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55.
Forbidden synonymous substitutions in coding regions 总被引:2,自引:0,他引:2
In the evolution of highly conserved genes, a few "synonymous"
substitutions at third bases that would not alter the protein sequence are
forbidden or very rare, presumably as a result of functional requirements
of the gene or the messenger RNA. Another 10% or 20% of codons are
significantly less variable by synonymous substitution than are the
majority of codons. The changes that occur at the majority of third bases
are subject to codon usage restrictions. These usage restrictions control
sequence similarities between very distant genes. For example, 70% of third
bases are identical in calmodulin genes of man and trypanosome. Third-base
similarities of distant genes for conserved proteins are mathematically
predicted, on the basis of the G+C composition of third bases. These
observations indicate the need for reexamination of methods used to
calculate synonymous substitutions.
相似文献
56.
Human alpha-galactosidase A (alpha-Gal A) is the lysosomal glycohydrolase
that cleaves the terminal alpha-galactosyl moieties of various
glycoconjugates. Overexpression of the enzyme in Chinese hamster ovary
(CHO) cells results in high intracellular enzyme accumulation and the
selective secretion of active enzyme. Structural analysis of the N -linked
oligosaccharides of the intracellular and secreted glycoforms revealed that
the secreted enzyme's oligosaccharides were remarkably heterogeneous,
having high mannose (63%), complex (30%), and hybrid (5%) structures. The
major high mannose oligosaccharides were Man5-7GlcNAc2 species.
Approximately 40% of the high mannose and 30% of the hybrid
oligosaccharides had phosphate monoester groups. The complex
oligosaccharides were mono-, bi- , 2,4-tri-, 2,6-tri- and tetraantennary
with or without core-region fucose, many of which had incomplete outer
chains. Approximately 30% of the complex oligosaccharides were mono- or
disialylated. Sialic acids were mostly N -acetylneuraminic acid and
occurred exclusively in alpha2, 3-linkage. In contrast, the intracellular
enzyme had only small amounts of complex chains (7.7%) and had
predominantly high mannose oligosaccharides (92%), mostly Man5GlcNAc2 and
smaller species, of which only 3% were phosphorylated. The complex
oligosaccharides were fucosylated and had the same antennary structures as
the secreted enzyme. Although most had mature outer chains, none were
sialylated. Thus, the overexpression of human alpha-Gal A in CHO cells
resulted in different oligosaccharide structures on the secreted and
intracellular glycoforms, the highly heterogeneous secreted forms
presumably due to the high level expression and impaired glycosylation in
the trans- Golgi network, and the predominately Man5-7GlcNAc2 cellular
glycoforms resulting from carbohydrate trimming in the lysosome.
相似文献
57.
R J Kulmacz 《The Journal of biological chemistry》1989,264(24):14136-14144
Prostaglandin H synthase catalyzes two reactions: the bis-dioxygenation of arachidonic acid to form prostaglandin G2 (cyclooxygenase activity), and the reduction of hydroperoxides to the corresponding alcohols (peroxidase activity). The cyclooxygenase activity can be selectively inhibited by many nonsteroidal antiinflammatory agents including indomethacin. In the native synthase, there is a single prominent protease-sensitive region, located near Arg253; binding of the heme prosthetic group makes the synthase resistant to proteases. To investigate the spatial relationship between the area of the synthase which interacts with indomethacin and the protease-sensitive region, the effects of indomethacin and similar agents on the protease sensitivity of the two enzymatic activities and of the synthase polypeptide were examined. Incubation of the synthase apoenzyme with trypsin (3.6% w/w) resulted in the time-dependent coordinate loss (75% at 1 h) of both enzymatic activities and the cleavage (85% at 1 h) of the 70-kDa subunit into 38- and 33-kDa fragments, indicating that proteolytic cleavage of the polypeptide at Arg253, destroyed both activities of the synthase simultaneously. Indomethacin, (S)-flurbiprofen, or meclofenamate (each at 20 microM) rendered both activities and the synthase polypeptide (at 5 microM subunit) resistant to attack by trypsin or proteinase K; these agents also inhibited the cyclooxygenase activity of the intact synthase. Two reversible cyclooxygenase inhibitors, ibuprofen and flufenamate, also made both of the activities and the synthase polypeptide more resistant to trypsin. Titration of the apoenzyme with indomethacin (0-3 mol/mol of synthase dimer) resulted in proportional increases in the inhibition of the cyclooxygenase and in the resistance to attack by trypsin. (R)-Flurbiprofen did not increase the resistance to protease or appreciably inhibit the cyclooxygenase. These results suggest that the same stereospecific interaction of these agents with the synthase that produced inhibition of the cyclooxygenase led to a decreased accessibility of the Arg253 region to proteases. Aspirin treatment made the synthase less resistant to trypsin; aspirin-treated synthase became more resistant to trypsin when it was incubated with indomethacin before addition of the protease. The presence of 50 microM arachidonate during digestion of apoenzyme or aspirin-treated apoenzyme with trypsin did not decrease the cleavage of the synthase subunit.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
58.
Hydroperoxides are known to induce the formation of tyrosyl free radicals in prostaglandin (PG) H synthase. To evaluate the role of these radicals in cyclooxygenase catalysis we have analyzed the temporal correlation between radical formation and substrate conversion during reaction of the synthase with arachidonic acid. PGH synthase reacted with equimolar levels of arachidonic acid generated sequentially the wide doublet (34 G peak-to-trough) and wide singlet (32 G peak-to-trough) tyrosyl radical signals previously reported for reaction with hydroperoxide. The kinetics of formation and decay of the doublet signal corresponded reasonably well with those of cyclooxygenase activity. However, the wide singlet free radical signal accumulated only after prostaglandin formation had ceased, indicating that the wide singlet is not likely to be an intermediate in cyclooxygenase catalysis. When PGH synthase was reacted with 25 equivalents of arachidonic acid, the wide doublet and wide singlet radical signals were not observed. Instead, a narrower singlet (24 G peak-to-trough) tyrosyl radical was generated, similar to that found upon reaction of indomethacin-treated synthase with hydroperoxide. Only about 11 mol of prostaglandin were formed per mol of synthase before complete self-inactivation of the cyclooxygenase, far less than the 170 mol/mol synthase produced under standard assay conditions. Phenol (0.5 mM) increased the extent of cyclooxygenase reaction by only about 50%, in contrast to the 460% stimulation seen under standard assay conditions. These results indicate that the narrow singlet tyrosyl radical observed in the reaction with high levels of arachidonate in this study and by Lassmann et al. (Lassmann, G., Odenwaller, R., Curtis, J.F., DeGray, J.A., Mason, R.P., Marnett, L.J., and Eling, T.E. (1991) J. Biol. Chem. 266, 20045-20055) is associated with abnormal cyclooxygenase activity and is probably nonphysiological. In titrations of the synthase with arachidonate or with hydroperoxide, the loss of enzyme activity and destruction of heme were linear functions of the amount of titrant added. Complete inactivation of cyclooxygenase activity was found at about 10 mol of arachidonate, ethyl hydrogen peroxide, or hydrogen peroxide per mol of synthase heme; maximal bleaching of the heme Soret absorbance peak was found with 10 mol of ethyl hydroperoxide or 20 mol of either arachidonate or hydrogen peroxide per mol of synthase heme. The peak concentration of the wide doublet tyrosyl radical did not change appreciably with increased levels of ethyl hydroperoxide. In contrast, higher levels of hydroperoxide gave higher levels of the wide singlet radical species, in parallel with enzyme inactivation.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
59.
Richard J. Kulmacz Alemka Kisic George J. Schroepfer 《Chemistry and physics of lipids》1979,23(4):291-319
The following compounds were prepared by chemical synthesis from tribenzoylsphingosine: (2S,3S,4S)-2-acetamido-1,3,4-trihydroxyoctadecane, (2S,3S,4R)-2-acetamido-1,3,4-triydroxyoctadecane, (2S,3S,5S)-2-acetamido-1,3,5-trihydroxyoctadecane, and (2S,3S,5R)-2-acetamido-1,3,5-trihydroxyoctadecane. These compounds were characterized by melting point determination, low and high resolution mass spectra, infrared, optical rotation, chromatographic, and chemical degradation studies. In addition, each of the compounds was converted to the corresponding free base and N-benzoyl derivative. (2S,3S,4R)-2-Benzylamino-1,3,4-trihydroxyoctadecane was prepared from the N-benzoyl derivative of authentic phytosphingosine. 相似文献
60.
Liu W Kamensky Y Kakkar R Foley E Kulmacz RJ Palmer G 《Protein expression and purification》2005,40(2):429-439
Bovine adrenal chromaffin granule cytochrome (cyt) b561 is a transmembrane hemoprotein that plays a key role in transporting reducing equivalents from ascorbate to dopamine-beta-hydroxylase for catecholamine synthesis. We have developed procedures for expression and purification of functional bovine adrenal cyt b561 in insect and yeast cell systems. The bovine cyt b561 coding sequence, with or without a hexahistidine-tag sequence at the C-terminus, was cloned into the pVL1392 transfer vector under the control of the polyhedrin promoter to generate recombinant baculovirus for protein expression in Sf9 insect cells (approximately 0.5 mg detergent-solubilized cyt b561/L culture). For the yeast system, the cyt b561 cDNA was modified with a hexahistidine-tag sequence at the C-terminus, and inserted into the pPICZB vector under the control of the alcohol oxidase promoter. The recombinant plasmid was transformed into Pichia pastoris GS115 competent cells to give methanol-inducible cyt b561 expression (approximately 0.7 mg detergent-solubilized cyt b561/L culture). Recombinant His-tagged cyt b561 expressed in Sf9 or Pichia cells was readily solubilized from membrane fractions with dodecyl maltoside and purified to electrophoretic homogeneity by one-step chromatography on Ni-NTA affinity resin. The purified recombinant cytochrome from both systems had a heme to protein ratio close to two and was fully functional, as judged by comparison with the spectroscopic and kinetic parameters of the endogenous cytochrome from chromaffin granules. A novel procedure for isolation of chromaffin granule membranes was developed to utilize frozen adrenal glands instead of fresh tissue. 相似文献