ACTIVE DOMAINS IN MUNG BEAN TRYPSIN INHIBITOR

In our experimentes, it has been demonstrated that the N-terminal fragment of the mungbean inhibitor could be isolated by affinity chromatography with immobilized trypsin aftertreating the inhibitor with CNB_r and pepsin. The properties of this active fragment have beenstudied. The C-terminal fragment of the hihibitor, however, will lose its activity during CNB_rcleavage because of the scission of the methionyl peptide bond near the active center. It isdeemed desirable to find out a more suitablo method for the cleavage of these two domains,both retaining inhibitory activity. Ths aim has new been achieved by using peptic digestion only, and the active centers ofthese two domains are identified as Lys and Arg by chemical modification. In alkaline pH,these two active fragments could be easily separated by affinity chromatography with immobi-lized trypsin. This amkes it possible to study and compare these two active fragmentsseparately. It is worth pointing out that such a C-terminal fragment with trypsin inhibitory activityhas not yet been reported among the Bowman-Birk inhibitors. The activities of the mung bean trypsin inhibitor decrease to about 50% during thecourse of modification with maleic anhydride and phenylglyoxal respectively. The two activecenters seem to be Lys nd Arg. The mung bean inhibitor could endure peptic digestionwithout any loss of activyty. On Sephadex gel filtration, two smaller active fragments with asize approximately one half of the original molecule are found. At pH 11.4 these two activefragments could be separated from each other by affinity chromatography with immobilizedtrypsin. The active fragment with Lys as the active center could be completely inhibitedwith maleic anhydride and the activity is completely restored after incubation at pH 3.5.This fragmeut consists of two paptide chains with about 35 amino acil residues. The N-termini are found to be Ser and Phe, and the C-termini to be Leu and Met. The activityof the fragment with Arg as the active center could be inhibited completely by phenylglyoxal.This fragment is a single peptide chain with about 27 amino acid residues. The N- and C-terminis are shown to be Asn and Asp respectively.     

THE STRUCTURE BASIS OF THE POOR FIBRIN SPECIFICITY OF UROKINASE(Ⅱ)——THE INHIBITION OF UROKINASE A CHAIN 149--157 ON THE FIBRIN STIMULATED ACTIVATION OF PLASMINOGEN BY TISSUE TYPE PLASMINOGEN ACTIVATOR

In view of the similarity of the charge distribution between fibrin A_α148--161 and Achain 149--157 of urokinase,the latter might compete with fibrin A_α148--161 when singlechain pro-urokinase is converted to double chain urokinase.To test this, the stretch of uro-kinase A chain 135--157 was separated from the low molecular weight urokinase, a competi-tive binding between this stretch and fibrin to tPA kringle-2 was shown by radio-bindingassay. The inhibition of the stretch on the fibrin stimulated activation of plasminogen wasdemonstrated in the caseinolytic system. The synthesized novapeptide urokinase A chain 149--157 (R-peptide) showed a significant inhibition on the activation of plasminogen in the pres-ence of fibrin. By contrasting finely with R-peptide, a synthesized novapeptide in which Arg154and Arg156 were replaced by Asp (D-peptide) did not show any inhibition effect on the fi-brin stimulated activation of plasminogen by tPA. These results suggest that the positivelycharged residues in the     

Purification and Characterization of β-Glucosidase from a Newly Isolated Strain Tolypocladium cylindrosporum Syzx4

A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer(ITS) rDNA gene sequence.The present study is to ferment,purify and characterize a β-glucosidase from T.cylindrosporum gams.The enzyme was purified to homogeneity by sulfate precipitataion,diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27...     

A NEW SYSTEM FOR THE SYNTHESIS OF HIGH LEVELS OF HBsAg SEQUENCE IN Escherichia coli

Using a system to study promoter activity, we have obtained a promoter fragment from E. coli chromosomal DNA. The HBsAg geno under the control of this promoter could be expressed in E. coli. The expression products are isolated and purified by means of a column of anti-HBs cross-linked to Sepharose 4 B. The pure products are characterized through the double diffusion method on 0.6% agarose and polyacrylamido-SDS gel electrophoresis. The synthesis of high levels of HBsAg sequences in E. coli is confirmed.     

THE CRYSTALLOGRAPHIC STUDY OF THE COM PLEX OF THE LYS ACTIVE FRAGMENT OF THE MUNG BEAN TRYPSIN INHIBITOR WITH BOVINE TRYPSIN

The Lys fragment of mung bean trypsin inhibitor can combine with bovine trypsin to form a complex at an equal molar ratio. The single cxystals of the complex were obtained by using the micro-still-setting method and the X-ray diffraction extended to 1.8 resolution. Its space group is P2_12_12_1 wlth cell dimensions α=62.9(1), b=63.4(1) and c=69.7(2). There is one complex molecule in a crystallographic asymmetric unit.     

STUDIES ON THE KINETICS OF PROPYLENE POLYMERIZATION WITH THE COMPLEXTYPE TiCl_3 CATALYST

The active center concentration C_p, the rate constant k_p, and the activation energy of chain propagation E_p in the polymerization of propylene with complex-type TiCl_3-(C_2H_5)_2AlCl catalyst system were studied. The Mn was corrected by (?) value determined by GPC. The values thus obtained for C_p, k_p, and E_p at 50℃were 3.01 mol/mol Ti, 6.27 1/mol·sec, and 5.10 Kcal/mol respectively.The kinetic parameters were compared with those obtained from conventional TiCl_3·AlCl_2 catalyst, showing that the higher activity of the complex-type catalyst over the conventional catalyst is not only due to the higher C_p of the former, but to a greater extent due to the increase of the k_p value.     

PURIFICATION AND KINETICS OF MOUSE LIVER FRUCTOSE 6-PHOSPHATE, 2-KINASE

The mouse liver fructose 6-phosphate, 2-kinase was purified by ultracentrifugation, polyethylene glycol precipitation, and subsequently by chromatography on DEAE-Sephadex, Blue-Sepharose and phasphocellulose columnS. Gel filtration and SDS polyacrylamide electrophorcsis showed that the enzyme has a molecular weight of 110,000 with two identical subunits. Mg~(2+) is essential for its activity. The activation of the enzyme by Mg~(2+) showed a positive cooperativity. The substrate saturation curve for fructose 6-phosphate was sigmoidal and for ATP was hyperbolic. The K_m's for ATP increased with decrease in concentrations of fructose 6-phosphate indicating that the sequence for the substrates binding was in an ordered mechanism with respect to fructose 6-phosphate prior to ATP. An ionizable residue at the active site with pKa 9.5 was essential for the ATP binding and the pKa shifted to 9.8 after the binding of ATP.     

STUDIES ON THE STRUCTURE OF FORKED CONJUGATIVE SYSTEMS——THE CONFORMATION OF 3-ACETYL-8-PHENYL-3,5,7-OCTATRIENONE-2

The conformational properties of the title molecule in the isolated state have been investigated by the PCILO method and compared with those found from X-ray crystallographic determination. The results indicate that, in this type of forked conjugative series, because of steric hindrance, only one branch of the forking groups is basically coplanar with the conjugated trunk chain and plays the role of a terminal group, while the other one is not coplanar with the conjugated trunk chain and acts as an ordinary substitutent. Referring to the phenyl group, the acetyl group on the different sides of ethylenic chain exhibits the properties of a terminal group.     

Purification and Characterization of Cyclic AMP-Binding Protein from Ganoderma lucidum

Cyclic AMP-binding protein was purified 30 fold from the mycelia of Ganoderma lucidum by the methods of ammonium sulfate precipitation, DEAE-cellulose, phospho-cellulose ion exchange chromatography and Sephacryl S-100 gel filtration. The molecular mass of the purified protein is 34.5 kDa and 17 kDa by Sephacryl S-100 gel filtration and SDS-ployacrylamide gel electrophoresis, respectively. From these results it is suggested that the protein has a homometric dimmer structure. The pI of the purified protein is pH 8.2 by native isoelectric focusing gel. The half-life of the protein activity in 10% glycerol at 4 ℃ is 7 d in crude extract, but its half-life is only 3 d under purifying conditions. The optimal conditions of the protein activity are at 1 ℃ and pH 7.5. Its activity is increased 6 times by 1 mmol/L Zn^2 and is slightly inhibited by cGMP,Cu^2 and Mn^2 .     

Preparative Purification and Bioassay of Bt Toxin from CrylAb Transgenic Rice

A method of extracting and purifying CrylAb protein(Bt toxin) from CrylAb transgenic rice was established. Most of the Bt toxin present in the tissue of CrylAb transgenic rice was extracted effectively with a solution of 50 mmoi/LNa2CO3 and NaHCO3. The crude protein containing Bt toxin was obtained after the pretreatment of CrylAb transgenic rice with ultra-filtration, ammonium sulfate precipitation and centrifugation. The dialysed crude protein was futher separated on DEAE Sephadex A-50 columns and Sephadex G-150 columns. The protein bound on DEAE Sephadex A-50 gel was eluted with buffer solution B(10 mmol/L tris-HCI buffer 1.0 mmol/L EDTA, pH=8.0) mixed with 0. 1, 0. 3, 0.5 and 0.8 mol/L NaCI in a discontinuous gradient elution mode. The peak of the Bt toxin eluted from the columns was identified by ELISA and bioassayed with larvae of tobacco hornworms and silkworms. The purity and the bioactivity of the Bt toxin were determined by means of SDS-PAGE and larvicidal assay, respectively. The purity of the Bt toxin obtained by this method is high, and its insecticidal activity is retained after the toxin is purified.     

Crosslinking reaction in the cationic polymerization of 1, 3-pentadiene

The cationic polymerization of 1,3-pentadiene (PD) initiated by AlCl_3 in n-hexane was carried out. Effects of arenes, alkyl halides and ethers on the gel formation resulting from crosslinking reaction were investigated. The erosslinking was reduced by various arenes through a chain transfer mechanism. Alkyl halides such as tert-butyl chloride and allyl chloride could complex with AlCl_3 to generate an initiating system giving rise to a gel-free polymerization, while benzyl chloride reduced the formation of gel by chain transfer. Ethers exerted two effects on the polymerization system: giving a complex initiating system with AlCl_3 to produce a relatively high molecular weight polymer, or reducing crosslinking by lowering activity of carbocations.     

BIOLOGICAL FUNCTION OF MODIFIED NUCLEOTIDES IN tRNA MOLECULES——SYNTHESIS AND BIOLOGICAL ACTIVITY OF THE ANALOGUES OF YEAST ALANYL tRNA WITH I_(34) REPLACED BY A_(34) OR G_(34)

Analogues of yeast alanyl tRNA with I_(34) replaced by A_(34) or G_(34) were synthesized. Synthetic analoguesof yeast alanyl tRNT occupy the same position as the natural yeast alanyl tRNA on polyacrylamide gelelectrophoresis, and their purity is about 95% after electrophoresis on a 10% or 20% polyacrylamide gel.The two terminal and nearest neighbour nucleotides of the analogues are all correct. The accepting acti-vity of the synthetic analogues is similar to that of the reconstituted natural yeast alanyl tRNA. The in-corporation activity of alanine into proteins of the synthetic analogues is about 30% of that of the naturalof reconstituted natural yeast alanyl tRNA when I_(34) is replaced by A, and is 90% when I_(34) is replaced byG.The reason of the variation in biological function of the analogues of yeast alanyl tRNA after I_(34) re-placed by A or G was discussed.     

ELECTROCARBOXYLATION OF ORGANIC COMPOUNDS WITH CARBON DIOXIDE CATALYZED BY METALLOPORPHYRINS(Ⅲ)——EFFECTS OF AXIAL LIGANDS ON CATALYTIC ACTIVITY OF CoTPP

The effects of peptides,amino acids and organic bases as an axial ligand on reaction ac-tivities in the electrocarboxylation of benzyl chloride with CO_2 catalyzed by CoTPP are reported.The imidazole organic base,peptide containing —SH and amino acid containing imidazolyl en-hance the catalytic activity.The effect of imidazole amounts on the catalytic activity of CoTPP isstudied.     

INHIBITORY PROPERTY CHARACTERIZATION AND REACTIVE SITE EXPLORATION OF THE ARROWHEAD PROTEINASE INHIBITOR

Affinity chromatography was used to separate two components A and B of the crystalline arrowhead proteinase inhibitor. Both A and B are double-headed and multifunctional proteinase inhibitors. Inhibitor A is capable of inhibiting equimolarly trypsin and chymo-trypsin simultaneously, and has a weak inhibitory activity toward kallikrein; whereas inhibitor B can inhibit two molecules of trypsin simultaneously, and shows rather higher inhibitory activity toward kallikrein than inhibitor A, but its inhibitory activity toward chymo-trypsin is much weaker than that of inhibitor A. The results of chemical modification and the competitive binding of trypsin and chymotrypsin with inhibitor A showed that the two reactive sites of both inhibitors A and B are Lys and Arg residues. Among them the Lys reactive site is specific for inhibiting mainly trypsin, whereas the active domain composed of the Arg reactive site appears to be multifunctional and capable of inhibiting many different Ser proteinases. Based on the st     

THE PHOSPHORYLATION SITE OF SNAKE MUSCLE FRUCTOSE 1,6-BISPHOSPHATASE AND ITS PHOSPHORYLATION CONDITIONS

From the tryptic digests of phosphorylated snake muscle FruP_(2ase), a phosphoryl peptide has beenisolated, its amino acid sequence was Gly-Ala-Gly-Ser-Arg and the phosphorylation site was consideredto be on the serine residue. Effectors of the enzyme such as FruP_2, F6P and AMP did not affect thephosphorylation. The effect of pH on phosphorylation was consistent with that on the activity of theenzyme. The activity of phosphorylated enzyme was slightly lower than that of the native enzyme, thisdifference in activities between the two forms of the enzyme increased with decreasing the substrateconcentration. Results further support that a phosphorylated intermediate is involved in the catalytic reac-tion of FruP_(2ase).     

STRUCTURAL STUDY OF BENZYLOXYCARBONYL-PROLYL-ALANYL-THREONYL-TERT-BUTYL ESTER

The crystal structure and molecular conformation of the synthetic oligopeptide (Z-Pro-Ala-Thr (But)_2) have been determined by X-ray analysis. The crystal is orthorhombic, spacegroup P2_12_12_1, with four molecules per unit cell of dimensions: a = 29.557, b = 11.583 andc = 8.830A. The diffraction data were collected using a PW-1100 four-circle diffractometer.The strueture was solved by direct methods, using the MULTAN-80 system of computerprograms and refined by the block-diagonal least-squares method. 7he final R value is 0.088(for non-hydrogen atoms). The Ala residue in this peptide is nearly in an eclipsed conformation. The peptide bondbetween Z and Pro is cis. The two atoms, C~β aud C~γ, are displaced on opposite sides of thepyrrolidine ring plane. The dihedral augles between the neighbouring peptide or carboxylgroups are all close to 90°. Hydrogen bonds link the peptides forming a chain in the zdireetion. The stacking of the peptides in the x and y directions is the result of the van derWaals fo     

CATION SUBSTITUTION ON PEROVSKITE-TYPE OXIDES FOR THE OXIDATIVE COUPUNG OF METHANE

The catalysis by a series of partially substituted CaTiO_3 and LaAlO_2 perovskites for theoxidative coupling of methane has been studied with the aid of a fixed bed reactor.The activity ofCaTiO_3 and LaAlO_3 perovskites could be improved if certain cations were substituted on the A and/or Bsites.The substitution on the B sites was more effective.A C_z-yield of 17% was achieved at 1073 Kwith a molar ratio of CH_4:O_2,=2.5:1 over a Ca_(0.9)Sr_(0.1)TiO_2 catalyst with good stability.     

THE CRYSTALLIZATION BEHAVIOR OF URETHANE SUBSTITUTED POLYDIACETYLENE

The crystalline behavior of urethane substitute polydiacetylene was studied by using pohrized light and electron microscopy. The lamellar morphological structure was observed in the crystallized films. The thickness of lamellae is about 300A, being independent of the crystalline temperature. But the size and density of lamellae were dependent on the crystallization temperature. If the molten film was sheared during the crystallzation process the oriented lamellae grew with their long axes perpendicular to the direction of shear and the chain direction was normal to the lamellar surface.     

THE LINKAGE OF ELECTRON TRANSPORT CHAINS IN FUSED MEMBRANE PROVED BY MONITORING THE REDUCTION OF CYTOCHROME C

The redox states of cytochrome c (cyt. c) of the respiratory electron transport carriers, in the deficient thylakoid of spinach chloroplast, crista vesicles of liver mitochondria and their combined system were monitored. The reconstituted system prepared by combining deficient thylakoid with crista vesicles showed a higher reductive effects of cyt. c than the system prepared by combining chloroplast and mitochondria. The less inhibition effects of photosynthetic chain and respiratory chain on light-induced reduction of cyt. c in combined system had been observed. Further, with cooperation between photosynthetic chain inhibitor and respiratory inhibitor, the combined system still showed the light-induced cyt, c reductive effects.These results indicated that the deficient thylakoid can combine with crista vesicles in the hypotonic solution to form a fused membrane in which the electron carriers of both energy transfer membranes can link together, and that the linkage of electron carriers may be in multipl     

STUDIES ON NATURAL AND MODIFIED PEPTIDE Trichosanthes TRYPSIN INHIBITORS

A peptide trypsin inhibitor was isolated and purified from the roots of Trichosanthes kirilowii (a Chinese medical herb) by using immobilized anhydro-trypsin affinity chromatography and HPLC C_(18) column reverse chromatography. It contains two major components, both consisting of 27 amino acid residues with three pairs of disulfide bonds. The sequence determination indicated that the difference between them is only in the ninth position, being Gln and Lys, respectively. The peptide bond of the inhibitor reactive site Arg-Ile (3--4) is easy to cleave at low pH by trypsin, resulting in a modified inhibitor. It might be the smallest naturally occurring protein inhibitor so far known. The modification reaction of the Trichosanthes inhibitor with trypsin is similar to the catalytic enzyme-substrate reaction. The dissociation constant of the modified inhibitor with trypsin is around fourfold that of the natural inhibitor.