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G Houen

Publications and source records attributed to G Houen.

At least 19 recordsLinked to original sources
PubMed1998

Affinity chromatography of thiol ester-containing proteins.

A method is described for the affinity chromatographic purification of thiol ester proteins. These comprise the complement proteins C3 and C4 and the protease inhibitor alpha 2-macroglobulin (alpha 2M) and are known to contain an internal beta-cysteinyl-gamma-glutamyl thiol ester. The method employs aminoalkyl ligands coupled to a divinylsulfonyl-derivatized agarose matrix, and the length of the aminoalkyl spacer arm was found to be important for the effectiveness of the matrix. Optimal results were obtained with diaminododecyldivinylsulfonyl-agarose. Employing this matrix the thiol ester proteins C3, C4 and alpha 2M were isolated from human pregnancy serum. Application of the method to chicken and rainbow trout serum gave rise to isolation of several proteins including chicken and rainbow trout alpha 2M.

Animals↗
PubMed1998

Structure and tissue-specific expression of genes encoding bovine copper amine oxidases.

A cDNA coding for an enzyme belonging to the family of copper amine oxidases was cloned from a bovine lung cDNA library using a PCR approach. The nucleotide sequence of this cDNA was found to be different from that of the previously published liver cDNA encoding bovine serum amine oxidase, another copper amine oxidase. Analyses using reverse transcription followed by PCR of RNA extracted from different bovine tissues confirmed that the copper amine oxidase gene expressed in bovine liver is closely related to, but different from, the copper amine oxidase gene expressed in bovine lung, kidney, spleen and heart. Northern blotting data showed that the level of copper amine oxidase expression in liver is considerably higher than in the other tissues tested. Southern blotting analyses of bovine chromosomal DNA suggested the existence of at least three copper amine oxidase genes. Two of these genes are apparently expressed in a tissue-specific manner as outlined above. A fragment of a third copper amine oxidase gene is identified. The exon-intron organization of the bovine copper amine oxidase genes analyzed is similar to that of the related human diamine oxidase gene, except that no intron in the position equivalent to that of the third intron in the human gene is found. In the third gene, a complete replacement of the third intron of the bovine copper amine oxidase gene (equivalent to the fourth intron of the human gene) has occurred.

Amine Oxidase (Copper-Containing)↗
PubMed1998

Polyamine-stimulated binding of diamine oxidase to DNA.

Diamine oxidase is a Cu-containing enzyme which intracellularly participates in the regulation of the levels of putrescine, spermidine and spermine and in this process produces growth inhibitory amino aldehydes and hydrogen peroxide. Extracellularly, the enzyme participates in the inactivation of biogenic amines, notably histamine. Here we present evidence that in the presence of polyamines, diamine oxidase has the ability to bind DNA and to oxidise DNA-bound polyamines. The enzyme associates with chromosomal DNA since it can be released from human placental DNA by treatment with DNase I and it may be involved in the degradation of DNA. Thus, diamine oxidase may belong to a new class of DNA-binding proteins.

Amine Oxidase (Copper-Containing)↗
PubMed1998

Chaperone properties of calreticulin.

Calreticulin is a highly conserved protein with a relative molecular weight of 46,000, and is mainly located in the endoplasmic reticulum. Calreticulin was first characterized as a calcium-binding protein in the endoplasmic reticulum, but since then other functions of calreticulin have been characterized, including chaperone and lectin properties, and regulation of integrin and nuclear hormone receptor activity. We have investigated the interactions between purified human placental calreticulin and native and denatured proteins. Our results show that calreticulin binds to denatured proteins in a time- and pH-dependent manner, which at physiological pH is dependent on divalent cations. The binding was dependent on the state of the denatured protein, and was highly sensitive to the ionic composition of the environment, being strongly inhibited by phosphate-containing compounds. Calreticulin did not seem to distinguish between denatured glycosylated and non-glycosylated proteins, and was found to bind to native basic proteins, presumably by sheer electrostatic forces.

Animals↗
PubMed1997

Interference of sugars with the binding of biotin to streptavidin and avidin.

Streptavidin and avidin have found widespread use as detection reagents in immunology, biochemistry and cell biology due to their high affinity binding to biotin, but the cellular functions of these proteins are not known. We have found that various sugars interfere with the binding of streptavidin and avidin to biotin. Mannose was most effective in inhibiting the binding to biotin followed by other saccharides. The inhibitory effect is most probably due to interactions of the sugars with residues in the binding pocket of streptavidin and avidin for biotin. These results show that great caution has to be exercised in the evaluation of experiments conducted with these detection reagents in the presence of sugars.

Avidin↗
PubMed1997

Conjugation to preadsorbed preactivated proteins and efficient generation of anti peptide antibodies.

A solid phase conjugation method is described based on the preadsorption of proteins to aluminium hydroxide adjuvant followed by activation of the adsorbed carrier proteins with iodoacetic acid N-hydroxysuccinimidester or other conjugation reagents. Cysteine-containing peptides were coupled to the iodoacetic acid-activated carrier-adjuvant particles through their SH groups. No dialysis is required since the reaction product is isolated at each step of the procedure by a simple centrifugation and can easily be extensively washed between individual manipulations. The method generates peptide-carrier-adjuvant particles with sterically defined presentation of the peptides at the surface of the particles. When used for immunization of mice and rabbits the conjugates elicited high-titered specific anti-peptide sera, which reacted well with the parent protein in ELISA. The strongest reactions were with the denatured form of the parent protein. On immunoblots antisera to the N- and C-terminus of calreticulin recognized the same M, 52,000 protein.

Adsorption↗
PubMed1997

A non-denaturing enzyme linked immunosorbent assay with protein preadsorbed onto aluminum hydroxide.

A method has been developed which prevents denaturation of proteins used for coating of plastic surfaces in enzyme linked immunosorbent assays (ELISA). The system takes advantage of the use of aluminum hydroxide (Al(OH)3) as an adsorbent for proteins. A model protein has been analyzed. and monoclonal antibodies specific for either the native form or the denatured form of the protein were used to monitor the extent of denaturation. Adsorption of the proteins to Al(OH)3 in carbonate buffer, pH 9.3, before coating the ELISA plate abolished the denaturation otherwise observed after direct adsorption of protein to plastic surfaces. The protection against denaturation was dependent on the buffer system and was not observed when phosphate buffers were used, due to elution of protein from Al(OH)3 or lack of binding to Al(OH)3 in the presence of phosphate. There is evidence that protein adsorbed onto the Al(OH)3 is required for binding of Al(OH)3 onto the plastic surface. This system may be useful in assay systems where discrimination between the native and denatured forms of proteins is important.

Adsorption↗
PubMed1997

Combined immunostaining and Coomassie Brilliant Blue staining of polyvinylidene difluoride membranes without organic solvent.

A method for staining proteins on polyvinylidene difluoride membranes without using organic solvent is described. The method uses preblocking of the membrane with either Tween 20 or polyethylene glycol followed by staining with 0.01% Coomassie Brilliant Blue. No destaining of the membrane is needed afterwards. Preblocking with polyethylene glycol is compatible with microsequencing while Tween 20 leads to very low initial yields. Preblocking with Tween 20 has the additional advantage of allowing immunostaining followed by Coomassie Brilliant Blue staining for total protein on the same membrane.

Electrophoresis, Polyacrylamide Gel↗
PubMed1996

Lactoferrin: similarity to diamine oxidase and purification by aminohexyl affinity chromatography.

A protein reacting with a monoclonal antibody against human placental diamine oxidase was purified from the specific granules of human neutrofil granulocytes using affinity chromatography on aminohexyl-divinylsulfonyl-agarose. The protein had an M(r) determined by SDS/PAGE, corresponding to diamine oxidase, but had other properties which indicated that it might be a different protein. A combination of protein chemical techniques, including N-terminal sequencing, identified the protein as lactoferrin, an iron-containing protein with an M(r) of approximately 800000, a high isoelectric point and ferroxidase activity. Purified commercial lactoferrin was shown to bind to aminohexyl-divinylsulfonyl-agarose, and to be eluted in a heterogenous way from the matrix by amines and salt. Alignment of the sequences of diamine oxidase and lactoferrin showed that they are similar, indicating a common ancestry for these two different classes of metallo-oxidases.

Amine Oxidase (Copper-Containing)↗
PubMed1996

Structural requirements for alpha-mating factor activity.

The sexual hormone of S. cerevisiae, alpha-mating factor (alpha-MF, WHWLQLKPGQPMY) has structural homology with mammalian luteinizing hormone releasing hormone (LHRH, pEHWSYGLRPG-NH2) and has been shown to exhibit LHRH activity [Loumaye et al. (1982) Science 218, 1323-1325]. We have tested whether LHRH has alpha-MF activity in yeast and found that it does not. We therefore synthesized a series of hybrid peptides of alpha-MF and LHRH to study the structural features which determine alpha-MF and LHRH activities. A hybrid peptide consisting of the LHRH sequence with the C-terminal tetrapeptide (QPMY) of alpha-MF did not exhibit alpha-MF activity. Thus, the lack of alpha-MF activity of LHRH is not due solely to the absence of the C-terminal residues. Substitution of Lys7 in alpha-MF with Arg, as is found in LHRH, did not affect the alpha-MF activity, nor did an additional substitution of Trp1 with pGlu. However, the C-terminal four amino acids of alpha-MF were necessary for alpha-MF activity. Our results indicate that insertion of a Ser residue in position 4 as found in LHRH abolishes alpha-MF activity. These results suggest that, in addition to an intact C-terminus, correct spacing of the N-terminal His2 and the C-terminus is required for alpha-MF activity. The hybrid peptides all exhibited less LHRH activity than either LHRH or alpha-MF. These structure-function studies indicate that the structural homology between these two reproductive hormones may not reflect an evolutionary relationship between them.

Amino Acid Sequence↗
PubMed1996

In situ detection of diamine oxidase activity using enhanced chemiluminescence.

In need of a simple and sensitive method for detection of diamine oxidase (EC 1.4.3.6) activity in connection with diamine oxidase purification from human placenta, we have developed an enhanced chemiluminescence method using putrescine as substrate and horseradish peroxidase and luminol for the detection of the H2O2 produced by diamine oxidase. The method allows direct detection of small amounts of diamine oxidase in serum samples after agarose gel electrophoresis and allows visualization of diamine oxidase activity in tissue sections. Employing this method we have detected diamine oxidase in sera from cow, horse, monkey, rabbit, and pregnant women. On tissue sections from term human placenta diamine oxidase activity was exclusively localized to the maternal side and was concentrated in vessels and fibrinoid areas.

Amine Oxidase (Copper-Containing)↗
PubMed1996

The primary structure and enzymic properties of porcine prochymosin and chymosin.

Preliminary investigations by N-terminal sequence analysis showed that pig and calf chymosin possessed 80% amino acid sequence identity but showed considerable differences in their enzymatic properties. A comparison of their structures may therefore contribute to an understanding of the significance of the amino acid residues responsible for the differences in these properties. Pig chymosis was extracted from the stomachs of pigs of less than 3 weeks of age, and was purified by ion exchange chromatography. Half of the primary structure was determined by amino acid sequencing and the complete structure was deduced from a cloned chymosin cDNA. Results showed that the zymogen showed 81% sequence identity with calf prochymosin and 57% identity with pig pepsinogen A. The size of the propart and location of the residue which becomes the N-terminus in the active molecule were the same in the prochymosins. The maximum general proteolytic activity at pH 3.5 of pig chymosin was 2-3% of that of the activity of pig pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of pig chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.3 of stomach extracts of individual pigs showed the existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or N-terminal sequencing, and no differences in the enzymatic properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and pig chymosin differ in the following positions (pig pepsin numbering, subsites in parentheses): Ser 12 Thr (S4), Leu 30 Val (S1/S3), His 74 Gln (S'2), Val 111 Ile (S1/S3), Lys 220 Met (S4). With regard to the low general proteolytic activity of pig chymosin, the substitution Asp 303 Val relative to calf chymosin may contribute to an explanation of this.

Amino Acid Sequence↗
PubMed1996

A comparison of the immunogenicity of the native and denatured forms of a protein.

The effect of heat denaturation on the physicochemical and immunological properties of a model protein, ovalbumin, and its formaldehyde/lysine-treated form was investigated. Polyacrylamide gel electrophoresis and gel filtration showed that heat denaturation converted ovalbumin to high Mr polymers, whereas formaldehyde/lysine-treated ovalbumin remained monomeric with only a small proportion forming oligomers. NMR analysis demonstrated that non-denatured structures could easily be differentiated from the denatured structures. Intraperitoneal immunization of rabbits and mice showed that both native and denatured forms of ovalbumin induced an immune response, but denatured forms of ovalbumin were found to be less immunogenic and to have a lower epitope density than native ovalbumin. Analysis of the antisera in crossed immunoelectrophoresis showed that they were specific for either native or denatured forms of ovalbumin. These findings were further investigated by ELISA and immunoaffinity chromatography, and the high specificity and low cross-reactivity was confirmed. We conclude that the immunogenic epitopes on denatured ovalbumin are different from those on ovalbumin, and that these epitopes reflect a continuum of denatured conformations.

Animals↗
PubMed1996

Cation-dependent interactions of calreticulin with denatured and native proteins.

Calreticulin has sequence homology with the molecular chaperone calnexin, which is known to control folding and assembly of nascent proteins in the endoplasmic reticulum in a calcium-dependent manner. We have investigated the interaction between human placental calreticulin and denatured placental and serum proteins under various incubation conditions. The interactions with denatured proteins differed significantly from the interactions with native proteins. The interactions were highly dependent on divalent metal ions or polyamines, but were not influenced by detergent and sulfhydryl agents. Our results indicate that calreticulin might have a similar role in protein folding as the chaperone calnexin.

Biotin↗
PubMed1995

Conjugation to preactivated proteins using divinylsulfone and iodoacetic acid.

Two methods for the preactivation of proteins and conjugation of peptides to proteins under mild conditions are presented. Preactivation of proteins with divinylsulfone (DVS) permits peptide conjugation through either amino, hydroxyl or sulphydryl groups depending on the coupling pH used, while preactivation with iodoacetic acid (IAA) N-hydroxy-succinimide ester permits selective conjugation through sulphydryl groups. In addition, the latter method allows quantitation of the conjugation ratio through determination of carboxymethyl cysteine after acid hydrolysis. The divinylsulfone activated proteins can be stored for extended periods of time at -20 degrees C until required for conjugation, while the iodoacetic acid activated protein can be stored for a few days at -20 degrees C. These conjugation methods were investigated with respect to obtaining peptide/protein conjugates for immunization purposes and for use as reagents in immunoassays. The DVS activated proteins permitted direct conjugation of luteinizing releasing hormone (LHRH) through its tyrosine side chain and allowed synthesis of well defined conjugates. The DVS derivatives of bovine serum albumin (BSA), reduced and carboxymethylated BSA and purified protein derivative (PPD) were compared with respect to their potential value as carriers for obtaining antibodies to LHRH (M(r) 1000) and epidermal growth factor (EGF, M(r) 5000). IAA-PPD was evaluated as a carrier for the conjugation of glutathione specifically through its cysteine side chain and for obtaining antibodies to glutathione. The antisera obtained were specific and of high titer, and the methods described here will thus allow the convenient synthesis of carrier conjugates with well defined characteristics.

Amines↗
PubMed1995

Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis.

A novel immunogenic antigen, the 6-kDa early secretory antigenic target (ESAT-6), from short-term culture filtrates of Mycobacterium tuberculosis was purified by hydrophobic interaction chromatography and anion-exchange chromatography by use of fast protein liquid chromatography. The antigen focused at two different pIs of 4.0 and 4.5 during isoelectric focusing, and each of these components separated into three spots ranging from 4 to 6 kDa during two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent differences in molecular masses or pIs of these isoforms were not due to posttranslational glycosylation. The molecular weight of the purified native protein was determined by applying gel filtration and nondenaturing polyacrylamide gel electrophoresis and found to be 24 kDa. ESAT-6 is recognized by the murine monoclonal antibody HYB 76-8, which was used to screen a recombinant lambda gt11 M. tuberculosis DNA library. A phage expressing a gene product recognized by HYB 76-8 was isolated, and a 1.7-kbp fragment of the mycobacterial DNA insert was sequenced. The structural gene of ESAT-6 was identified as the sequence encoding a polypeptide of 95 amino acids. The N terminus of the deduced sequence could be aligned with the 10 amino-terminal amino acids derived from sequence analyses of the native protein. N-terminal sequence analysis showed that the purified antigen was essentially free from contaminants, and the amino acid analysis of the antigen was in good agreement with the DNA sequence-deduced amino acid composition. Thus, the heterogeneities observed in the pI and molecular weight of the purified antigen do not derive from contaminating proteins but are most likely due to heterogeneity of the antigen itself. Native and recombinant ESAT-6 are immunologically active in that both elicited a high release of gamma interferon from T cells isolated from memory-immune mice challenged with M. tuberculosis. Analyses of subcellular fractions of M. tuberculosis showed the presence of ESAT-6 in cytosol- and cell wall-containing fractions. Interspecies analyses showed the presence of ESAT-6 in filtrates from M. tuberculosis complex species. Among filtrates from mycobacteria not belonging to the M. tuberculosis complex, reactivity was observed in Mycobacterium kansasii, Mycobacterium szulgai, and Mycobacterium marinum.

Amino Acid Sequence↗
PubMed1994

HPLC and NMR investigation of the serum amine oxidase catalyzed oxidation of polyamines.

In the presence of amine oxidases polyamines arrest cell proliferation owing to the generation of hydrogen peroxide and amino aldehydes. In this investigation the bovine serum amine oxidase catalyzed oxidation of polyamines has been studied by HPLC analysis of dansylated reaction products with or without NaBH4 reduction and by NMR spectroscopy of the reaction products and 3-aminopropanal was found to be a major reaction product. These findings were further substantiated by analysis of the reaction products by ion-exchange chromatography and by analysis of the products formed by oxidation of polyamines by the cofactor of Cu amine oxidases, 6-hydroxydopa. 3-Aminopropanal is unstable and can give rise to acrolein by beta-elimination.

Acrolein↗