[Effect of ventral prostate secretory proteins on oviductal fluid glycoproteins in golden hamsters].

OBJECTIVE: To investigate the effect of the secretory proteins of the ventral prostate on the glycoproteins in the oviductal fluid of golden hamsters. METHODS: Male golden hamsters were divided into four groups: sham operation (SH), total removal of accessory sex glands (TX), and retainment of the ventral prostate only (VP). Oviductal fluid was collected from female hamsters at 0.5, 2, 4 and 6 h after mating with the males of different operated groups with or without ventral prostate. Glycoproteins were probed with a panel of lectins and their changes in the oviductal fluid were analyzed by Western blot. RESULTS: The 47 000, 52 000, 81 000 and 128 000 WGA-binding proteins were observed in the oviductal fluid of the 6 h TX group, the 32 000, 35 500, 47 000 and 52 000 WGA-binding glycoproteins noted in the 6 h VP group, the 47 000, 68 000, 95 000 and 128 000 pisum sativum agglutinin (PSA)-binding glycoproteins shown in the 6 h TX and VP groups, two extra 32 000 and 37 500 bands detected in the 6 h VP group, the 47 000 and 52 000 dolichos biflorus agglutinin (DBA)-binding glycoproteins present in the 6 h VP but absent in the 6 h TX group. CONCLUSION: Ventral prostate secretory proteins affect acetylglucosamine, N-acetylgalactosamine/galactose and mannose in the oviductal fluid collected 6 hours after mating. And these glycoproteins may play an important role in the development of embryos.

[Identification of sperm-binding proteins in the ventral prostate of the golden hamster].

OBJECTIVE: To investigate the binding of secretory proteins in the ventral prostate to the surface of sperm. METHODS: We used different techniques to demonstrate the possibility of ventral prostate secretory proteins binding to sperm in golden hamsters. Polyclonal antibodies against crude secretion of the ventral prostate cultured in rabbits were used to detect the antigens in hamster epididymal, uterine and oviductal spermatozoa by indirect immunofluorescence technique. The uterine and oviductal spermatozoa were collected after mating with the males with or without ventral prostate glands. The ventral prostate secretory proteins were isolated and transblotted to the membrane, which was incubated with the biotinylated epididymal sperm membrane proteins, and then the biotinylated binding proteins were stained. RESULTS: An immunoreaction restricted to the middle piece was observed in the sperm incubated with the ventral prostate secretion and ejaculated sperm recovered from the uteri and oviducts. The rate of the epididymal sperm bound with the ventral prostate secretory proteins was (80 +/- 5) %, and the rats of the sperm binding to the ventral prostate secretory proteins were (30.0 +/- 4.6) % from the uterus and (16.0 +/- 3.6) % from the oviduct after mating with the males with ventral prostate glands, significantly higher than after mating with those without prostate glands (P < 0.01). Five bands were identified by Western blot analysis in vitro of the ventral prostate secretory proteins incubated with biotinylated epididymal sperm membrane proteins. CONCLUSION: The present data indicate that ventral prostate secretory proteins bind to the middle piece of sperm in golden hamsters.

beta-Agkistrodotoxin inhibition of Ca2+-dependent release of glutamate, aspartate, glycine and gamma-aminobutyric acid from cerebrocortical synaptosomes following its binding to synaptic membrane.

The binding properties of beta-AgTX, a snake pre-synaptic toxin, membranes and its effect on transmitter release from cerebrocortical synaptosomes were investigated. Assay of [125I]-beta-AgTX binding to rat synaptic membrane revealed a high affinity binding site for the toxin within the synaptic membrane. Preincubation with beta-AgTX inhibited K+-evoked Ca2+-dependent glutamate release from synaptosomes in a concentration-dependent manner, as determined by an on-line enzyme-linked fluorometric assay. The toxin also blocked the Ca2+-dependent release of other transmitters, aspartate, glycine, and GABA induced by K+-depolarization. However, Ca2+-ionophore, ionomycin-stimulated Ca2+-dependent transmitter release was not significantly affected by beta-AgTX, indicating that the toxin inhibits transmitter release by reducing the entry of Ca2+ into cytoplasm. It is suggested that beta-AgTX-binding site in synaptic membrane is related to the release of a variety of transmitters.

[Purification of a Storage Protein from Aphis craccivora Koch and Its Subunit Characterization].

By using of DE52 Cellulose, Sephadex G-150 and FPLC Q Sepharose chromatography, a storage protein (SP) was purified from Aphis craccivora Koch and its subunit was characterized. Its molecular weight was about 60 kD as determined by SDS-PAGE analysis under both reducing and non-reducing conditions, and its pI was about 5.0. It was a glycoprotein. The protein accumulated in larval hemolymph, and decreased when host turned to adult, and could be detected in adults in very low concentration. According to the molecular weight, amino acids composition, and its dynamic alteration of concentrations, the protein should be a persisting storage protein of hemimetabolous insects.

Isolation and Characterization of a Neurotrophic Factor-like Substance from Venom of Agkistrodon acutus.

A neurotrophic factor-like substance from Agkistrodon acutus was isolated by ion exchange and gel filtration chromatography and was found to be homogenous by electrophoresis. Itsmolecular weight was estimated to be approximately 26 kD by gel filtration. A characteristic of the substance was that the protein consisted of two subunits which were bound one to another noncovalently. The analytical isoelectric focusing revealed its isoelectric point of 7.8. The biological activity of the substance was comparable to that of mouse 2.5 S NGF. It rescued PC12 cells from apoptosis at the concentration interval of 1 100 &mgr;g/L and could induce PC12 cells differentiate to sympathetic-like neurons.

Cloning and Expression of cDNA for Thrombin-like Enzyme from Agkistrodon halys Pallas Snake Venom.

Total RNAs were extracted from the venom gland of the Agkistrodon halys Pallas snake. The thrombin-like enzyme gene was amplified by RT-PCR, cloned, and its nucleotide sequences have been determined. The enzyme called pallase cDNA encodes 708 nucleotides, namely 236 amino acids. Based on the homology, the catalytic residues and disulfide bridges of pallase were deduced as follows catalytic residues, His(41), Asp(86) and Ser(182) and disulfide bridges, Cys(7)-Cys(139), Cys(26)-Cys(42), Cys(74)-Cys(234), Cys(118)-Cys(188), Cys(150)-Cys(167) and Cys(178)-Cys(203). The pallase expression plasmids under the control of the T7 promoter was constructed and the pallase was expressed in E. coli.

Cloning and Functional Expression of Neurotoxin cDNA from Naja naja atra.

Three new cDNAs named NL1, NL2 and NL3 were cloned from the total RNA of Naja naja atra by RT-PCR. The protein sequences encoded by them showed 77%, 72% and 98% structure identity to cobrotoxin which is a postsynaptic neurotoxin from Taiwan cobra (Naja naja atra), respectively. The five conservative residues, Tyr(25), Lys(27), Trp(29), Arg(33) and Lys(47), essential for the function of cobrotoxin were also found in the three cDNAs. The NL3, the most homologous to cobrotoxin, was expressed in E.coli BL21(DE3) by cloning into pET28b+. The expressed product was insoluble inclusion bodies and could be purified up to 90% purity in the range of 6 mg per liter culture cells by a single affinity step. The purified protein was refolded in vitro and the toxicity was assessed to be less than the native cobrotoxin in mice.

Structure Determination of Basic Phospholipase A(2) from the Venom of Agkistrodon halys Pallas in A New Monoclinic Crystal Form.

Basic phospholipase A(2) from the venom of Agkistrodon halys Pallas ( Agkistrodin blomhoffii brevicaudus ) exhibits hemolytic and anti-coagulant activities. A new monoclinic crystal form with four molecules per asymmetric unit was grown in the absence of n-octyl beta-o-glucopyranoside (beta-OG). The enzyme structure was determined by the molecular replacement method. The combined analysis of self- and cross- rotation function was used and non-crystallographic symmetry restraints were imposed to the structure refinement. The final model gave an acceptable crystallographic R factor and reasonable stereochemistry. Two molecules formed an interfacial-recognition-site linked dimer and two such dimers constituted a tetramer having pseudo 222 symmetry. Structural comparison with previously reported monoclinic forms, in which beta-OG was bound, showed that the variation of crystallization conditions had effects on the crystal packing, leading to significant changes of the cell parameters. Nevertheless, the structures of both the dimer and tetramer in the two crystal forms closely resembled to each other, indicating that the oligomers found in the monoclinic crystal forms were stable.

Purification, Crystal Growth and Preliminary X-ray Analysis of a Phospholipase A(2) from Venom of Agkistrondon acutus.

An acidic phospholipase A(2) from Agkistrondon acutus venom has been purified to homogeneity via four steps using CM-Sepharose, two times DEAE-Sepharose, and Mono Q FPLC. The molecular weight of the protein was about 16.5 kD and the isoelectric point was 4.3. The purified enzyme showed a potent inhibitory effect on platelet aggregation induced by ADP in human platelet-enriched plasma. The enzyme was then crystallized by hanging drop diffusion method using 2-methyl-2, 4-pentanediol as a precipitant. Two kinds of single crystals suitable for X-ray crystallographic studies were obtained. X-ray crystallographic analysis showed that both crystal forms belong to monoclinic system and space group P2(1). The cell dimensions of form I crystals were a = 43.48 Aring;, b = 71.49 Aring;, c = 43.85 Aring; and beta = 116.32 deg; Those of form II crystals were a = 49.25 Aring;, b = 38.33 Aring;, c = 70.25 Aring; and beta = 99.20 deg;. Complete diffraction data sets have been collected to medium resolution for the two crystal forms.

Cloning of the BPLA(2) Gene from Agkistrodon halys Pallas.

First strand cDNA synthesis was primed with synthetic oligonucleotide from total RNA extracted from the gland of the snake Agkistrodon halys Pallas. The pro-BPLA(2) gene was then amplified by PCR and cloned into the pBS-ks vector. Its nucleotide sequence has been determined by analyzing the DNA sequence of three colonies containing the pro-BPLA(2) gene. The deduced amino acid sequence consists of 138 amino acids and agrees with the partly known amino acid sequence except for seven amino acid residues. The successful cloning of the BPLA(2) gene not only has made the determination of its total amino acid sequence possible, but also provided a good basis for further research work in the protein engineering of functional peptides from snakes.

Purification and Characterization of Phospholipase A(2) from the Venom of Snake Trimeresurus stejnegeri Schmidt.

A phospholipase A(2)(PLA(2)) was purified from the venom of the snake Trimeresurus stejnegeri Schmidt by Sephadex G-75 gel filtration, Mono Q ion exchange chromatography and Superose-12 gel filtration with FPLC and proved to be homogeneous as shown in SDS-PAGE and IEF. Its molecular weight is around 18 000 and isoelectric point is 4.7. Amino acid composition of PLA(2) was determined. Apart from hydrolyzing phosphatidylcholine, the enzyme has a potent inhibitory effect on platelet aggregation induced by ADP or collagen with half-inhibitory concentration of 10-15 &mgr;g/ml and 50-80 &mgr;g/ml respectively.

Crystal Structure of Ca(2+) Ion-saturated Acidic Phospholipase A(2) From Agkistrodon halys Pallas.

The Ca(2+) ion is a cofactor for the catalysis of phospholipase A(2). The crystals of Ca(2+)-saturated acidic phospholipase A(2) from Agkistrodon halys Pallas were obtained by adding CaCl(2) during the crystallization to ensure the complete binding of Ca(2+). The synchrotron diffraction data were collected at 1.6 Aring; resolution. The structure was determined by difference Fouriers methods. The refined structure of Ca(2+)-saturated acidicPLA(2) resembles closely that of the native acidicPLA(2) with, however, some small conformational differences in the Ca(2+)-binding site and the C-terminal loop. The pentagonal bipyramidal configuration consisting of seven oxygen ligands of Ca(2+) ion appears more regular than that of the native acidicPLA(2). The small conformational changes induced by Ca(2+) implies that the main role of Ca(2+) ion is to stabilize the oxyanion in the tetrahedral intermediate formed during the catalysis by electrophilic interaction.

Expression of Nerve Growth Factor from Agkistrodon halys Pallas in Silkworm Larvae.

The cDNA encoding nerve growth factor (NGF) precursor from Agkistrodon halys Pallas (a Chinese snake species) was cloned into pBacPAK8 a baculovirus shuttle vector. The recombinant shuttle vector pBacPAK-NGF was coinfected with linear Bm-BacPAK6 DNA into BmN cells. The recombinant virus was screened and plaque-purified. The silkworm larvae were infected with the recombinant virus and collected 5 days later. The SDS-PAGE and the NGF activity by bioassay of PC12 cells have shown a high expression level of NGF of good biological activity in the larvae.

Cloning and Sequencing of Genes Encoding Phospholipase A(2) from Agkistrodon acutus.

Synthetic oligonucleotides were used to amplify phospholipase A(2) (PLA(2)) gene by RT-PCR from total RNA of snake Agkistrodon acutus venom gland. The PCR products were subcloned and positive clones were screened with acidic PLA(2) gene from Agkistrodon halys Pallas. Finally, four cDNAs of PLA(2) isoenzymes were isolated. Their complete sequences were determined by bidirectional sequencing and their amino acid sequences were deduced. They were designated as A.aAPLA(2)I A.aAPLA(2)II A.aBPLA(2) and A.aLys(49)-PLA(2) according to their isoelectric points calculated by computer and special structure characteristics respectively. The amino acid sequence of 1 10 residues of A.aAPLA(2)I deduced from the cDNA is identical to that of acidic PLA(2) which had been isolated from Agkistrodon acutus. A.aLys(49)-PLA(2) is unique because of the usual Asp(49) is replaced by Lys(49), which may lower its enzymatic activity. Their similarity scores were calculated and compared by computer. The successful cloning of these isoenzymes genes may provide more information for the study on structure-function relationship of PLA(2) family.

Modeling and Analysis of Structures of Phospholipase A(2)'s from Venom of Agkistrodon haly Pallas.

We have modeled three-dimensional structures of basic-acidic hybrid phospholipase A(2)-II and neutral phospholipase A(2) from venom of snake Agkistrodon halys Pallas, based on the known structures of basic and acidic phospholipase A(2)'s from the same source. We have compared these structures of phospholipase A(2)'s, explained the results of fluorescent spectrum study on the phospholipase A(2)'s and calculated the electrostatic potential maps on the catalytic active site. We suggest that the electrostatic potential around the catalytic active site of PLA(2) containing a calcium ion favors the binding of the PLA(2) to its substrate with negative charge.

Research on the Diversity of PLA(2) Gene from Agkistrodon halys Pallas.

We used degenerate primers to amplify phospholipase A(2)(PLA(2)) gene by RT-PCR from venom total RNA of Agkistrodon halys Pallas, and screened with B-PLA(2) gene as probe. Finally we isolated three cDNAs containing A-PLA(2) and two other genes showing similar characteristic structure--Asn(49)-PLA(2) and BA-PLA(2). Their complete sequence was determined by bidirectional sequencing and their amino acid sequence was deduced. The amino acid sequence of A-PLA(2) deduced from the cDNA agreed with that determined by protein sequencing except for four residues; Asn(49 )-PLA(2) and B-PLA(2) are very alike (up to 95%), but the Asp(49)-PLA(2) of B-PLA(2) involved in the enzyme reaction is replaced by Asn(49) of Asn(49)-PLA(2). Therefore, it possibly affects the enzyme reaction of Asn(49)-PLA(2). The N-terminus sequence of BA-PLA(2) is highly homologous to B-PLA(2), but its C-terminus sequence is almost the same as A-PLA(2). The successful cloning of these isoenzyme genes not only discloses the structure diversity of PLA(2)(namely DNA modification and recombination), but also provides excellent material for the study of structure-function relationship in PLA(2).

Expression and Biochemical Characterization of Acidic Phospholipase A(2)I from Agkistrodon acutus.

A cDNA encoding acidic phospholipase A(2)I(A.aAPLA(2)I)from Agkistrodon acutus was inserted into a bacterial expression vector and effectively expressed in E.coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column Superose(TM)12. The enzymatic acti-vity and platelet aggregation inhibiting effect of the expressed A.aAPLA(2)I is close to those of denatured-refolded native acidic PLA(2) from Agkistrodon halys Pallas, and has the same hemolytic activity as denatured-refolded basic phospholipase A(2) from Agkistrodon halys Pallas. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.

Cloning and Expression of Actin Gene of Schistosoma japonicum Chinese Strain.

A 1 131 bp cDNA fragment was amplified by RT-PCR from adult Schistosoma japonicum(Chinese strain) mRNA with primers designed according to published SmAct2 encoding Schistosoma mansoni actin. Sequence analysis indicated that this fragment, with 92% homology to SmAct2, was a complete open reading fragment (ORF) of actin gene of Schistosoma japonicum (Chinese strain). This gene was cloned into the expression vector pET28a( ) and subsequently expressed in Escerichia coli. SDS-PAGE revealed that the molecular weight of this expressed product was 47 kD. Western blotting showed that the recombinant protein had good reactivity with the rabbit serum immunized with Sj worm antigen, indicating that this gene encode actin of Schistosoma japonicum(Chinese strain).

The crystallization and Preliminary Crystallographic Analysis of the Monoclinic Crystal Form of Basic Phospholipase A(2) from the Venom of Agkistrodon halys Pallas.

The basic phospholipase A(2) from the venom of Agkistrodon halys Pallas exhibits strong hemolytic activity. The enzyme has been crystallized by vapour diffusion techniques. Diffraction data of the crystal have been collected up to 2.5 Aring; resolution using the synchrotron radiation-imaging plate-Weissenberg camera system. The crystal parameters were calculated with an auto-indexing program. The crystal belongs to C2 space group with unit cell dimensions of alpha=100.38 Aring;, b=54.37 Aring;, c=117.38 Aring; and beta=120.71 deg;. Each asymmetric unit probably contains four or five molecules.

Expression of the APLA(2) Gene from Agkistrodon halys Pallas.

The APLA(2) gene from Agkistrodon halys Pallas has been cloned into the expression plasmid pBLMVL2 and expressed in E. coli RR1. The molecular weight of the expressed product is approximately 14 kD as shown by SDS-PAGE, its expression level is about 30% of the total cellular proteins. The protein was produced as insoluble inclusion bodies. After partially purified by washing the inclusion bodies, the product was denatured and refolded into active form. Then, the expressed APLA(2) was purified by FPLC Superose (TM) 12 and was a single band as shown by SDS-PAGE. The purified expressed protein had specific activity as the native enzyme and cross-reacted with antisera prepared against the native enzyme. The successful expression of the APLA(2) gene from Agkistrodon halys Pallas provides a good basis for further structure-function studies.