Summary Four antifreeze proteins (AFPs) were purified from larvae of the beetle Dendroides canadensis. The AFPs are similar in amino acid compositions, having high contents of hydrophilic amino acids (45–55 mol%) and cysteine (16 mol% Cys). Approximately half of the Cys residues form disulfide bridges, and both the disulfide bridges and free sulfhydryls are essential for activity. The N-terminals of the AFPs are blocked. The pH optimum of the AFPs is 7.8, but major loss of activity occurred only at very high pH (12.0). The detergents SDS and Triton X-100 did not inactivate the AFPs. Circular dichroism spectra indicate the presence of both and secondary structures in the AFPs, in addition to a large random structure component.Abbreviations
AFP
antifreeze protein
-
CD
circular dichroism
-
DTT
dithiothreitol
-
HPLC
high pressure liquid chromatography
-
PAGE
polyacrylamide gel electrophoresis
-
PAS
periodic acid Schiff
-
SDS
sodium dodecyl sulfate
-
TFA
trifluoroacetic acid 相似文献
We have further characterized the 5-HT3 receptors in rat and rabbit tissues by evaluating the binding of the 5-HT3 receptor ligand, [3H]GR67330 to homogenates of rabbit ileum, rat ileum and rat brain (entorhinal cortex). In each tissue specific [3H]GR67330 binding represented a single saturable, high affinity site (Kd = 0.14, 0.18, 0.076 nM in rabbit ileum, rat ileum and rat brain respectively). The densities of sites present in rabbit and rat ileum were similar to that present in rat brain (Bmax = 63, 47, 72 fmol/mg protein in rabbit ileum, rat ileum and rat brain respectively).
In each tissue, 5-HT3 receptor agonists and antagonists potently competed for [3H]GR67330 binding. Derived inhibition constants were similar in rat ileum and brain. However marked differences in IC50s were apparent for rabbit ileum compared with rat brain or ileum. These were most apparent with agonists. Thus, mCPBG [1-(meta-chlorophenylbiguanide)], phenylbiguanide, 5-HT and 2-methyl 5-HT were at least 5 times less potent to inhibit [3H]GR67330 binding in rabbit ileum than rat brain. The most pronounced differences were evident with phenylbiguanide and mCPBG which were 70 and 300 times less potent in the rabbit ileum respectively compared with the rat tissues. These differences were unlikely to be due to depletion effects because tissue combination experiments (rabbit ileum and rat brain) yielded biphasic inhibition curves for phenylbiguanide with affinities for each component similar to those in the individual tissues. Antagonist affinities also varied between the rabbit and rat tissues, although less markedly. Amongst the antagonists, the most marked differences were apparent with SDZ 206–830 and quipazine each being 10 times less potent to inhibit binding to rabbit than rat tissue.
Hill coefficients for inhibition of binding varied with tissue. In rat brain, as previously described for [3H]GR67330, Hill coefficients for agonist (and quipazine) inhibition of binding were greater than unity. This was less marked in rat and rabbit ileum tissues.
The present studies provide further evidence for species variation in 5-HT3 receptors. 相似文献
The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately expressed: (i) unlike TIS11, the TIS11b and TIS11d mRNAs are detectable in quiescent Swiss 3T3 cells and are not dramatically induced by 12-O-tetradecanoylphorbol-13-acetate; (ii) cycloheximide superinduction does not occur for TIS11b and TIS11d; and (iii) unlike TIS11, TIS11b expression is extinguished in PC12 pheochromocytoma cells. 相似文献
A human subtelomeric repeat (designated as the HST repeat) has been isolated and characterized from a yeast artificial chromosome containing one human telomere. This repeat is located immediately adjacent to the telomeric T2AG3 repeats at the extreme termini of the human chromosomes. The DNA sequence of 3.6 kb of the HST repeat has been determined. The HST repeat spans over 3.6 kb in length, and contains one evolutionarily conserved CpG-rich region. The copy number of the HST repeat varies among telomeres. Genomic hybridization experiments suggest that the HST repeat consists of two distinct segments, and the distal portions of the HST repeat are also distributed elsewhere in the genome. In HeLa cells, the HST repeat sequence appears to be transcribed into a 6 kb polyadenylated RNA and a variety of non-polyadenylated RNA species. 相似文献
Isolated ovarian corpus luteal cells and granulosa cells of rat were employed to investigate the effect of alpha-ANP on the secretion of progesterone and estradiol. The contents of the steroid hormones are determined by RIA. The results showed that 0.1-10 ng/ml ANP promoted progesterone production in a dose dependent manner. alpha-ANP also enhanced progesterone production by granulosa cells, but not estradiol. It seems that the effect of alpha-ANP on ovarian steroidogenesis is a direct one. 相似文献
We have previously characterized a cellular thyroid hormone-binding protein (p55) that is found concentrated on the lumenal face of the endoplasmic reticulum and nuclear envelope (Cheng, S.-y., Hasumura, S., Willingham, M.C., and Pastan, I. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 947-951). To understand the role p55 plays in thyroid hormone action, we examined the regulation of p55 by 3,3',5-triiodo-L-thyronine (T3). Rat pituitary tumor GH3 cells cultured in regular medium, thyroid hormone-depleted medium (Td medium), or Td medium supplemented with 50 nM T3 (Td + T3 medium) were metabolically labeled with [35S]methionine and immunoprecipitated with antibodies against p55. Treatment with T3 caused a fall in p55 levels. Poly(A+) RNA from cells cultured in regular, Td, or Td + T3 medium was hybridized to a cDNA from p55. T3 withdrawal or addition had no effect on p55 mRNA levels. Furthermore, the initial rates of synthesis of p55 from cells cultured in regular, Td, and Td + T3 were found to be similar. However, analysis of the decay curves from cells in which p55 was pulse-labeled with [35S]methionine indicated that p55 is 2-fold less stable in T3 containing medium. These results indicated that down-regulation of p55 by T3 occurs at the post-translational level. Since DNA sequence analysis indicates that p55 is identical to protein disulfide isomerase and the beta-subunit of prolyl-4-hydroxylase, T3 may mediate its effects on the synthesis, secretion, and/or transport of proteins via p55. 相似文献
Incubation of [1-13C]-5-phosphoribosyl pyrophosphate ([1-13C]PRPP) and glutamine with PRPP amidotransferase results in rapid production and disappearance of two new resonances at 89.3 and 85.9 ppm. These resonances coincide with two of the products produced upon incubation of [1-13C]ribose 5-phosphate with NH3. Extensive NMR studies (15N and 1H-13C chemical shift correlation spectra) have allowed assignment of these resonances to beta- and alpha-phosphoribosylamine. These studies represent the first spectral observations of this chemically reactive intermediate. The rate of interconversion of alpha- to beta-phosphoribosylamine as a function of pH has been determined by saturation and inversion-transfer NMR methods. The rate of formation of 5-phosphoribosylamine (PRA) from ribose 5-phosphate and NH3 and its rate of decomposition as a function of pH have been determined with a glycinamide ribonucleotide synthetase trapping system fashioned after earlier studies of Nierlich and Magasanik [Nierlich, D. P., & Magasanik, B. (1965) J. Biol. Chem. 240, 366]. Phosphoribosylamine has a t1/2 = 38 s at 37 degrees C and pH 7.5. The pH-independent equilibrium constant for ribose 5-phosphate and NH3 with phosphoribosylamine has been established, 2.5 M-1, by use of these rate constants as well as by NMR methods. This equilibrium constant and the rates of nonenzymatic interconversion of alpha- and beta-PRA provide essential background for studying the mechanism of glycinamide ribonucleotide synthetase and investigating the possibility of channeling phosphoribosylamine between this enzyme and the first enzyme in the purine pathway. 相似文献
The standard model of epithelial cell renewal in the intestine proposes a gradual transition between the region of the crypt containing actively proliferating cells and that containing solely terminally differentiating cells (Cairnie, Lamerton and Steel, 1965 a, b). The experimental justification for this conclusion was the gradual decrease towards the crypt top of the measured labeling and mitotic indices. Recently, however, we have proposed that intestinal crypts normally undergo a replicative cycle so that at any time in any region of the intestine, crypts will be found to have a wide range of sizes. We show here that if this intrinsic size variation is taken into account, then a sharp transition between the proliferative and nonproliferative compartments of individual intestinal crypts is consistent with the labeling and mitotic index distributions of mouse and rat jejunal crypts. Thus there is no need to invoke the region of gradual transition from proliferating to nonproliferating cells as is done in the standard model. The position of this sharp transition is estimated for both the mouse and rat. Experiments to further test our model are suggested and the significance of the results discussed. 相似文献
Herpes simplex virus type I (HSV-I)-induced thymidine kinase has been shown to catalyze phosphoryl transfer from adenosine 5'-[gamma-(S)-16O,17O,18O]triphosphate to thymidine with inversion of configuration at phosphorus. The simplest interpretation of this result is that phosphoryl transfer occurs by a single in-line group transfer between ATP and thymidine within the ternary enzyme complex. 相似文献