cerevisiae: evidence for difference in receptor binding and functional activities.

Three recombinant homodimeric isoforms of platelet-derived growth factor (PDGF) were produced and purified in milligram quantities by expression of PDGF A- and B-chains in yeast cells. Structural analysis of the purified short and long variants of PDGF-AA (PDGF-AAS and PDGF-AAL) and PDGF-BB showed that they had been properly processed and assembled into dimers. PDGF-AAS and PDGF-AAL were found to bind only to the PDGF A-type receptor on human fibroblasts, with affinities of 0.1 and 0.2 nM, respectively. PDGF-BB bound to cells with A- and B-type receptors and to cells with B-type receptor only with affinities of 0.6 nM in both cases. Each fibroblast appeared to express about 4-5 times more B-type receptors than A-type receptors. The maximal mitogenic response to PDGF-BB of human fibroblasts was almost 2-fold higher than that induced by either of the two PDGF-AA forms. The three isoforms of PDGF also stimulated growth in soft agar of human fibroblasts with PDGF-BB inducing a higher maximal response.

Base Sequence↗
PubMed1988

A major part of platelet-derived growth factor purified from human platelets is a heterodimer of one A and one B chain.

Platelet-derived growth factor, PDGF, purified from human platelets is a disulfide-bonded dimer consisting of two homologous polypeptide chains denoted A and B; it has not been known whether it is a heterodimer or a mixture of homodimers. We present here evidence that a major part of PDGF has a heterodimer structure. A highly homogeneous, 31-kDa PDGF was purified in the presence of protease inhibitors and shown to contain both chains by means of immunoprecipitations with peptide antisera specific for the A and B chains, respectively. The susceptibility of PDGF to mild acid treatment and its chromatographic behavior in reversed-phase high performance liquid chromatography and immobilized metal ion affinity chromatography, as compared to A and B chain homodimers, is consistent with a heterodimer structure. Analysis of PDGF purified according to our routine, large scale procedure revealed the major part to have a heterodimer structure. In addition, B chain homodimers were also found. With the demonstration that a major part of PDGF purified from human platelets occurs as a heterodimer, all three dimeric forms of PDGF have been identified. The following nomenclature to distinguish the various forms is suggested: PDGF-AA, a homodimer of A chains; PDGF-AB, a heterodimer; PDGF-BB, a homodimer of B chains; PDGF, any dimeric form of A or B chains.

Antigen-Antibody Complex↗