Biomedical subjects

M J Hseu

Publications and source records attributed to M J Hseu.

6 recordsLinked to original sources

PubMed1999

Crocalbin: a new calcium-binding protein that is also a binding protein for crotoxin, a neurotoxic phospholipase A2.

Utilizing Marathon-ready cDNA library and a gene-specific primer corresponding to a partial amino acid sequence determined previously, the complete nucleotide sequence for the cDNA of crocalbin, which binds crotoxin (a phospholipase A2) and Ca2+, was obtained by polymerase chain reaction. The open reading frame of the cDNA encodes a novel polypeptide of 315 amino acid residues, including a signal sequence of 19 residues. This protein contains six potential Ca(2+)-binding domains, one N-glycosylation site, and a large amount of acidic amino acid residues. The ability to bind Ca2+ has been ascertained by calcium overlay experiment. Evidenced by sequence similarity in addition, it is concluded that crocalbin is a new member of the reticulocalbin family of calcium-binding proteins.

Amino Acid Sequence↗

PubMed1997

Purification and partial amino acid sequence of a novel protein of the reticulocalbin family.

Binding proteins in neuronal membranes for a phospholipase A2 with presynaptic neurotoxicity have been purified. Three polypeptides of 87, 65, and 50 K Da were obtained from the synaptic membrane fraction of guinea pig brain utilizing an immobilized crotoxin (a phospholipase A2) column. For large scale purification, porcine brain was used instead, and two polypeptides of 50 and 18 K Da were found. The 65 and 18 K polypeptides may represent hitherto unidentified components of the crotoxin-binding proteins. Partial N-terminal amino acid sequence and a partial sequence for an internal peptide fragment have been determined for the 50 K polypeptide. Search of protein data bank reveals that this polypeptide or protein is a novel member of the reticulocalbin family of calcium-binding proteins.

Amino Acid Sequence↗

PubMed1995

Conversion of bovine pancreatic phospholipase A2 at a single site into a competitor of neurotoxic phospholipases A2 by site-directed mutagenesis.

A 45-kDa polypeptide preferentially present in neuronal membranes was previously identified as a subunit of a binding (or receptor) protein for several phospholipase A2 variants with neurotoxicity, including crotoxin, by chemical cross-linking experiments (Yen, C.-H., and Tzeng, M.-C. (1991) Biochemistry 30, 11473-11477). The binding of crotoxin to this receptor protein was completely suppressed by sufficient F22Y, a mutated bovine pancreatic phospholipase A2 generated by site-directed mutagenesis of Phe22 of the wild-type enzyme to Tyr. The IC50 of this inhibition was estimated to be 1 microM. In sharp contrast, the wild-type enzyme gave no effect even at 50 microM. This mutation resulted in only minor and localized structural perturbations with little effect on enzymatic activity. Other phospholipase A2 molecules capable of competing with crotoxin for this binding invariably have Tyr at this position. It was concluded that this Tyr residue is an important determinant for the binding of a number of phospholipase A2 variants to the 45-kDa receptor.

Animals↗

PubMed1995

Binding proteins on synaptic membranes for crotoxin and taipoxin, two phospholipases A2 with neurotoxicity.

Crotoxin and taipoxin are both neurotoxic phospholipases A2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A2. Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca(2+)-free condition, taipoxin or crotoxin inhibits with IC50 of 20-1000 nM the Na(+)-dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Identification of a crotoxin-binding protein in membranes from guinea pig brain by photoaffinity labeling.

Crotoxin is a neurotoxic phospholipase A2 capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin with N-hydroxysuccinimidyl-4-azidobenzoate and with Na[125I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin, Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of about 10(-8)M. Mojave toxin, caudoxin, notexin, Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while beta-bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.

Affinity Labels↗

PubMed1989

Taipoxin-binding protein on synaptic membranes: identification by affinity labeling.

Affinity labeling techniques were used to identify the neuronal membrane molecules involved in the binding of taipoxin, a neurotoxic protein with phospholipase A2 activity. After [125I]taipoxin had bound to synaptosomes from guinea pig brain, treatment with disuccinimidyl suberate resulted in the formation of a predominant radioactive conjugate of 60,000 Da. Notexin and some other PLA2s are weakly inhibitory to this conjugation, while beta-bungarotoxin and some others are not inhibitory. The 60K conjugate was not detected when plasma membranes from several nonneuronal tissues were used. We concluded that a 45,000 Da protein specifically present in neuronal membranes is (a subunit of) the major molecule responsible for taipoxin binding.

Affinity Labels↗