ALTERATION IN CHROMATIN CONFORMATION OF PROLIFERATING AND DIFFERENTIATING ENZYME GENES IN NORMAL MOUSE LIVER AND ASCITES HEPATOMA CELLS AND ITS RELATION TO THE GENE EXPRESSION OF ENZYMES

Nuclei from the normal mouse liver were partially digested with micrococcal nuclease, followed by DNA extraction, agarose gel clectrophoresis and dot blot hybridization with ~(32)P-labeled cDNA probes of CPS_1 and ACT complex, It was clearly shown that the CPS_1 genes were distributed on the monomer, dimer. and trimer of nucleosomes, while the genes coding for ACT complex were distributed on the condensed oligonucleosomes. An opposite manner of distribution of CPS_1 and ACT complex genes was, however, noted in the case of ascites hepatoma cells, in which the specific activity of ACT was 13 times higher than that in the normal liver, while that of CPS_1 was remarkably reduced. Similar patterns of change in mRNA level of CPS_1 and ACT complex were observed in the normal mouse liver and ascites hepatoma cells, indicating a close relationship between chromatin structure and gene expression of these enzymes.     

STUDIES ON THE SYMMETRY OF QUATERNARY STRUCTURE OF PHOSPHOENOLPYRUVATE CARBOXYLASE FROM SORGHUM LEAVES

Studies on the symmetry of quaternary structure of PEP carboxylase from sorghum leaves were carried out by means of chemical crosslinking with bifunctional reagents (diimidoesters) and subsequent SDS polyacrylamide gel electrophoresis.Results showed that the four identical subunits of the enzyme are associated isologously: i.e. the quaternary structure of the enzyme has a D_2 symmetry.The patterns of PEP carboxylase crosslinked with suberic dimethylimidate varied in the presence of various ligands.Substrate PEP, Mg~(2+) (required for catalysis of the enzyme), PEP plus Mg~(2+) and the inhibitor malate increased the extent of crosslinking of the enzyme in varying degrees, indicating that binding of these ligands to the enzyme gave rise to changes in the subunit interactions.However,the extent of crosslinking of PEP carboxylase with suberic dimethylimidate decreased greatly in the presence of the activator G6P. Meanwhile, the enzyme remains a tetrameric form either in the absence or in the presence of G6P.     

SCANNING ELECTRON MICROSCOPY STUDY OF FILLED SILICONE RUBBER

The fracture surfaces of a number of silicone vulcanizates were investigated by the use of scanning electron microscopy (SEM). It was found that the difference in the presence and absence of filler, the variation of its surface modification as well as the history of thermal aging of the vulcanizates, all of these factors made difference in surface morphology of the fractured surface. This was correlated with the strength of the vulcanizates. The reinforcing effect of filler and the process of fracture were discussed.     

KINETICS OF IRREVERSIBLE MODIFICATION OF ENZYME ACTIVITY IN A SYSTEM OF COUPLED ENZYME ASSAY (Ⅱ)——BOTH PRIMARY AND AUXILIARY ENZYMES MODIFIED BY REAGENT Y

Following the previous paperr, we examined the kinetics of the irreversible modification of enzyme activity in coupled assay when the modifier reacted with both primary and auxiliary enzymes. It was shown that Tsou's kinetic method can also be used in the present situation in some conditions.     

SURFACE OF GELATIN MODIFIED POLY(L-LACTIC ACID)FILM

In this paper, the surface structure of poly(L-lactic acid) (PLLA) film modified with gelatin was investigated. ThePLLA film specimens were treated directly with aqueous alkali solution to provide their surfaces with carboxyl groups, sothat these functional groups could become the reactive sites for gelatin immobilization. The functional groups of the PLLAfilms were identified by ATR-FTIR spectra and XPS spectra, the changes in surface morphology were observed by usingenvironmental scanning electron microscopy (ESEM), and the hydrophilicity of modified PLLA films was examined bywater contact angle measurement. Experimental results showed that the gelatin was immobilized with water-solublecarbodiimide (EDC) onto the PLLA film's surfaces, and the gelatin content on the polymer surface was related to carboxylicgroup formed in the controlled hydrolysis process. Rough surfaces caused by hydrolysis will predominantly favor the adhesion and growth of cell; and the hydrophilicity of these surfaces after the modification procedure is enhanced.     

LIMITED PROTEOLYSIS OF SNAKE MUSCLE FRUCTOSE 1, 6-BISPHOSPHATASE

Limited proteolysis of snake muscle fructose 1,6-bisphosphatase (EC. 3. 1. 3. 11) with subtilisin resulted in a larger fragment of 29,000 daltons and a smaller fragment of 7000 daltons, and limited tryptic digestion for fructose 1, 6-bisphosphatase mainly produced a 32,000 daltons fragment. Both of the modified enzymes have a 4 to 5 fold increase in activity at pH 9.2 as well as a twofold increase in activity at pH 7.5. At 6μM of AMP more than 90% of the activity was inhibited for the native enzyme but not for the modified enzymes. In case of subtilisin, only 50% of activity was inhibited and in case of trypsin, non inhibition was observed. Results indicated the cleavage sites for the two proteases were apparently different.The above results and the fact, as reported previeusly, that snake muscle fructose 1, 6-bisphosphatase modified with 5, 5'-dithiobis(2-nitrobenzoic acid) also caused a twofold increase in its activity lead us to propose that the catalytic site of snake muscle fructose 1, 6-bis-phosp     

CLONING AND SEQUENCE ANALYSIS OF 3''''-END REGION OF POTATO VIRUS Y GENOME

The entire coat protein (CP) gene and part of the 3'-noncoding sequence of the potatovirus Y (PVY, the Chinese isolate) genome were synthesized with polymerase chain reaction(PCR) using cDNA of its genomic RNA as a template. A restriction endonuclease site Ncoland the initiation codon AUG were included in primer Y5 while the SalI site was includedin primer Y3. After being double digested with Ncol and SalI enzymes, the PCR product wascloned into a pGEM derivative plasmid, and the CP gene in one of the clones, pPCY6, wassequenced. Several clones were selected from the cDNA library by using the CP gene frag-ment of pPCY6 as a probe and the sequences of these clones were determined. These se-quences included part of the NIb gene, entire CP gene and 3'-noncoding region, 1317 bp alltogether.Sequence analysis indicated that the nucleotide sequence homology of the CP geneof this strain with that of the 0 strain (94.2%) was a little higher than with that of the Nstrain (89.6%), but the homology of amino acid se     

STUDIES ON THE RNA POLYMERASES FROM THE CELL NUCLEI OF VICIA FABA L.LEAVES

Isolated cell nuclei from young leaves of Vicia faba L. have a high level of RNA polymorose activity and can serve as a good source for purification of higher plant RNA polymerases. The nuclei were lysed in a solution of high (NH_4)_2SO_4 concentration and the lysate was subjected to sonication to liberate RNA polymerases, which then were fractionated on DEAE-cellulose and DEAE-Sephadex columns. Three enzyme peaks were detected in the eluate fractions. According to their sensitivity to α-amanitin they are the RNA polymerases Ⅰ, Ⅱ and Ⅲ. Divalent cation Mn or Mg is essential for the enzyme activity. The Mn optimum concentration is 3mM and the Mg optimum concentration is 5mM for the RNA polymerases Ⅰ and Ⅱ. Calf thymus DNA and the DNA of Vicia faba have equal efficiency as template for RNA synthesis by these enzymes.     

CHANGES OF QUATERNARY CLIMATE OF LOESS DISTRICT IN CHINA

On the basis of the data of sporo-pollen analysis, the weathering index of loess and paleosol layers and the climatic differences between the periods of the formation of loess and paleosol as reflected by intensities of natural remanent magnetization of loess and paleosol, it may be confirmed that the loess layers represent the climate of glacial stages, while paleosol was formed during interglacial stages. The climatic changes in the loess area of China have undergone 23 cycles which were combined with the paleomagnetic dating. TL ages of paleosol layers and the average ages of termination could be excellently correlated with the δ~(18)O stages of deep sea core V_(28-239). Thus, it may be recognized that the Luochuan and Xi'an loess-paleosol sequences are the typical climatostratigraphy in China and even in the world.     

EFFECTS OF DEOXYCORTICOSTERONE ACETATE ON MUSCLE ELECTROLYTES,RESTING POTENTIAL AND MITOCHONDRIA IN RATS

Deoxycorticosterone acetate, injected daily for 5, 10, 20 or 30 days, reduced the serum potassium levels of rats by about 25%, a decrease that was independent of the treatment period employed. Serum sodium concentrations were unchanged with treatment duration. The potassium concentrations in extensor digitorum longus (EDL) and soleus (SOL) muscles were significantly decreased, and those of sodium increased, after treatment. Accompanying these changes of electrolyte concentration, the resting membrane potentials of treated EDL and SOL were hyperpolarized in vivo, and showed depolarization with the decreases of external potassium concentrations in vitro. The mitochondria in the muscles of treated rats were damaged. The degree of damage was more serious in EDL than in SOL and was dependent on the duration of deoxycorticosterone acetate treatment.     

EFFECT OF SOLVENT ON THE COPOLYMERIZATION OF 2,2,6,6-TETRAMETHYL 4-PIPERIDINYL METHACRYLATE WITH STYRENE

The radical copolymerizations of 2,2,6, 6-tetramethyl 4-piperidinyl methacrylate (TMPM) with styrene in various solvents have been studied. The monomer reactivity ratios r_1 and r_2 were determined. It is observed that in all of these reaction systems, there are appreciable solvent effects on both r_1 and r_2, which can be correlated to the difference in chemical shifts of olefinic protons of TMPM. And the variance of the copolymer microstructure in various media was discussed.     

Covalent attachment of lysine to commercial restriction endonucleases

In the present work lysine was coupled through a water-soluble carbodiimide to several restriction enzymes. The work was carried out to assess the effects on enzyme activity of attaching a small molecule to the enzyme carboxyl groups, with the intent of using carboxyl groups for subsequent immobilization of restriction enzymes on solid supports. Lysine was coupled to Eco RI, Bam HI, and Bgl I with partial to complete retention of enzyme activity. The commercial enzymes contained a large relative concentration of bovine serum albumin (BSA). Therefore, commercial Eco RI, a sample of electrophoretically pure Eco RI, and some high purity BSA each were separately labeled with³H-lysine and the products separated by dialysis and polyacrylamide gel electrophoresis. For the commercial Eco RI preparation, 0.9 μmol of lysine was attached to each μmole of the enzyme fraction; lysine was attached to the BSA and enzyme fractions in the ratio 2.3. The results agreed reasonably well with the amount coupled to the high purity Eco RI and the high purity BSA. The results suggest that carbodiimide coupling through enzyme carboxylic acid groups may be a useful approach for subsequent immobilization of restriction enzymes on solid supports.     

PROPERTIES AND SYNTHESIS OF NEW SUPPORTS FOR IMMOBILIZATION OF ENZYMES BY COPOLYMERIZATION OF VINYLENE CARBONATE AND METHACRYLIC ACID

Methacrylic acid first was neutralized with an aqueous solution of NaOH to pH=6.0～7.0, vinylene carbonate(VCA) was added to the solution, then monomers were copolymerized in paraffin oil by means of reverse-phase suspensionpolymerization and hydrophilic copolymeric supports were prepared. The properties of the supports were determined usingtrypsin and results show that the amount of enzymes coupled to the supports and the specific activity of immobilized trypsinare related to the content of VCA structure units, reaction time and concentration of enzyme solution, etc.     

Screening the anti-gout traditional herbs from TCM using an in vitro method

The aim of this work was to evaluate the ability of 33 herbal extracts in inhibiting the acute inflammation and xanthine oxidase(XOD) activity.The anti-inflammation effects of the herbal extracts were detected by an in vitro cell model,which was established by stimulating human umbilical vein endothelial cells(HUVEC) using sodium urate(MSU).In this model,the intercellular adhesion molecule-1(ICAM-1) and interleukin-1 beta(IL-1β) were expressed,and the anti-inflammation effects of herbal extracts were evaluated by detecting the content changes of ICAM-1 and IL-1β in cell lysates and cell culture supernates using an enzyme-linked immunosorbent assay(ELISA).Moreover,an ultrahigh performance liquid chromatography and tandem mass spectrometry(UPLC-MS/MS) method was used for the detection of XOD activity and the screening of XOD inhibitors in this research.The amount of uric acid from each analyte was directly detected using the multiple reaction monitoring mode and the uric acid level could be reduced via the addition of an inhibitor.Results indicated that Salviae Miltiorrhizae Radix et Rhizome,Rhei Radix et Rhizoma,Polygoni Cuspidati Rhizoma et Radix,Selaginellae Herba,Paeoniae Radix Rubra,especially Ginkgo Folium seemed to be more effective in anti-inflammation and inhibiting XOD activity.The anti-inflammation and enzyme inhibitory activities of the herbal extracts may be correlated with their bioactive components.And the differences between the herbal extracts were correlated with the amount of flavonoid and anthraquinone components.In our study,we have investigated the potential anti-inflammation bioactivity of 33 herbal extracts in vitro,which could provide a reference for further in vivo research in the prevention and treatment of gout.     

PRELIMINARY CRYSTALLOGRAPHIC STUDIES ON THE COENZYME BINDING TO GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM Palinurus versicolor

The three forms of glyceraldehyde-3-phosphate dehydrogenase from Palinurus versicolor muscle, namelyapo native enzyme, apo calboxymethylated enzyme and the enzyme carrying two fluorescent NAD deriva-tives per tetramer have been crystallized. The space groups and unit cell dimensions of these crystals areisomorphous to each other and to those of three corresponding holo-enzymes saturated with NAD~+ reportedin a previous paper suggesting that binding of coenzyme to the apo enzymes does not lead to significantconformational changes involving domain movement as demonstrated in the case of glyceraldehyde-3-phos-phate dehydrogenase from Bacillus stearothermophilus.     

STUDIES ON SULFHYDRYL AND ALKALINE AMINO ACID RESIDUES OF FRUCTOSE-6-PHOSPHATE-2-KINASE

This Communication reports the roles of cysteine, lysine and arginine residues inchicken liver fructose-6-phosphate-2--kinase. Chemical modification of the enzyme withDTNB demonstrates that there are nine SH groups in the enzyme. Among these SH groups, atneutral pH six SHs can be titrated; in the presence of Fru6P four can be titrated; and inthe presence of ATP seven can be titrated. During the process of titration, the activity ofthe enzyme increases first and subsequently decreases gradually to the original level. NENIhas an effect similar to DTNB for the activity changes of the enzyme. On the contrary,PCMB or iodoacetic acid has a little effect on the activity of the enzyme. The pH profileof the activity shows that there is an essential ionizable group with a pK_a of 9.0, mostprobably the lysyl residue, which responds to the catalytical reaction. PLP or phenylglyoxalinactivates the enzyme. For PLP inactivation, Fru6P, one of the substrates, most effectivelyprotects the enzyme. Both products Fru2,6P_2     

Influence of counteranions on catalytic ability of immobilized laccase in Cu-alginate matrices： Inhibition of chloride and activation of acetate

Laccase is a promising oxidase with environmental applications, such as lignin degradation and chlorophenol detoxification. Laccase immobilization can significantly improve physiochemical stability and reusability compared to the free enzymes. In this work, anion effect was investigated in entrapment of Cu-alginate matrix with five types of anions, including perchlorate(ClO4à), nitrate(NO3à), sulfate(SO42à), chloride(Clà), and acetate(CH3CO2à). Accordingly, chloride inhibition and acetate activation were detected in the o-tolidine kinetic experiments, while effects of the other three anions were much smaller. Such counteranion effects were also observed in the laccase-catalyzed biodegradation of 2,4-dichlorophenol. The results indicated that counteranions in the enzyme immobilization process are crucial for catalytic capacity, probably due to the competition with the carboxylate groups in alginate.Our results also imply that these anions might coordinate the copper cations in laccase.     

Conventional and microwave irradiated synthesis,biological activity evaluation and molecular docking studies of highly substituted piperazine-azole hybrids

Azole derivatives(3,6) obtained starting from 1-(2-methoxyphenyl) piperazine were converted to the corresponding Mannich bases containing β-lactame or flouroquinolone core via a one pot three component reaction.The synthesis of conazole analogues was carried out starting from triazoles by three steps.Reactions were carried out under conventional and microwave mediated conditions.All the newly synthesized compounds were screened for their antimicrobial,enzyme inhibition and antioxidant activity,and most of them displayed good-moderate activity.Binding affinities and non-covalent interactions between enzyme-ligand complexes were predicted with molecular docking method at molecular level.Docking results complemented well the experimental results on α-glucosidase and urease inhibitory effects of the compounds.Higher binding affinities and much more interaction networks were observed for active compounds in contrary to inactive ones.It was predicted with the docking studies that triazole and anisole moieties in the structure of the synthesized compounds contributed to the stabilization of corresponding enzymes through noncovalent interactions.     

Wild-type and molten globular chorismate mutase achieve comparable catalytic rates using very different enthalpy/entropy compensations

The origin of the catalytic power of enzymes with a meta-stable native state,e.g.molten globular state,is an unsolved challenging issue in biochemistry.To help understand the possible differences between this special class of enzymes and the typical ones,we report here computer simulations of the catalysis of both the well-folded wild-type and the molten globular mutant of chorismate mutase.Using the ab initio quantum mechanical/molecular mechanical minimum free-energy path method,we determined the height of reaction barriers that are in good agreement with experimental measurements.Enzyme-substrate interactions were analyzed in detail to identify factors contributing to catalysis.Computed angular order parameters of backbone N–H bonds and side-chain methyl groups suggested site-specific,non-uniform rigidity changes of the enzymes during catalysis.The change of conformational entropy from the ground state to the transition state revealed distinctly contrasting entropy/enthalpy compensations in the dimeric wild-type enzyme and its molten globular monomeric variant.A unique catalytic strategy was suggested for enzymes that are natively molten globules:some may possess large conformational flexibility to provide strong electrostatic interactions to stabilize the transition state of the substrate and compensate for the entropy loss in the transition state.The equilibrium conformational dynamics in the reactant state were analyzed to quantify their contributions to the structural transitions enzymes needed to reach the transition states.The results suggest that large-scale conformational dynamics make important catalytic contributions to sampling conformational regions in favor of binding the transition state of substrate.