Biomedical subjects

U Hellman

Publications and source records attributed to U Hellman.

At least 145 records · Page 8Linked to original sources

PubMed1988

Latent high molecular weight complex of transforming growth factor beta 1. Purification from human platelets and structural characterization.

Human transforming growth factor beta 1 (TGF-beta 1) was purified as a latent high Mr complex from human platelets by a six-step procedure. Analysis by sodium dodecyl sulfate (SDS)-gel electrophoresis under reducing conditions revealed that the complex was composed of at least three components with apparent Mr values of 13,000, 40,000, and 125,000-160,000. The 13-kDa subunit was part of a disulfide-bonded dimer and was identified by amino acid sequencing as TGF-beta 1. The 40-kDa subunit was identified as the amino-terminal part of the TGF-beta 1 precursor lacking the hydrophobic signal sequence. Partial sequencing of the 125-160-kDa protein revealed that it is distinct from known proteins. The 40-kDa and the 125-160-kDa subunits are linked by disulfide bonds, forming a complex with an apparent Mr of 210,000 on SDS gels under nonreducing conditions. Experiments with partial reduction revealed that each complex contains two 40-kDa components linked by disulfide bonds; in addition, the dimer is disulfide-linked to one 125-160-kDa binding protein. TGF-beta 1 binds noncovalently to the 210-kDa complex, and in bound form, TGF-beta 1 is inactive. Incubations of the latent form of TGF-beta 1 at extreme pH values, in 0.02% SDS or in 8 M urea, lead to activation of TGF-beta 1, whereas the complex was resistant to treatment with 5 M NaCl or heat (3 min at 95 degrees C).

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of the rubella virus capsid protein gene reveals an unusually high G/C content.

The nucleotide sequence of the rubella virus capsid protein (C) gene has been determined from a cDNA clone derived from the 40S genomic RNA. The sequence covers the coding region of the C protein (831 nucleotides), 70 nucleotides of the 5' untranslated region, and the 5' end of the downstream E2 membrane protein gene. The capsid gene is unusually rich in C (41.6%) and G (31.2%) residues (G + C 72.8%), and poor in A (15.4%) and U residues (11.8%). There are regions with long runs of up to 45% C or 35% G residues. The codon usage is non-random, with a strong preference for C and G residues in the third position. Starting from two in-frame AUG codons (seven amino acid residues apart) an open reading frame (ORF) was identified that extended in frame into the ORF coding for the downstream E2 membrane protein gene. Since the amino terminus of the capsid protein is blocked, we could not determine which of the AUGs serve as the initiating codon. To verify that the deduced ORF was correct, we have determined the amino acid sequence of 13 tryptic peptides corresponding to one-third of the C protein. Our data show that the C protein is about 277 residues in length (Mr about 30750). It is very hydrophilic and rich in prolines (14.1%) and arginines (14.4%). Clusters of these amino acids are concentrated in the amino-terminal third of the C protein. No sequence homology to the capsid protein of several alphaviruses was observed. Together with our previous sequence data we have now completed the sequence of the genes coding for the structural proteins C, E2 and E1 of rubella virus.

Amino Acid Sequence↗

PubMed1987

Identification of a polypeptide growth inhibitor from bovine mammary gland. Sequence homology to fatty acid- and retinoid-binding proteins.

A polypeptide growth inhibitor purified from bovine mammary gland (mammary-derived growth inhibitor) has been shown to reversibly inhibit proliferation of mammary carcinoma cells at concentrations of about 10(-10) M. The carrier of inhibitory activity has been identified biochemically as an about 13-kDa polypeptide and chemically by elucidating the amino acid sequence. No homology to any of the hitherto structurally investigated growth inhibitors (transforming growth factor beta, interferons) has been observed. The data revealed extensive sequence homology of mammary-derived growth inhibitor to a family of low molecular mass hydrophobic ligand-binding proteins, among them a fatty acid-binding protein from rat heart, myelin P2, a differentiation associated protein in adipocytes (p422) and the cellular retinoic acid-binding protein. Interaction with as yet unknown hydrophobic ligands might play a functional role in the mechanism of growth inhibition excerted by mammary-derived growth inhibitor.

Animals↗

PubMed1987

Structure and evolution of the repetitive gene encoding streptococcal protein G.

The complete sequence of the structural gene encoding the immunoglobulin G binding protein from Streptococcus G148 has been determined, as well as its 5' and 3' flanking sequences. The sequence reveals an open reading frame encoding a putative preprotein with a relative molecular mass of 63294. N-Terminal sequencing of the mature protein, spontaneously released from streptococcal cells, demonstrates that the signal peptide consists of 33 amino acids. The DNA sequence reveals extensive internal homologies similar to other cell-wall-bound receptors from gram-positive bacteria. Comparisons with a related gene previously isolated from another strain of streptococci revealed large differences in size, due to variations in the number of internal repeats. The structure of the gene suggests an evolution through multiple duplications.

Amino Acid Sequence↗

PubMed1987

Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.

Amino Acid Sequence↗

PubMed1986

Hydrophobic interaction chromatography of incompletely methylated transfer RNA from Escherichia coli on octyl-sepharose.

Phenylalanine-specific transfer RNA from methionine-starved relaxed Escherichia coli K12 separates into two components when chromatographed on Octyl-Sepharose. The difference in elution between the two tRNAs has been shown to depend on the methyl group in the highly modified 2-methylthio-N-6-isopentenyladenosine. The first eluted tRNAPhe lacks this methyl group, while the last eluted tRNAPhe is fully methylated. Other differences in the modification patterns have no effect on the elution from Octyl-Sepharose. The elution pattern of tyrosine- and serine-specific tRNAs, also normally containing ms2i6A, is similar.

Amino Acyl-tRNA Synthetases↗

PubMed1986

Staphylococcal protein A consists of five IgG-binding domains.

A genetic approach is described to clarify the IgG-binding properties of the N-terminal portion of staphylococcal protein A (region E). Several gene fragments, encoding region E or B or protein A, have been cloned and expressed in Escherichia coli. The gene products were purified by IgG-affinity chromatography and subjected to structural and functional analyses. Both fragments can be efficiently purified using this method, suggesting that region B as well as region E has Fc-binding activity. In addition, gene fusions were assembled giving fragments EB and EE, which both showed a divalent Fc-binding. These results demonstrate that protein A consists of five IgG-binding domains. The implications of these findings for the structure of protein-A--immunoglobulin-G complexes are discussed.

Amino Acids↗

PubMed1985

Analysis of signals for secretion in the staphylococcal protein A gene.

Different constructs of the gene encoding staphylococcal protein A were introduced in Staphylococcus aureus and S. xylosus as well as Escherichia coli. The product of the gene without the cell wall anchoring domain was efficiently secreted in all three hosts. N-terminal sequencing of the affinity-purified mature protein revealed a common processing site after the alanine residue at position 36. In contrast, when an internal IgG-binding fragment of protein A (region B) was inserted after the protein A signal sequence, the product was poorly secreted and N-terminal sequencing revealed no processing at the normal site. This demonstrates that the structure of the polypeptide chain beyond the signal peptide cleavage site can affect cleavage. Another construct, containing the N-terminal IgG-binding part of the mature protein A (region E) followed by region B, gave correct processing and efficient secretion. Unexpectedly, the gene product, EB, was not only secreted and correctly processed, but was also excreted to the culture medium of E. coli. Secretion vectors containing the protein A signal sequence were constructed to facilitate secretion of foreign gene products. Insertion of the E. coli gene phoA, lacking its own promoter and signal sequence, led to efficient secretion of alkaline phosphatase both in E. coli and S. aureus.

Amino Acid Sequence↗

PubMed1985

Analysis of protein A encoded by a mutated gene of Staphylococcus aureus Cowan I.

The protein A (spa) genes from Staphylococcus aureus Cowan I and a mutant strain of Cowan I called V-1 earlier suggested to produce a monovalent IgG-binding protein A have been cloned in Escherichia coli. The DNA sequences coding for the IgG-binding part of the spa genes from both strains have been determined and compared with each other and with a partial amino acid sequence of purified protein A from strain V-1. The nucleotide sequence of the spa gene from strain V-1 reveals an NH2-terminally located IgG-binding region homologous to region E first reported for strain 8325-4, region D and the major portion of region A. The amino acid sequence analysis of the purified protein A from this strain also shows the presence of regions E and D but only a minor part of region A. Reversed-phase high-performance liquid chromatography fractionation of purified protein A from strain V-1 revealed that the preparation was heterogeneous, containing mainly two peptides with different abilities to bind IgG molecules. A shuttle vector containing the cloned protein A gene from V-1 was constructed and transformed into different strains of S. aureus and the produced protein A was purified and analysed using sodium dodecyl sulfate/polyacrylamide gel electrophoresis.

Amino Acid Sequence↗

PubMed1985

Amino acid sequence of the trypsin-generated C3d fragment from human complement factor C3.

Human C3d (try-C3d), prepared from trypsin-digested C3, was fragmented by cleavage with CNBr. Eight peptides were defined and separated by h.p.l.c. on reversed-phase columns. By automatic Edman degradation the complete sequences of five peptides and partial sequences of three peptides were determined. To obtain overlapping peptides the latter three fragments were digested with trypsin, chymotrypsin or Staphylococcus aureus V8 proteinase, after which the fragments were separated on reversed-phase columns. Two of the CNBr-cleavage peptides were completely sequenced, and 70% of the sequence of the remaining CNBr-cleavage peptide was determined. The non-sequenced part represents a very hydrophobic segment of try-C3d. The sequence data obtained represent 90% of the primary structure of try-C3d. Alignment of the CNBr-cleavage fragments was made easier by comparison with the cDNA sequence of mouse pro-C3 [Wetsel, Lundwall, Davidson, Gibson, Tack & Fey (1984) J. Biol. Chem. 259, 13857-13862]. Comparison of try-C3d with the equivalent part of human C4B revealed an extensive sequence homology in the N-terminal half of the molecules.

Amino Acid Sequence↗

PubMed1985

Site specificities of three transfer RNA methyltransferases from yeast.

The site specificities of two distinct tRNA(m1G)methyltransferases and one tRNA(m2G)methyltransferase from yeast have been investigated by heterologous methylation and analysis of purified Escherichia coli tRNAs. The two tRNA(m1G)methyltransferases were found to be specific for sites 9 and 37, respectively. The tRNA(m2G)methyltransferase was specific for site 10. Two of the enzymes were purified by affinity chromatography on tRNA-Sepharose.

Base Sequence↗

PubMed1985

Characterization of tryptic fragments of human complement factor C3.

C3c and C3d fragments were prepared in pure form from trypsin-digested human C3, and the individual chains of tryptic C3c were isolated by gel filtration on Sepharose 4B in 6M guanidinium hydrochloride. No low mol. wt (Mr) fragments were identified. The polypeptide chains were characterized with regard to Mr, amino acid composition and N-terminal amino acid sequence. Tryptic C3c consisted of one fragment from the beta-chain (Mr 64,000) and two from the alpha'-chain (Mr 40,000 and 23,000). The beta-chain fragment was derived from the C-terminal part of the chain, and the 23,000-Mr component constituted the amino terminal end of the alpha-chain. The 40,000-Mr fragment emanated from the C-terminal end of the alpha-chain. Tryptic C3d displayed microheterogeneity on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, but possessed a homogeneous N-terminal, identical to that described by Tack et al. (1980) (Proc. natn. Acad. Sci. U.S.A. 77, 5764-5768). By utilization of antisera against subunits of C3 and C3c in immunoblotting a degradation scheme for C3 by trypsin was proposed and the positions of the fragments in the intact molecule indicated.

Amino Acid Sequence↗

PubMed1985

Genetic variation of haemoglobin alpha- and beta-chains in rabbits detected by isoelectric focusing and reversed-phase chromatography.

Previous studies on the amino acid sequences and on the amino acid composition of peptides revealed genetic polymorphism both of the haemoglobin alpha-chain (Hb alpha) and beta-chain (Hb beta) in rabbits. In this study, rabbit haemolysates were analysed by isoelectric focusing in a narrow pH range (6.7-7.7) and by reversed-phase chromatography. Two variants were found for both Hb alpha and Hb beta. The two methods detected the same variants in this material. Inheritance data were consistent with the hypothesis that the observed Hb alpha and Hb beta variants were each controlled by two codominant, autosomal alleles. Haemoglobin polymorphism appears to be frequent in domestic rabbits since both variants of each chain were observed in all the three breeds studied.

Animals↗

PubMed1984

Purification and characterization of pancreatic colipase from the dogfish (Squalus acanthius).

Pure colipase from dogfish (Squalus acanthius) was obtained from an extract of pancreatic gland. It has a high isoelectric point (10.2) and the molecular weight was calculated to be 9108-9383. The N-terminal sequence was shown to be Gly-Leu-Phe-Leu-Asn-Leu-Ser-Ala-Gly-Glu-Leu-Cys-Val-Gly-Ser-Phe-Gln -Cys-Lys-Ser-Ser-Cys-Cys-Gln-Arg-Glu-Thr-Gly-Leu-Ser-Leu-Ala -Arg-Cys-Ala-. This sequence shows great homology with colipases from man, horse, pig and hen. There were indications of the existence of a proform of dogfish colipase. The propeptide was found to be Ala-Pro-Glu-Arg.

Amino Acid Sequence↗

PubMed1984

Primary sequence differences between Chido and Rodgers variants of tryptic C4d of the human complement system.

Human tryptic C4d of the Chido and Rodgers variant was fragmented by cyanogen bromide and trypsin. The fragments were characterized by amino acid analysis and sequence determination. Polymorphism between the two genetic variants was detected in 5 positions. Four were closely located (residues 141, 142, 145, 146), where Leu, Ser, Ile, His occurred in the Chido variant and Pro, Cys, Leu, Asp in the Rodgers variant, respectively. In position 94 Gly was found in Chido and Asp in Rodgers. Alignment of the fragments was performed and it is concluded that tryptic C4d of both variants contains 346 residues.

Amino Acid Sequence↗

PubMed1984

Chemical characterization of cyanogen bromide fragments from the beta-chain of human complement factor C3.

The isolated beta-chain of human complement factor C3 (C3 beta) was fragmented by cyanogen bromide. Nine fragments were defined by gel filtration and high-pressure liquid chromatography, and characterized with respect to their Mr, amino acid composition and N-terminal amino acid sequence. Approx. 30% of the primary structure of C3 beta was determined. Alignment of the 3 N-terminal fragments allowed determination of 61 of the amino terminal residues of C3 beta. This region demonstrated 40% homology with the sequence in the N-terminal segment of the alpha-chain of the cobra venom factor.

Amino Acid Sequence↗

PubMed1984

The primary sequence of human pancreatic colipase.

The amino acid sequence of an activated colipase purified from human pancreas was determined. The protein consists of a single polypeptide chain of 86 amino acids (human colipase86) and has a molecular weight of 9289. The sequence was determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of two CNBr peptides. Sequence determination of porcine procolipase II was also performed, which showed that in the original sequence determination apparently two residues were missed. These residues were determined to be a leucine at position 37 and a serine in position 50. For comparison with porcine and equine procolipases, the residues composing human colipase are numbered from 6 to 91. No human procolipase has been isolated so far. The colipases from man, pig, horse and chicken show a high degree of homology: human colipase differs from the other proteins by substitutions of 19 (porcine), 24 (equine A) and 21 (equine B) residues, respectively.

Amino Acid Sequence↗

PubMed1983

Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from the Indian cobra (Naja naja).

The presence of a factor immunologically related to cobra venom factor (CVF) was demonstrated in serum and plasma from the Indian cobra (Naja naja kaoutia). The factor was purified from cobra plasma by affinity chromatography on an anti-CVF gel and was found to consist of a protein composed of two polypeptide chains similar in size to those of human C3. With use of immunoblotting technique, common antigenic determinants were found in the smaller chain of the prepared material and the beta-chain of human C3; the larger chain may display antigenic determinants present in the alpha-chain of human C3. These findings suggest that this molecule represents the C3 of the cobra complement system. Common antigenic determinants were also demonstrated in the alpha-chain of CVF and the beta-chains of human and cobra C3. No reactions were observed between the beta- and gamma-chains of CVF and any antiserum against human C3 or its subunits. Upon immunodiffusion analysis, cobra serum was found to contain a factor besides C3 sharing antigens specific for CVF, while cobra C3 was antigenically deficient compared to CVF. This suggests that cobra C3 physiologically is degraded to a molecule very similar to or identical with CVF.

Animals↗