Enzyme & protein最新文献

We have applied a HPLC method to separate and quantitate UDPgalactose (UDPGal) and UDPglucose (UDPGlu) in human white blood cells (WBCs). Trichloroacetic acid-treated, protein-free filtrates were chromatographed on an anion-exchange column (Dionex CarboPac PA-1) using a gradient of 20-40% potassium phosphate buffer (pH 4.5). Recoveries of UDPGal and UDPGlu ranged from 93 to 106%, and the method was linear over a wide range of WBC protein concentrations. Volumes of blood as low as 2.5 ml (2.2 mg WBC protein) could be used to achieve quantitative recovery of the sugar nucleotides. The mean values and standard deviations of UDPGal and UDPGlu in 33 normal individuals ranging in age from 1 day to 65 years were 12.4 +/- 4.2 and 31.5 +/- 9.3 mumol/100 g protein, respectively. No statistical differences in UDPGal and UDPGlu values were observed between children and adults. No correlation was established between the concentrations of UDPGal and UDPGlu and either total WBC count or the number of lymphocytes obtained from Coulter counter analysis. There was no relationship between the concentrations of UDPGal and UDPGlu in WBCs and RBCs which were prepared from the same blood specimen.

The distributional and activity changes of hypoxanthine guanine phosphoribosyltransferase (HGPRT) were investigated in the developing mouse brain. The HGPRT activity level was low at birth, increased rapidly during the first 7 days of life, and underwent a gradual increase thereafter to the mature level. Polyclonal antibody against HGPRT purified from mouse brain was prepared for immunohistochemical demonstration of the enzyme during brain development. In the cerebellum, part of the Purkinje cells was consistently immunostained throughout growth, and the presence of HGPRT was observed in the dendrites of mature Purkinje cells. The most dominant change in HGPRT localization was observed in the hippocampus. Little HGPRT was detectable in the newborn mouse hippocampus. At postnatal day 7, cytoplasmic HGPRT appeared sporadically in the granular cells independently of the region of the hippocampus. The number of positive immunoreactive cells increased with growth, and the dendrites of granular cells were also immunostained on postnatal day 28. Further immunostaining was noted in the granule cells of the dentate gyrus on postnatal day 35. The above results suggest that HGPRT may play an important role in the developing hippocampus. Further investigations of the HGPRT in the human hippocampus may help to clarify the mechanism underlying the neurological disorders encountered in the Lesch-Nyhan syndrome.

Outer membrane (OM) proteins of beta-lactam-susceptible and -resistant strains of Pseudomonas aeruginosa were analyzed by 2-D polyacrylamide gel electrophoresis. Carrier ampholytes, pH 4-8, and immobilized pH gradient (IPG), pH 3.5-10.0, procedures were used. An acidic-protein spot (pI = 5.2) detected in susceptible but not in an imipenem-resistant strain was sequenced and twenty-five N-terminal amino acids had total homology with the OM protein D, the imipenem-specific porin of P. aeruginosa. A basic-protein spot (pI = 9.0) detected in ceftazidime-resistant, but not in a susceptible strain was sequenced and fourteen N-terminal amino acids had homology with a beta-lactamase encoded by the ampC gene of P. aeruginosa. The IPG procedure allows identification of more than one hundred proteins of the OM fraction from a single gel. Detection of beta-lactamase in OM fractions might reflect a periplasmic contamination, but its anchorage within the OM cannot be ruled out.

The kinetic constants for irreversible enzyme inhibition are determined by non-linear least-square regression using a new optimisation technique. An analysis is given of how calculation of the dissociation constant and the unimolecular rate constant is affected both by the inherent error involved in fitting an exponential curve to the plot of product concentration against time, and by the ill-conditioned nature of the equations relating these constants to the parameters of the exponential. The analysis is applied to simulated sets of product concentration curves.

The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation and has a highly conserved structure from slime molds to humans. The elongated molecule which has a molecular mass of approximately 2,000 kD is formed by a barrel-shaped 20S core complex and two polar 19S complexes. The 20S complex has C2 symmetry and is built by four seven-membered rings of which the outer rings are rotated by 26 degrees relative to the inner rings while the inner rings are in register. The 19S cap complex is asymmetric and therefore considerably less well understood on a structural level. From a comparison of the activity and regulation of the 26S and 20S particles, it can be deduced that the 20S particle contains the protease activity while the 19S complex is supposed to contain isopeptidase, oxidoreductase, ATPase and protein-unfolding activities. In this article we describe the structure of various proteasome complexes as determined by electron microscopy and discuss structural implications of their subunit sequences.

An Hinf1 associated restriction length polymorphism pattern is reported for the catalase gene of Hungarian normocatalasemic individuals and acatalasemic patients. The 2.4-kb pCAT 10 probe revealed 9 bands (2.1, 1.5, 1.2, 1.1, 0.9, 0.8, 0.6, 0.5 and 0.4 kb) with 9 distinct patterns for the controls. The same patterns were detected for the Hungarian acatalasemic patients. The examination of the A to T mutation of the Hungarian acatalasemic patients and their relatives at position -21 in the flanking region with Hinf1 polymorphism could not reveal any difference between the acatalasemic and the normocatalasemic catalase gene.

In our search for potential inhibitors of casein kinase II (CKII) in Artemia, we have shown that dinucleoside polyphosphates are a novel class of effectors for this ubiquitous protein kinase. P1,P4-di(guanosine-5')-tetraphosphate (Gp4G) is a better CKII inhibitor than P1,P4-di(adenosine-5')-tetraphosphate (Ap4A). The inhibition by both effectors is more potent when GTP is used as phosphate donor instead of ATP. The inhibition of CKII increases with the number of phosphates linking the guanosine/adenosine moieties (for n = 2-6). Ap4A does not compete with the protein substrate and causes an increase in the apparent KmATP and a decrease in the apparent VmATP, indicating a mixed type of inhibition with respect to ATP.

The behavior of the catalytic activities of the enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and glutathione reductase was tested in rat liver and kidney homogenates stored at 4, -20 and -70 degrees C and in corresponding tissue samples stored at -70 degrees C. The stabilities of enzymes were different for various enzymes and were dependent on the organ (liver, kidney) and the storage temperature. The storage temperature of -70 degrees C guaranteed the best stability and the five enzymes investigated were sufficiently stable in preserved tissue samples or in homogenates prepared with conventional mannit/sucrose homogenization solution. Under such conditions, these enzymes were stable over at least 4 or 1 weeks, respectively.

Extracts from the parenchymous leaf-gel of the Aloe vera plant (Aloe barbadensis Miller) were shown to contain glutathione peroxidase (GSHPx) activity. The activity was purified to homogeneity by ion exchange and gel filtration (FPLC) chromatography in the presence of 0.5 mM glutathione. The native enzyme has an apparent molecular weight of 62 kD as determined by gel filtration. In the presence of sodium dodecylsulfate (SDS), the molecular weight was estimated to be about 16 kD as determined by polyacrylamide-gel electrophoresis (SDS-PAGE). The native enzyme is proposed to be constituted of four identical subunits; it also contains one atom of selenium per subunit, as found with most glutathione peroxidases from animal sources. The Km values were determined to be 3.2 mM for glutathione and 0.26 mM for the hydroperoxide substrate, cumene hydroperoxide. The enzyme is competitively inhibited by N, S, bis-FMOC glutathione (Ki = 0.32 mM), a potent inhibitor of glyoxalase II. Inhibitors of glyoxalase I (e.g. S-octylglutathione) have no effect on the peroxidase activity.

Guinea pigs were given a single intraperitoneal injection of 1.35 mg/kg body weight of mercuric chloride; then various kidney enzymes and extracellular DNA were assayed in urine. Dramatic increases of all studied markers were observed on the first day following treatment. Sequential collection of urines allowed for kinetic studies: membrane markers alkaline phosphatase and gamma-glutamyltransferase were first released, then cytosolic lactate dehydrogenase and mitochondrial glutamate dehydrogenase, finally extracellular DNA; DNA release is equated with cell death. The features of kidney damage revealed by comparative and quantitative studies of these noninvasive markers suggest that brush border erasure was more extensive than cell necrosis.