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B Havsteen

Publications and source records attributed to B Havsteen.

At least 19 recordsLinked to original sources
PubMed1998

Elucidation of a new biological function of an old protein: unique structure of the cobra serum albumin controls its specific toxin binding activity.

Although few proteins have been studies as thoroughly as serum albumin, a new biological property of this evolutionary ancient protein was recently discovered: The ability of cobra serum albumin (CSA) to specifically sequester lethal endogenous toxins. A study of the structural basis of this property is reported in this contribution. Two independent approaches were used to alter the structure of the CSA at defined positions: Directed mutagenesis and limited proteolysis. The conserved pattern of the disulfide linkages in the primary structure of the serum albumins showed in the case of the cobra snake (Naja naja kaouthia) an anomaly at C11 and C502, which suggested the existence of a unique spatial structure in this protein. The two cysteine residues were singly replaced with the consensus residue, i.e. C11-->F and C502-->T. The former substitution increased the specific neurotoxin binding capacity of the CSA by the factor 1.7 +/- 0.2, whereas the latter replacement reduced it to (25 +/- 2)%. The limited proteolysis yielded the large tryptic peptides T60, T40, T30 and T18, which after isolation by PAGE followed by HPLC had retained a strong toxin affinity. The location of these peptides in the amino-acid sequence was identified by Edman degradation and suggested the order of their release. On the basis of these data, a model of the unfolding and of the activity changes of the CSA caused by the structural perturbations was composed and the kinetic parameters associated with the process were evaluated. The results support the hypothesis of the existence of a structure of multiple homologous domains with a disulfide linkage between C11 and C502 in the native CSA that joins the chain ends to form a dense conformation.

Animals↗
PubMed1997

Attractor control of the binding of digoxin to a specific antibody.

The characteristics of attractor control of the changes in the molecular vibrations of a protein have previously been detected when an enzyme (chymotrypsin) reacted with a specific substrate and when myoglobin interacted with oxygen. Similar studies have now been carried out on the binding of a hapten, digoxin, to an antibody. The temperature factors of the Fab-fragment of a specific anti-digoxin antibody with and without the bound antigen were used in this analysis. The integral correlation function of the difference in the temperature factor between the free and the loaded state of the antigen binding site indicated the existence of a regular attractor of the dimension 4.0+/-0.1 in the light chain and one of the dimension 5.7+/-0.7 in the heavy chain, the former under the control of 11+/-1 factors and the latter by 12+/-2 factors. This result was corroborated by Poincaré plots showing the cross-section of attractors and by a positive Liapunov exponent. The power spectrum was, as expected, broad, but the autocorrelation function showed only significant damping in the case of the L-chain. The spacing of the temperature factors resembled a "Devil's Staircase" suggesting the operation of a stochastic attractor. Its dimension, which was determined by the methods of the correlation between the step-gag lengths and that of the Farey tree was found to be near one. Repetition of the calculation using data for the second antigen-antibody complex in the unit cell yielded similar results. However, the dimensions of the attractors in the second complex (6.0+/-0.1 for the L- and 7.6+/-0.1 for the H-chain) are somewhat larger than that of the first, probably reflecting the lower degree of order of the latter. In all cases, the saturation of the integral correlation coefficient with increasing number of phase-space dimensions strongly indicates the existence of an attractor. The evidence of attractors in the molecular dynamics of proteins raises doubt about the value of trajectories calculated by integration of equations of atomic movement of the prediction of folding pathways since the stochastic element in the dynamics can eliminate leading equations in the set, thus influencing the folding pathway.

Antibodies↗
PubMed1996

[The molecular cloning and sequencing of the cobra serum antitoxic protein gene].

In the previous study the authors found at least one peptide of 39 amino acid residues of several purified tryptic-fragments of the cobra serum antitoxic protein (CSAP), whose sequence had shown certain correlation with the rat serum albumin. For further clarification of the toxin-binding activity of CSAP in relation with its corresponding structure, the cDNA gene library of cobra liver cells was established and DNA primers were designed according to the known amino acid sequences of the tryptic peptides of CSAP. The specific probes were prepared and the specific clone was screened out from the library by PCR. Finally, the CSAP cDNA was sequenced from its sub-clones. Then the complete amino acid sequence of CSAP was elucidated. It is a polypeptide chain of 614 amino acid residues with a molecular size of 69.8 KDa. In comparing the whole sequence of CSAP, especially its signal peptide, with mammalian serum albumins, the authors have come to realize that CSAP is just the cobra serum albumin (CSA).

Amino Acid Sequence↗
PubMed1995

A zinc metalloproteinase is responsible for the release of CD30 on human tumor cell lines.

The activation marker CD30 is expressed on the cell surface of the malignant cells in Hodgkin's disease and a few non-Hodgkin lymphomas. We have analyzed the regulation of membrane-bound CD30 and found that the binding of a variety of anti-CD30 antibodies induced down-regulation of CD30 on cell lines. In addition, such down-modulation was also observed after treatment of the cell surface proteins with the sulfhydryl reagent iodoacetamide or after stimulation of the second messenger pathway with phorbol ester or calcium ionophore. This modulation was abolished at 4 degrees C and strongly inhibited by chelators like EDTA or 1,10-phenanthroline, whereas EGTA, a selective inhibitor of Ca(2+)-dependent proteinases and other inhibitors of serine, thiol and acid proteinases, showed no effect. The down-modulation was strengthened by Zn2+ or Cd2+, but not by other divalent cations such as Fe2+, Mn2+, Mg2+, Ca2+ or Co2+, thus indicating the involvement of a zinc metalloproteinase in CD30 modulation which can be activated by protein kinase C and by alkylation of sulfhydryl groups. Pulse-chase experiments, analysis of the CD30 glycosylation and specific measurement of the 90-kDa soluble form of CD30 (sCD30) with a sandwich radioimmunoassay revealed that CD30 down-modulation results from enhanced release of 90-kDa sCD30 by the site-specific cleavage of CD30 accomplished by a zinc metalloproteinase. This release occurs at the cell membrane without prior endocytosis.

Antibodies, Monoclonal↗
PubMed1995

Potential linkage disequilibrium between schizophrenia and locus D22S278 on the long arm of chromosome 22.

Locus D22S278 at 22q12 has been implicated in schizophrenia by sib-pair analysis. In order to replicate these results, we performed the transmission test for linkage disequilibrium (TDT) in 113 unrelated schizophrenic patients and their 226 parents. Evidence for potential linkage disequilibrium was obtained between schizophrenia and allele 243 of the marker AFM 182xd12 at the locus D22S278 (P = 0.02). The results of our study suggest a detectable oligogenic gene in a multigene system for schizophrenia closely linked to D22S278 on the long arm of chromosome 22. If confirmed by others, this finding could lead to the identification of a schizophrenia susceptibility gene.

Adolescent↗
PubMed1995

General linear compartment model with zero input: I. Kinetic equations.

The derivation of kinetic equations is described for n-compartment linear models, in which the substance may be simultaneously introduced into one or more compartments at t = 0 and eliminated from any compartment. For a given zero-input, general formulas are derived which describe the amount of tracer in any of the compartments as a function of time and the model parameters. New algorithms have been developed which allow the expression of the kinetic equations.

Algorithms↗
PubMed1995

General linear compartment model with zero input: II. The computerized derivation of the kinetic equations.

The final equations obtained in the first article of this series describing the concentrations of substances in any of the compartments of a model are here cast in an easily programmable form. A computer program with an easy input method and the ability to expand all of the coefficients in the kinetic equations in terms of the model parameters has been developed. The program has been written in the BASIC language.

Computer Simulation↗
PubMed1995

Evidence of the coevolution of a snake toxin and its endogenous antitoxin cloning, sequence and expression of a serum albumin cDNA of the Chinese cobra.

A full-length cDNA of the serum albumin (CSA) of the cobra (Naja naja kaouthia) was cloned from a lambda gt 11 library. It encodes a mature protein of 614 amino-acid residues homologous to the precursor of mammalian serum albumins. The 1 degree and 2 degrees structures of the CSA resemble those of the human variety. The putative toxin binding sites are mainly located in the subdomains IIA and IIIA. The relation between structural homology and function of the serum albumins (SA) is discussed. An analysis of their evolutionary tree revealed that anti-toxicity arose by < 90 amino-acid exchanges. The rate of substitution is much higher in the SA than in cytochrome C, which probably reflects the difference in evolutionary driving forces. The evolutionary period of the SA (6.7 +/- 0.1 M.Y.) significantly exceeds that of hemoglobin (5.8 M.Y.). Eight tripeptides in the nicotinic acetylcholine receptor (ACR), all flanking the putative toxin binding site, are also found in the CSA where they join to form 1 octa-, 1 penta- and 4 tripeptides, thus indicating the concerted evolution of two functionally linked proteins: toxin and antitoxin (CSA).

Amino Acid Sequence↗
PubMed1993

Purification, characterization and binding interactions of the Chinese-cobra (Naja naja atra) serum antitoxic protein CSAP.

The characterization of the single-chain protein in Chinese-cobra (Naja naja atra) blood serum, which yields strong specific protection against the venom of the same snake, is reported. The protein, CSAP (cobra serum antitoxic protein), was purified to electrophoretic homogeneity. Over the pH range 5-9 it formed stable complexes with the neuro- and the cardio-toxin of the snake. The molecular size of the CSAP was estimated to be 70.3 +/- 0.3 kDa. Tryptic hydrolysis of CSAP yielded several peptides that were able to bind to the toxin. The native CSAP maximally bound 8 +/- 1 toxin molecules/molecule. Six tryptic fragments, containing 5-39 residues, were sequenced. The longest of these displayed sequence similarity to rat serum albumin. The protective effect of the CSAP was demonstrated in vivo on mice and in vitro by measurement of the rate of haemolysis. Kinetic and thermodynamic parameters of the binding interactions of the neurotoxin and the CSAP were determined from the rates of displacement of 125I-labelled toxin from its complexes with the CSAP by unlabelled toxin by using a DEAE-cellulose filter assay for CSAP-toxin complexes. The toxin molecules rapidly dissociated from one type of site and slowly from a second. The binding capacity and concentration of the CSAP suffice to explain the protective effect of the latter against the toxin.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of the human liver ethanolamine kinase.

Ethanolamine kinase was purified from human liver to apparent homogeneity and its properties were studied. The minimum molecular weight of the purified enzyme was estimated by SDS-PAGE to 42,000 +/- 2000 Da. The molecular weight of the native enzyme determined by Superose 12 (FPLC) gel filtration was 87,000 +/- 4000 Da, suggesting that ethanolamine kinase in human liver consists of two chains of the same or almost the same molecular weight. The titration of the active site normality showed 1.03 +/- 0.05 active sites/chain. The Km value for ethanolamine was 0.25 +/- 0.01 mM.

Adenosine Triphosphate↗
PubMed1990

Protein kinase activity of the intracellular but not of the membrane-associated form of the Ki-1 antigen (CD30).

The Hodgkin-associated Ki-1 antigen (CD30) consists of a 120-kDa membrane-associated glycosylated phosphoprotein (Ki-1/120) and a 57-kDa non-glycosylated phosphoprotein (Ki-1/57) which only occurs intracellularly. Both molecules are phosphorylated at serine residues. An analysis of the peptide fragments resulting from staphylococcal V8-protease digestion of the Ki-1/57 molecule revealed identical bands irrespective of the cell source. Only a few bands of the Ki-1/57 digests appeared among the peptide fragments of the Ki-1/120 membrane antigen. The protein kinase activity was tested for both forms of the Ki-1 antigen. The Ki-1/120, devoid of the Ki-1/57 molecule, was immunoprecipitated from cell lysates of Hodgkin-analogous cell lines L428 or L540, which had been loaded with the Ki-1 or the Ki-1-analogous antibodies Ber-H2, HSR-1, -2 and -3 (method 1). These other antibodies reacted with the Ki-1/120, but not with the Ki-1/57 antigen. The latter, devoid of the Ki-1/120, was isolated from L540 cells after removal of the membrane form by method 1 or from U266/Bl myeloma or Raji Burkitt lymphoma cells which only contain the smaller form. Effects of non-specific adsorption were eliminated by various control precipitates. The Ki-1/57 intracellular antigen showed autophosphorylation and could phosphorylate histones as well. In contrast, a protein kinase activity of the membrane-associated Ki-1/120 could not be demonstrated.

Amino Acids↗
PubMed1989

Oxazolones as substrates in active site titrations of hydrolases.

Active site titrations provide essential information for studies of enzymes, e.g., functional purity and half-site reactivity. The use of a hitherto overlooked class of hydrolase substrates, the oxazolones, for an easy synthesis of reagents possessing desirable properties, e.g., specificity, solubility, and optical parameters, is described. The operational normality may be determined in aqueous media or organic solvents, e.g., dimethyl sulfoxide, which stabilize the enzymes for extended periods at room temperature. Absorbance or fluorescence measurements may be used for detection. The lower limit permitting analyses in this series was found to be 60 pmol of enzyme.

Binding Sites↗
PubMed1989

The Hodgkin-associated Ki-1 antigen exists in an intracellular and a membrane-bound form.

The Hodgkin-associated Ki-1 antigen occurs in two different molecular forms. The 120-kDa membrane-associated form is a phosphorylated glycoprotein, which is derived from a non-phosphorylated intracellular 84-kDa apoprotein that is co-translationally N-glycosylated with a carbohydrate portion of 6 kDa. The other form of the Ki-1 antigen is a non-glycosylated phosphoprotein of 57 kDa which only occurs intracellularly. Both forms of the antigen are phosphorylated at serine residues. Enzymatic cleavage with sialidase reduced the 120-kDa membrane antigen by about 15 kDa, while its 90-kDa precursor and the 57-kDa intracellular form of the Ki-1 antigen remained unaltered. Pulse-chase experiments revealed that the 57-kDa and 90/120-kDa molecules are synthesized independently of each other. Four to eight hours after synthesis, the degradation of the 120-kDa molecule to a 105-kDa membrane-associated intermediate begins. This is further processed and appears in the cell supernate as a 90-kDa molecule. Hodgkin's disease-derived, Epstein-Barr virus-transformed cell lines and the acute T cell leukemia line MOLT-4 contain both forms of the Ki-1 antigen, whereas only the 57-kDa intracellular antigen is expressed in U266/B1 myeloma cells, in the Burkitt lymphoma cell lines Raji and Daudi and in acute promyelocytic HL-60 leukemia cells.

Amino Acids↗
PubMed1987

Biochemical characterization and biosynthesis of the Ki-1 antigen in Hodgkin-derived and virus-transformed human B and T lymphoid cell lines.

The Hodgkin-associated Ki-1 antigen was analyzed in different cell lines. In Hodgkin analogous L428 cells, biosynthetically labeled with radioactive amino acids, the Ki-1 antibody precipitated three glycoproteins with 90, 105, and 120 kDa, respectively. Surface-labeling revealed that the two larger components were membrane-associated forms of the Ki-1 antigen, although the 90-kDa molecule was shown in pulse-chase experiments to be the precursor of the 105- and 120-kDa forms. All three forms of the Ki-1 antigen possess a tunicamycin-sensitive 6-kDa N-linked carbohydrate moiety. O-Linked oligosaccharides could not be detected. Thus, the differences in m.w. are probably not due to glycosylation. The ionophore monensin prevented the appearance of the membrane-associated molecules, which demonstrated that they are assembled between the transcompartment of the Golgi complex and their insertion into the cell membrane. The 90-kDa precursor molecule cannot be generated by disulfide reduction from the two larger forms. After internal labeling with P-32, only the 105- and 120-kDa bands became visible, indicating that the Ki-1 molecule is phosphorylated after its processing into the two larger membrane-associated forms. Analysis of the Ki-1 antigens from other cell lines demonstrated that after external labeling of two other Hodgkin-derived cell lines, six Epstein-Barr virus lymphoblastoid cell lines and one human T leukemia virus I-positive T cell line, both the 105- and the 120-kDa membrane molecules could be detected, regardless of the presence or type of virus integrated.

Antigens, Neoplasm↗
PubMed1986

High-resolution 2-dimensional protein mapping of "diploid" and "aneuploid" mammary adenocarcinomas.

Two-dimensional-electrophoretic analysis has been applied to non-neoplastic mammary epithelium from eight healthy women, tumor tissue from eight "diploid" mammary adenocarcinomas, and tumor tissue from eight "aneuploid" mammary adenocarcinomas. Compared with non-neoplastic mammary epithelium, a slight numerical net non-neoplastic mammary epithelium, a slight numerical net increase of the protein spots was detected in "diploid" tumors and a marked increase in "aneuploid" tumors. Two prominent spots were present in all 16 malignant tissues examined and absent in all eight non-neoplastic tissues (silver staining method). The results suggest that a difference in the composition of cellular proteins exists both between non-neoplastic mammary cells and malignant tumor cells, and between "diploid" and "aneuploid" tumors.

Adenocarcinoma↗
PubMed1986

Characterization of the porcine ACTH receptor with the aid of a monoclonal antibody.

Monoclonal antibodies were raised against the ACTH receptor by immunization of BALB1 c mice with porcine adrenal cortex cell membranes. Competition and binding experiments confirmed that one of these antibodies, IV/14/9, reacted with the ACTH receptor in competition with ACTH and caused a definite ACTH-like effect. This antibody was used to characterize the hormone receptor of the porcine adrenal cortex. The number of antibody binding sites per cell was calculated from a Scatchard plot to be 124,100 +/- 10,000. The curve was linear indicating the existence of a single class of receptors. The finding that a high concentration of IV/14/9 totally suppressed maximally ACTH-induced steroidogenesis confirms the view that only a single class of ACTH receptors exists. The antibody IV/14/9 neither reacted with intact porcine thymus cells nor with normal porcine lymphocytes but was bound to the mouse adrenal tumor cell line Y1 and to normal rat adrenal cortex cells with a low affinity. Two dimensional electrophoresis of lysates of porcine adrenal cortex cells and subsequent blotting of the proteins on nitrocellulose, using IV/14/9 as a primary and anti-mouse Ig as a secondary reagent, permitted the detection of three forms of the receptor, two of which had an identical apparent molecular mass of about 85,000 Da (isoelectric points: 6.14 and 6.27). The third was somewhat larger (94,000 Da and pI = 6.21).

Adrenal Cortex↗
PubMed1985

Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

Animals↗