Phylogenetic relationships of someMicrosporum andArthroderma species inferred from mitochondrial DNA analysis

Thirty-eight strains of 12Microsporum and 10Arthroderma (Nannizzia) species were investigated by analysis of mitochondrial DNA with 6 restriction enzymes, and classified into 13 genetic groups. The phylogenetic tree of the 13 groups thus established was constructed. On the tree,M. audouinii, M. langeronii, M. rivalieri, M. distortum, M. equinum, M. ferrugineum andA. otae comprise one genetic group and are suggested to be the same species.A. gypseum, A. fulvum, M. duboisii, M. ripariae, A. incurvatum, A. persicolor andA. obtusum are clustered on one of five boughs of the tree indicating their close relation.A. racemosum andA. cajetani are also closely related.     

Human DNA helicase IV is nucleolin, an RNA helicase modulated by phosphorylation

The cDNA encoding human DNA helicase IV (HDH IV), a 100-kDa protein which unwinds DNA in the 5′ to 3′ direction with respect to the bound strand, was cloned and sequenced. It was found to be identical to the human cDNA encoding nucleolin, a ubiquitous eukaryotic protein essential for pre-ribosome assembly. HDH IV/nucleolin can unwind RNA-RNA duplexes, as well as DNA-DNA and DNA-RNA duplexes. Phosphorylation of HDH IV/nucleolin by cdc2 kinase and casein kinase II enhanced its unwinding activity in an additive way. The Gly-rich C-terminal domain possesses a limited ATP-dependent duplex-unwinding activity which contributes to the helicase activity of HDH IV/nucleolin.     

Characterization of crystals of the thermostable DNA polymerase I from thermus aquaticus

Thermus aquaticus DNA polymerase I is an enzyme that is of both physiological and technological interest. It carries out template-directed polymerization of DNA at elevated temperatures and is widely used in polymerase chain reaction (PCR). We have obtained crystals of the enzyme that diffracts X-rays to at least 3.0 Å resolution in a cubic space group. Determination of the three-dimensional structure of the native enzyme along with those of relevant complexes will greatly enhance our knowledge of molecular events involved in DNA replication, will permit improvements in PCR, and will add to our knowledge of the structural bases of thermo stability in proteins. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray analysis of fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase.

Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.     

一种免受乙醇干扰的GOD的酶电极

将酶电极应用于发酵糖的测定时。与糖共存的乙醇常常会影响测糖的准确性,通过对葡萄糖氧化酶(GOD)电极的研究,探讨了乙醇对测糖酶电极测定影响,进而研制出抗干扰的GOD酶电极,若是GOD电极的酶膜通过夹心法制备．在乙醇含量为0．1％(V／V)时·即产生显著的影响,使测定结果偏大4．3%,且乙醇的影响随浓度的升高而增大,若用尼龙网固定GOD膜,GOD电极在测定20mmol／L和5mm01／L左右的葡萄糖溶液时,含量高达9％的乙醇仍未对测定产生显著的影响．表现出良好的不受乙醇干扰的特性,并且,该尼龙网GOD电极具有良好的重复性、稳定的响应活性及较长的保存期．     

Selection of a proteolytic enzyme to solubilize lean beef tissue

Many proteases are available for the hydrolysis of various protein substrates. The qualitative effect of most experimental variables on reaction progress is known, so it is possible to devise a rational procedure for selecting the best enzyme. Reaction time and enzyme concentration should be chosen in the region where they have little effect on reaction progress. Substrate concentration should be low to avoid possible product inhibition. Each enzyme should be tested at its optimum pH, and at a range of temperatures around (mainly below) the reported temperature optimum. Enzyme cost and other relevant factors should also be considered in the enzyme selection. Using this selection procedure Alcalase was chosen as the most appropriate enzyme for solubilizing lean beef tissue.     

Surface immobilization and entrapping of enzymes on glutaraldehyde crosslinked gelatin particles

A mild and reproducible method has been developed for the surface-immobilization of enzymes on glutaraldehyde crosslinked gelatin beads. In this method glutaraldehyde is used in a dual capacity, as crosslinking agent and as the enzyme coupling agent. Glucoamylase (exo-α-1,4-d-glucosidase, EC 3.2.1.3), β-d-fructofuranosidase (invertase, EC 3.2.1.26) and β-d-glucoside (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) have been successfully immobilized by this method, on the surface of the crosslinked gelatin particles. The method can be combined with the existing technology for the production of gelatin-entrapped enzymes. Thus, dual immobilized enzyme conjugates of glucoamylase and invertase have been prepared using this method, by entrapment of one enzyme in, and surface-binding of the other to, the gelatin matrix. The coupling of glucoamylase onto cross-linked gelatin particles by precipitation with poly(hexamethylenebiguanide hydrochloride) was also tested.     

Properties of thermolysin immobilized in polymer matrix by radiation polymerization

Thermolysin (Bacillus thermoproteolyticus neutral proteinase, EC 3.4.24.4) has been immobilized by radiation polymerization of hydrophilic and hydrophobic monomers, and its properties, such as enzyme activity, thermal stability and durability, have been studied. The activity of the immobilized enzymes increased with an increase in the hydrophilicity of the polymer matrix and with a decrease in monomer concentration. Immobilization with hydrophilic monomers increased the thermal stability of the enzymes, but the thermal stability of the enzymes immobilized with hydrophobic monomers was comparable with that of native enzymes. The durability of the immobilized enzymes was examined by continuous hydrolysis of casein; enzymes immobilized with a high concentration (90%) of hydrophilic monomers appeared to be stabilized and could be used for long times.     

Firefly luciferase: Purification and immobilization

The state of the art of firefly luciferase research is reviewed with special emphasis on its purification and immobilization. The notion of bioluminescence and its role in APT monitoring is described. The need to purify luciferase and the advantages of immobilization are discussed. An insight into the existing methods of luciferase purification and immobilization is given. The scope of the bioluminescent assay is underlined.     

Effect of gonadal steroids on hypothalamus and anterior pituitary endo-oligopeptidase B (proline-endopeptidase) activity in castrated female rats

In the present study we investigated the possible participation of endo-oligopeptidase B (poline-endopeptidase) in the control of gonadotrophin secretion through the control of LH-RH inactivation. This enzyme selectively hydrolyzes the Pro9-Gly10-NH2 peptide bond of LH-RH, thereby inactivating this substance. The enzyme activity was evaluated using a specific colorimetric substrate, i.e., Z-Gly-Pro-SM. Female adult Wistar rats were submitted to castration, experimental situations that are known to produce changes in gonadotrophin secretion. Hypothalamic and pituitary endo-oligopeptidase B activity was shown to be present predominantly in the soluble fraction of the enzyme preparations. The results also indicated that endo-oligopeptidase B activity adult female rat pituitary decreased after castration and increased after administration of estradiol and progesterone to castrated animals. The present results lead us to suggest that anterior pituitary endo-oligopeptidase B may be related to the control gonadotrophin secretion in female rats.