Biomedical subjects
Publications and source records attributed to A Henschen.
Variable subgroups of both chains of a human monoclonal anti-RH(D) IgG1 (kappa), QA37C3G6, were determined from their N-terminal sequences. Sequence comparison with corresponding chains of other human antibodies indicated that the light chain belongs to the third subgroup of human kappa-light chains while the heavy chain belongs to the second subgroup of human heavy chains.
Antibody H2D5D2F5 is a human monoclonal anti-Rh(D) IgG1 (lambda) produced by Epstein-Barr virus-immortalized B lymphocytes from a healthy donor. The complete amino acid sequence of the light (L) chain, with the exception of positions 94-97, was determined by Edman degradation of the intact chain, containing 30 residues, and derived tryptic and thermolytic peptides. Sequences of the peptides were aligned by comparison with the sequences of previously reported L chains. H2D5D2F5 L chain belongs to the first variable subgroup of human chains. Its sequence does not reveal striking differences when compared to those of other human lambda chains issued from myeloma or hybridoma.
Acrosin is a disulfide-bonded two-chain glycoprotein, which belongs to the serine proteinase family and which plays a central role in mammalian fertilization. The amino acid sequence of acrosin from different species has been recently derived by cDNA analysis. Boar sperm acrosin contains twelve cysteine residues forming two interchain and 4 intrachain disulfide bonds. Protein-chemical and mass-spectroscopic analyses of fragments and subfragments obtained by proteolytic and chemical degradation of the isolated protein allowed the unambiguous localization of all disulfide bridges and glycosylation points in boar acrosin. The 12 cysteines and the glycosylated asparagines in the porcine enzyme are absolutely conserved in number and position within all known acrosin sequences. Thus, the disulfide bond and glycosylation patterns outlined here are conserved during evolution and may be important for enzyme function.
Human platelet glycoprotein IIb (GPIIb) and IIIa (GPIIIa) form a Ca2(+)-dependent heterodimer, the integrin GPIIb/IIIa, which functions as the fibrinogen receptor at the surface of activated platelets. GPIIB and GPIIIa are synthesized as single polypeptides from single messages and their amino acid sequences were derived from their cDNAs. The GPIIb precursor is proteolytically processed to yield the known disulphide-bonded two-chain (GPIIb alpha and GPIIb beta) covalent structure found in mature GPIIb. Our present protein chemical and mass spectrometric analyses indicate that the GPIIb precursor is proteolytically cleaved at two or three sites, to give rise to an homogeneous alpha-chain (GPIIb 1-856) single disulphide-bonded to one of the two beta-chains, which are present in a nearly 1:1 ratio: GPIIb beta 1 (860-1008), with pyroglutamic acid as its blocked N-terminal residue: and GPIIb beta 2 (872-1008), with the already known N-terminal sequence. These results satisfy the previously observed electrophoretic size-residue: and GPIIb beta 2 (872-1008), with the already known N-terminal sequence. These results satisfy the previously observed electrophoretic size-heterogeneity of the beta-chain, confirmed the potential cleavage sites in the junction region, and indicate a probable dual proteolytic processing of GPIIb, which may be relevant to the rest of the two-chain alpha-subunits of the integrin family.
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The primary structure of the beta-subunit (acyl lyase subunit) of citrate lyase from Klebsiella pneumoniae (ATCC 13,882) was determined with protein chemical methods. The polypeptide chain consists of 289 amino acid residues and has a molecular mass of 31,352 Da. The two half-cystine residues of the subunit are present as cysteines and not involved in disulfide bridges. The sequence shows no homology to known sequences of proteins or nucleic acids and reads (sequence; see text)
The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2-mercaptoethanol, the peptide chain components were separated by reverse-phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.
Glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the Ca2(+)-dependent GPIIb/IIIa complex, which acts as the fibrinogen receptor on activated platelets. GPIIb and GPIIIa are synthesized as single peptide chains. The GPIIb precursor is processed proteolytically to yield two disulphide-bonded chains, GPIIb alpha and GPIIb beta. The GPIIb/IIIa complex has two membrane attachment sites located at the C-termini of GPIIb beta and GPIIIa. The short cytoplasmic tails of GPIIb beta and/or GPIIIa become most likely associated to the cytoskeleton of activated platelets. In the present work the C-terminal amino acid residues of platelet GPIIb beta and GPIIIa have been analyzed by protein-chemical methods and compared with those predicted from cDNA analysis. We were able to confirm the positions of the C-termini in both glycoproteins and the identity of the C-terminus predicted for GPIIIa, i.e. threonine. However, glutamine, not glutamic acid as predicted for GPIIb beta from the human erythroleukemic cell line and megakaryocyte cells, was found to be the C-terminal amino acid of GPIIb beta. This indicates that the glutamic acid in the GPIIb precursor is posttranslationally modified to glutamine.
Tetanus toxin is a 151-kDa protein. The complete amino acid sequence is known. The mature toxin is made up of two peptide chains and contains 10 half-cystine residues. Treatment with 4-vinylpyridine in the presence of 6 M guanidine converted six of them into S-pyridylethyl cysteine residues as determined by amino acid analysis. When alkylation was preceded by mercaptolysis, all 10 half-cystine residues were recovered in the S-pyridylethylated form. It was therefore concluded that the toxin contains six sulfhydryl groups and two disulfide bonds. The positions of the residues carrying sulfhydryl groups and of those involved in disulfide bridges were determined by labelling of the toxin alternatively with 4-vinylpyridine or with 4-dimethylaminoazobenzene-4'-iodoacetamide (DABIA), directly or after mercaptolysis. The toxin derivatives were cleaved with cyanogen bromide and the elution patterns in reversed-phase HPLC compared. The chromatography components were identified by N-terminal amino acid sequence and amino acid composition. In the chromatography of the non-mercaptolysed, DABIA-treated sample four chromophore-carrying components were detected which could be demonstrated by N-terminal sequence analysis to correspond to six half-cystine-containing cyanogen bromide fragments. In the mercaptolysed, DABIA-treated sample three additional chromophore-carrying components were present, corresponding to two previously disulfide-linked cyanogen bromide fragments and one fragment which had contained an internal disulfide bridge. The HPLC patterns showed characteristic differences as the DABIA-labelled fragments were considerably more hydrophobic than the corresponding vinylpyridine-labelled fragments. It was established that the half-cystine residues in positions 26, 185, 198, 311, 868, and 1300 are present in the sulfhydryl form, that those in positions 438 and 466 are disulfide-bridged, thereby connecting the light and heavy chains of the toxin, and that those in positions 1076 and 1092 are disulfide-bridged, thereby giving rise to a loop in the heavy chain. During the progress of the investigations about 20% of the amino acid sequence previously predicted from DNA analysis was confirmed by protein-chemical methods.
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We have developed a model system in which the mechanisms of neuronal damage due to hyperexcitation can be studied in isolation and where extended observation periods can be used. Substantia nigra pars reticulata (SNPR) develops a hypermetabolic necrosis following status epilepticus (Nevander et al. 1985; Auer et al. 1986). We transplanted rat fetal nigral area alone or together with fetal frontal neocortex to the anterior chamber of the eye in adult rats. Following 3 months of transplant maturation the hosts were subjected to status epilepticus for 60 min. In single nigral transplants no sign of structural damage was found. In the double transplants of frontal cortex and the substantia nigra a tissue necrosis had developed in the nigral part. This was demonstrated by a total loss of glial fibrillary acidic protein (GFA) immunoreactivity within a circumscribed necrotic region in the nigral part of the double transplant. Such a loss of GFA immunofluorescence had also developed in the host SNPR, as we have earlier shown (Eriksdotter-Nilsson et al. 1987). Thus, intraocular brain tissue transplants provide a unique model for studies on the development of neuronal damage and functional dependence between different neuronal structures for the development of such damage.
The primary structure of a cytotoxin from Pseudomonas aeruginosa was determined by sequencing of the structural gene. The cytotoxin (31,700 Mr) lacks an N-terminal signal sequence for bacterial secretion but contains a pentapeptide consensus sequence commonly found in prokaryotic proteins which function in a TonB-dependent manner. The cytotoxin gene has a [G + C]-content of 53.8% which is considerably lower than generally observed for genes from Pseudomonas aeruginosa. The cytotoxin gene was exclusively detected in strain 158 but not in three other clinical isolates, as determined by Southern and Northern hybridization. The latter technique revealed that the toxin is translated from monocistronic mRNA. The promoter of the cytotoxin is inactive in Escherichia coli. Upon site-directed modification of the 5'-noncoding region by the polymerase chain reaction the gene was expressed under control of the trc-promoter. The gene product obtained in Escherichia coli was nontoxic. Toxicity was induced by subsequent treatment with trypsin. [35S]methionine-labeled cytotoxin with high specific radioactivity was obtained by in vitro transcription/translation. Like [125I] labeled material from Pseudomonas aeruginosa this polypeptide bound to membrane preparations from Ehrlich ascites cells, as evidenced by sedimentation through a sucrose gradient at neutral pH.
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Three variants of a Crotalus atrox venom enzyme that cleaves primarily the Arg42 - Ala bond of the B beta-chain of human fibrinogen were separated by high-performance liquid chromatography and were shown by N-terminal sequence analysis to be amino acid isoforms. All are single-chain glycoproteins whose approximate molecular weights lie between 26,000 and 28,000. The isoenzymes also cleaved the A alpha-chain at Arg491 - His, though much more weakly, with strengths for both bonds that varied among the isoforms.
Acrosin is a multifunctional enzyme combining several functional properties within a single molecule: the catalytic triad of the proteinase, hydrophobic domains responsible for the special membrane-associating character of the enzyme and the carbohydrate binding sites by which the molecule can bind to the zona pellucida. Acrosin occurs in the sperm acrosome as an inactive precursor, proacrosin, with a molecular mass of 53-55 kDa. Proacrosin is activated by a single proteolytic clip between Arg23 and Val24 generating the high molecular mass acrosin. The activation of proacrosin to the biologically active enzyme which occurs concomitantly with the acrosome reaction appears to be regulated on and by the zona pellucida. It is hypothesized that alternating cycles of binding to the zona, digestion of the zona and release from the zona together with the forward motility of the spermatozoon would be required to achieve penetration.
The flavin-containing monooxygenase has been purified from rabbit liver and lung microsomes. SDS-PAGE analysis shows that both enzyme forms migrate as a single band with an apparent Mr of 59000. The NH2-terminus of both forms is blocked. The liver oxidase contains a lower percentage of glutamine/glutamate and a greater amount of phenylalanine than does the lung flavoprotein. Polyclonal antibodies to a 14-amino-acid peptide obtained after CNBr cleavage of the liver oxidase cross-react with the microsomal and purified liver enzyme, but do not recognize the lung oxidase. HPLC profiles of tryptic digests of the liver and lung enzymes exhibit different patterns. Sequence alignment of selected peptides from the liver and lung oxidases reveals aberrant residues within homologous segments. These findings are interpreted to mean that both enzymes represent distinct gene products.
1. Single-chain toxin is enzymatically converted into two-chain isotoxins which differ from the precursor by their higher pharmacological activity, acidity and hydrophilicity. The interchain disulfide bridge and the disulfide loop within fragment C have been located at the amino acid level. 2. Independent of the enzymes used, the nicking sites are positioned within a region spanning no more than 17 amino acids. The N- and C-termini of the primary gene product are preserved in the two-chain toxin. The chains have been separated by isoelectric focussing and can be reconstituted to functionally intact toxin. 3. Light chain inhibits neurotransmitter release on different systems. First, permeabilized bovine adrenal chromaffin cells and rat pheochromocytoma (PC 12) cells release catecholamines when exposed to micromolar [Ca2+]. Inhibition is achieved with light chain or reduced two-chain toxin, but not with single-chain toxin or heavy chain. Washing away the light chain does not restitute the Ca2(+)-evoked release. The light chains of tetanus and botulinum A toxin act in a apparently similar, however not identical manner. Second, light but not heavy chain inhibits the release of acetylcholine when injected into Aplysia neurones. 4. The pharmacology of heavy chain is quite different. Ganglioside binding is mediated by its fragment C moiety, and modulated by the adjoining beta 2 piece and by light chain. Heavy chain and to a lesser degree its N-terminal beta 2-fragment promote the loss of calcein from liposomes indicating pore formation. Its C-terminal fragment C is inactive in this respect.(ABSTRACT TRUNCATED AT 250 WORDS)
Proteolytic enzymes released from granulocytes upon stimulation with the chemotactic N-formyl peptide FNLPNTL (in the presence of cytochalasin B) prevented activation of tumor cell single-chain urokinase-type plasminogen activator (pro-uPA) by plasmin. Elastase was identified by the use of eglin C (elastase inhibitor) and a monoclonal antibody to elastase as the functional proteolytic enzyme in granulocyte supernatants. Action of purified granulocyte elastase on pro-uPA generated enzymatically inactive two-chain uPA linked by disulfide bridges which was indistinguishable by SDS-PAGE from plasmin-generated HMW-uPA. The major elastase cleavage site in pro-uPA was located between Ile159 and Ile160. a minor one between Thr165 and Thr166. Elastase cannot substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. However, when pro-uPA was first activated by plasmin to form enzymatically active HMW-uPA, this enzymatic activity was not impaired by subsequent elastase treatment.