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1.
The structures of the colicin Ib plasmid (ColIb), the delta transfer factor and a plasmid determining kanamycin resistance and colicin Ib production called KColIb, were compared. Radiolabelled mini-ColIb plasmids and isolated DNA fragments of ColIb were used as probes for nitrocellulose blots of digests of the other two large plasmids. The structure of delta was consistent with it having one large deletion of about 10 MDa in the SB fragment and two insertions of approximately 6 MDa and 12 MDa in the SB and SA fragments of the ColIb plasmid. It was hypothesized that KColIb had six small insertions in SA, SB, SE and near the junction of the SB and SD fragments. However, ColIb, KColIb and delta were homologous for at least 70% of their lengths. The highly conserved regions in the three plasmids were the regions that corresponded to fragments SA, SC and SD of ColIb. In addition, delta and KColIb differed from ColIb at similar sites. The possible evolution of these plasmids is discussed. 相似文献
2.
The developing rat placenta expresses two placental lactogens at different stages of pregnancy: rat placental lactogen I from Days 11 to 13 of pregnancy and rat placental lactogen II (rPLII) from Day 12 to term. In this paper, we describe cDNA clones for rPLII, which have been isolated from a Day 18 rat placental cDNA library. The rPLII clones hybrid-select a mRNA which translates in vitro to a protein of 25,000 daltons. This protein is processed by dog pancreatic microsomes to a 22,000-dalton form, identical in size to rPLII isolated from pregnant rat serum. Both forms are precipitated by an anti-rPLII antiserum and an anti-ovine prolactin antiserum. The mRNA for rPLII is first expressed in Day 12 placenta and reaches a maximum at about Day 18 of pregnancy, in parallel with the appearance of the hormone in serum. Sequencing of the cDNA shows that, unlike human placental lactogen which is 85% homologous to human growth hormone at the amino acid level, rPLII is much more closely related to the prolactins. Thus, rPLII is 52% homologous to rat prolactin at the amino acid level, but only 34% related to rat growth hormone. This is the second placental lactogen to be fully characterized, and in the rat this hormone appears to have evolved by a route quite different from that which produced placental lactogen in humans. 相似文献
3.
Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels 下载免费PDF全文
An inhibitor of malted barley (Hordeum vulgare cv Conquest) α-amylase II was purified 125-fold from a crude extract of barley kernels by (NH4)2SO4 fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a protein with an approximate molecular weight of 20,000 daltons and an isoelectric point of 7.3. The protein was homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated the presence of about 9 half-cystine residues per mole. The neutral isoelectric point of the inhibitor suggested that some of the apparently acidic residues (glutamic and aspartic) existed in the amide form. The first twenty N-terminal amino acids were sequenced. Some homology appeared to exist between the α-amylase II inhibitor and trypsin inhibitor from barley. Complex formation between α-amylase II and the inhibitor was detected by the appearance of a new molecular weight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitor had to be preincubated for 5 min, prior to assaying for enzyme activity before maximum inhibition was attained. Inhibition increased at higher pH values. At pH 5.5, an approximately 1100 molar excess of inhibitor over α-amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1 molar ratio of inhibitor to enzyme produced the same degree of inhibition. 相似文献
4.
Preparation and properties of pure, full-length IclR protein of Escherichia coli. Use of time-of-flight mass spectrometry to investigate the problems encountered. 下载免费PDF全文
L. J. Donald I. V. Chernushevich J. Zhou A. Verentchikov N. Poppe-Schriemer D. J. Hosfield J. B. Westmore W. Ens H. W. Duckworth K. G. Standing 《Protein science : a publication of the Protein Society》1996,5(8):1613-1624
IclR protein, the repressor of the aceBAK operon of Escherichia coli, has been examined by time-of-flight mass spectrometry, with ionization by matrix assisted laser desorption or by electrospray. The purified protein was found to have a smaller mass than that predicted from the base sequence of the cloned iclR gene. Additional measurements were made on mixtures of peptides derived from IclR by treatment with trypsin and cyanogen bromide. They showed that the amino acid sequence is that predicted from the gene sequence, except that the protein has suffered truncation by removal of the N-terminal eight or, in some cases, nine amino acid residues. The peptide bond whose hydrolysis would remove eight residues is a typical target for the E. coli protease OmpT. We find that, by taking precautions to minimize Omp T proteolysis, or by eliminating it through mutation of the host strain, we can isolate full-length IclR protein (lacking only the N-terminal methionine residue). Full-length IclR is a much better DNA-binding protein than the truncated versions: it binds the aceBAK operator sequence 44-fold more tightly, presumably because of additional contacts that the N-terminal residues make with the DNA. Our experience thus demonstrates the advantages of using mass spectrometry to characterize newly purified proteins produced from cloned genes, especially where proteolysis or other covalent modification is a concern. This technique gives mass spectra from complex peptide mixtures that can be analyzed completely, without any fractionation of the mixtures, by reference to the amino acid sequence inferred from the base sequence of the cloned gene. 相似文献
5.
Vanadium (V) is an orally effective treatment for diabetes, but relatively little is known about the mechanisms controlling its normal metabolism nor the long term pharmacokinetics of oral administration. We have examined the accumulation of V in various organs from rats fed liquid diet for up to 18 days, containing no additional V, 1.6, 80, or 160 mole/kg/day as either sodium orthovanadate (SOV) or vanadyl sulfate (VS). V content was assayed using a sensitive neutron activation analysis method. The organs of the nonsupplemented animals contained widely varying concentrations (ng of V/g dry tissue weight) with brain < fat < blood < heart < muscle < lung < liver < testes < spleen < kidney. All organs accumulated V in a dose dependent manner. Not all organs showed steady state amount of V at 18 days, so additional rats were fed SOV or VS, switched to control diet, and assayed at 0, 4 and 8 days. From this data we calculated organ half lives of V. Insulin sensitive tissue tissues, such as liver and fat, had shorter half-lives than tissues that are relatively less insulin sensitive, such as spleen, brain and testes. SOV and VS fed rats showed similar patterns, but VS had somewhat shorter t1/2's. Additional studies of old and young rats fed control diet for 45 days show accumulation of V in spleen and testes. These results indicate that vanadium metabolism varies widely among different organs, and that insulin, either directly or indirectly has effects on the retention of vanadium. This may have impact on the therapeutic use of vanadium in Type I diabetics with no insulin, or Type II patients who may be relatively hyperinsulinemic. 相似文献
6.
In a series of 21 human fetal thyroid glands examined histologically in serial sections, seven ultimobranchial body cysts were found. The position of these cysts correlated well with the distribution of calcitonin-containing cells found by previous investigators in the adult thyroid gland. Ultimobranchial body cysts found external to the thyroid lobes may offer a developmental explanation for the paucity of calcitonin found in some adult thyroid glands. The close developmental relationship between the parathyroid gland and the ultimobranchial body could explain the presence of calcitonin found in these glands in some adults. 相似文献
7.
Microfilariae of Onchocerca sweetae are broadly distributed in the superficial layers of the dermis of the water buffalo (Bubalus bubalis). A total of 2855 insects representing 20 species were collected from O. sweetae-infected bait buffaloes. Only one species, Culicoides sp. "M", ingested microfilariae from buffalo skin. Larval development of O. sweetae was observed in the thorax of this species. Atotal of 829 insects, representing 7 species and including 749 parous Culicoides spp. were collected from light and Manitoba traps. Developing filarioid larvae were observed only in Culicoides sp. "M". It is concluded that Culicoides sp. "M" is a natural intermediate host of O. sweetae in the Northern Territory of Australia. 相似文献
8.
9.
W C Duckworth F G Hamel R Bennett M P Ryan R A Roth 《The Journal of biological chemistry》1990,265(5):2984-2987
The mechanisms of cellular insulin degradation remain uncertain. Considerable evidence now exists that the primary cellular insulin-degrading activity is a metallothiol proteinase. Two similar degrading activities have been purified and characterized. Insulin protease has been purified from rat skeletal muscle and insulin-degrading enzyme from human red blood cells. Whereas the two degrading activities share a number of similar properties, significant differences have also been reported; and it is not at all established that they are the same enzyme. To examine this, we have compared antigenic and catalytic properties of the two enzymatic activities. Monoclonal antibodies against the red blood cell enzyme adsorb the skeletal muscle enzyme; and on Western blots, the antibodies react with an identical 110-kDa protein. Immunoaffinity-purified enzymes from both red blood cells and skeletal muscle degrade [125I]iodo(B26)insulin to the same products as seen with purified insulin protease and with intact liver and kidney. Chelator-treated muscle and red blood cell enzymes can be reactivated with either Mn2+ or Ca2+. Thus, insulin-degrading enzyme and insulin protease have similar properties. These results support the hypothesis that these activities reside in the same enzyme. 相似文献
10.