共查询到20条相似文献,搜索用时 31 毫秒
1.
Molecular forms of immunoactive atrial natriuretic peptide in the rat hypothalamus and atrium 总被引:3,自引:0,他引:3
C C Glembotski G M Wildey T R Gibson 《Biochemical and biophysical research communications》1985,129(3):671-678
Acid extracts of rat hypothalamus and atrium were prepared by a procedure previously shown to minimize proteolytic degradation of peptides. The majority of the immunoactive material in the atrial extracts had a molecular weight of approximately 9,000 to 15,000 daltons, while that in the hypothalamic extracts had a molecular weight of about 1,500 to 1,800 daltons. The major molecular weight forms of atrial natriuretic peptide from each extract were further distinguishable when analyzed by RP-HPLC. These results suggest that small peptides such as atriopeptins I, II, and III, may not be authentic post-translational processing products in the atrium, and that the hypothalamus and atrium may differentially cleave pro-atrial natriuretic peptide to form tissue-specific products. 相似文献
2.
In addition to reducing the analysis time, the direct examination of proteolytic digests by fast atom bombardment mass spectrometry (FABMS) greatly extends the information that is available from peptide mapping experiments. Mass spectral data are particularly useful for identifying post-translationally modified peptides. For example, the molecular weight of a disulfide-containing peptide may be used to locate the disulfide bond in the protein from which the peptide was derived. This paper describes a new procedure, which is useful for identifying disulfide-bonded peptides. Peptides are treated with performic acid to modify certain residues and thereby cause a characteristic change in the peptide molecular weight. This change in molecular weight is determined by FABMS and used to help identify peptides. Results for a series of small peptides demonstrate that Cys, Met, and Trp are the only residues that undergo a change in molecular weight under the conditions used here. Furthermore, these changes in molecular weight are diagnostic for each of the residues. Cysteinyl-containing peptides are of particular interest, because their identification is essential for locating disulfide bonds. The molecular weight of a peptide increases by 48 mu for each cysteinyl residue present. This approach is used to identify peptides that contain both cysteinyl and cystinyl residues in the peptic digest of bovine insulin. The method is extended to the analysis of a tryptic digest of cyanogen bromide-treated ribonuclease A. A computer-assisted analysis procedure is used to demonstrate the specificity with which peptide molecular weight is related to specific segments of the protein.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
3.
An ultramicroprocedure is described for determining picomole concentrations of small peptides and amino acids in microliter quantities of plasma. A micro Cu-Sephadex G-25 column is used to separate the small molecular weight peptides from the amino acids. The procedure is relatively simple, rapid, reliable, accurate, and very sensitive. The limits of sensitivity depend upon the specific activity of the dipeptides or amino acids used; increasing the specific activity gives greater sensitivity. 相似文献
4.
The application of an ultrafiltration procedure for fractionation of molecular weight of dissolved organic matter (DOM) extracellularly released by phytoplankton is described. Seven ultrafiltration membranes Diaflo (Amicon Corp. USA), with range 500--300,000 molecular weight (MW), were used for separation of different molecular weight compounds released by phytoplankton in Rhode River estuary of Chesapeake Bay, and their composition was determined. Percentage of extracellular release of DOM by phytoplankton varied from 3.92--68.07% of total carbon fixed in photosynthesis. The composition of algal extracellular products varied with different phytoplankton populations. However, two fractions of molecular weight compounds dominated in the composition of DOM released, i.e. a low molecular weight fraction of less than 1,000 MW and the fraction between 10,000--30,000 MW. The ultrafiltration procedure is effective for studying the composition of DOM released by phytoplankton in natural waters. 相似文献
5.
A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from 1000-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns allowed a reliable molecular weight determination in the range from 800 to 3500. 相似文献
6.
Further Characterization of an Enkephalin-Generating Enzyme from Adrenal Medullary Chromaffin Granules 总被引:8,自引:5,他引:3
An adrenomedullary protease capable of generating Met5-enkephalin from endogenous precursor(s) has been purified 1,000-fold using affinity chromatography in combination with gel filtration. This trypsin-like enzyme has an apparent molecular weight of 20,000 daltons by gel filtration. The reactivity of the enzyme toward several fluorogenic peptides, Peptides E and F, and the heptapeptides, Met5-enkephalin-Arg6-Phe7 and Met5-enkephalin-Arg6-Arg7, was examined. The two heptapeptides and the fluorogenic compounds were poor substrates for the adrenal enzyme; in contrast, Peptides E and F were cleaved. The low molecular weight products of Peptide F digestion were identified by HPLC as Arg1-Met6-enkephalin, Met5-enkephalin, and Met5-enkephalin-Lys6, while digestion of Peptide E resulted in the production of Leu5-enkephalin and Met5-enkephalin-Arg6-Arg7. [3H]-beta m-Lipotropin was not hydrolyzed by the adrenal enzyme. These results indicate that this adreno-medullary protease is capable of cleaving adrenal opioid peptides at the paired basic sites and thus represents a possible candidate for a proenkephalin-converting enzyme. 相似文献
7.
Amino acid sequence analysis of bitter peptides from a soybean proglycinin subunit synthesized in Escherichia coli 总被引:1,自引:0,他引:1
Kim MR Choi SY Kim CS Kim CW Utsumi S Lee CH 《Bioscience, biotechnology, and biochemistry》1999,63(12):2069-2074
The cDNA encoding A1aB1b proglycinin was expressed in E. coli, for the efficient isolation of a single peptide responsible for the bitterness. The 55-kD proglycinin was highly purified, hydrolyzed, and further purified through a series of chromatographic steps to yield fractions with the major bitter peptides. The most bitter-tasting fractions contained peptides with average molecular weights lower than 1,700 Da. An analysis of the amino acid sequences indicated that many small bitter peptides (< 1,000 Da) are composed of uncharged polar amino acids as well as hydrophobic amino acids, with a charged residue often being present at either end. This suggests the involvement of a certain structural requirement in taste perception. 相似文献
8.
A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond). 相似文献
9.
A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond). 相似文献
10.
There was a rapid transfer of radioactive peptides to other lipoprotein fractions during the first 30 min after the intravenous injection of 125I-labeled rat very low density lipoprotein (VLDL) into rats. After this initial redistribution of radioactivity, label disappeared slowly from all lipoprotein fractions. The disappearance of 125I-labeled human VLDL injected into rats was the same as that of rat VLDL. Most of the radioactivity transferred from VLDL to low density (LDL) and high density (HDL) lipoproteins was associated with two peptides, identified in these studies by polyacrylamide gel electrophoresis as zone IVa and IVb peptides (fast-migrating peptides, possibly analogous to some human C apolipoproteins), although radioactivity initially associated with zone I (analogous to human apolipoprotein B) and zone III (not characterized) was also transferred to LDL and HDL. That the transfer of label from VLDL to LDL and HDL primarily involved small molecular weight peptides was confirmed in studies using VLDL predominantly labeled in these peptides by in vitro transfer from 125I-labeled HDL. Both zone I and zone IV radioactivity was rapidly removed from VLDL during the first 5 min after injection. However, although most of the zone IV radioactivity was recovered in LDL and HDL, only 12% of the label lost from zone I of VLDL was recovered in other lipoproteins, with the remainder presumably having been cleared from the plasma compartment. We have concluded that, during catabolism of rat VLDL apoprotein, there is a rapid transfer of small molecular weight peptides to both LDL and HDL. During the catabolic process, most of the VLDL is rapidly removed from the circulation, with only a small portion being transformed into LDL molecules. 相似文献
11.
N D Light 《Biochimica et biophysica acta》1979,581(1):96-105
The cyanogen bromide peptides from insoluble and pepsin solubilised type I collagen of bovine bone, dentine, meniscus, tendon, skin and cornea were compared by SDS-polyacrylamide gel electrophoresis. In each case alpha 1CB6 was shown to be the only peptide of molecular weight greater than 10 000 involved in cross-linking. The major helical peptides alpha 1CB3, alpha 1CB8, alpha 1CB7 and alpha 2CB4 were not implicated in cross-linking in any tissue either by end overlap or helix-helix interaction. The C-terminal alpha 2 chain peptide alpha 2CB3,5, which contains a large helical region, was not involved in cross-linking to any large peptides, although a slight increase in molecular weight in all tissues examined did suggest a possible interaction(s) with a very small peptide of molecular weight 4--5000. 相似文献
12.
J Gutkowska K Racz W Debinski G Thibault R Garcia O Kuchel M Cantin J Genest 《Peptides》1987,8(3):461-465
An atrial natriuretic factor-(ANF) like immunoreactivity (IR-ANF), is present in the posterior hypophysis of the rat. In order to obtain more direct information on the presence and biological activity of this new posterior hypophysis peptide, we applied a procedure similar to that described for rat atria, to extract an ANF-like material from the posterior hypophysis of the rat. An analysis of the tissue extracts by reverse-phase high performance liquid chromatography (RP-HPLC) suggested that, in this organ, the ANF-like peptides may be present in multiple forms: a low molecular weight peptide which had a RP-HPLC pattern similar to that of the synthetic rat 28 amino acid C-terminal (Ser 99-Tyr 126) ANF, and an unidentified higher molecular weight peptide. The partially purified low molecular weight peptide was found to have a potency similar to that of synthetic rat ANF in the inhibition of adrenocorticotropin-stimulated aldosterone secretion in dispersed zona glomerulosa cells, suggesting that the ANF-like peptide was biologically active. Immunohistochemical visualization of the ANF-like peptides revealed the distribution of the peptide within the posterior hypophysis. There was no immunohistochemical staining for ANF in the intermediate lobe. These results suggest the existence of biologically active ANF-like peptides within the posterior hypophysis of the rat. It is possible that these peptides may modulate locally the posterior hypophysis hormone secretion. 相似文献
13.
L J Fretto E W Ferguson H M Steinman P A McKee 《The Journal of biological chemistry》1978,253(7):2184-2195
The potential cross-link acceptor sites of fibrin were specifically labeled with the fluorescent, substitute cross-link donor monodansyl cadaverine (MDC). Several fluorescent alpha-chain peptides generated from enzymatic and cyanogen bromide (CNBr) cleavage of the labeled fibrin were identified by sodium dodecyl sulfate disc gel electrophoresis; they were isolated and then characterized by amino acid analysis, NH2-terminal sequence analysis, and chromatographic and electrophoretic analyses of their digestion products. Ancrod cleavage of MDC-labeled fibrin produced a series of six alpha-chain peptides of molecular weights 34,000 to 12,000, each of which contained an MDC-labeled acceptor site, and an NH2-terminal alpha-chain derivative of molecular weight 37,500. The latter remains disulfide bound in the residual fibrin and has two MDC-labeled sit-s which are separable by CNBr cleavage. Mild plasmin digestion of MDC-labeled fibrin generated fluorescent alpha-chain peptides of molecular weights 45,000, 42,000, 35,000, 23,000, 21,000, and 2,500 in the supernatant and a nonfluorescent NH2-terminal alpha-chain derivative of molecular weight 25,000 which remained in the insoluble residual fibrin. The alignment of these plasmic supernatant peptides was determined from NH2-terminal sequence analyses which indicated that an MDC acceptor site was located at approximately residue 255 of the Aalpha-chain. Cleavage of the MDC-labeled alpha-chain by CNBr, however, localized most of its fluorescence (approximately 80%) to a fragment of molecular weight 29,000 which had the same NH2-terminal sequence as the labeled plasmic peptide of molecular weight 21,000. Both peptides were cleaved by ancrod into two acceptor site-containing peptides of approximately equal fluorescence. The preliminary NH2-terminal sequence analyses of these peptides, when combined with the above findings, indicated that these two other cross-link acceptor sites are in a peptide segment which comprises the middle 17% of the Aalpha-chain. 相似文献
14.
15.
K. S. Raju 《Molecular and cellular biochemistry》1983,56(1):81-88
A tryptic cleavage procedure was used to obtain stable copper-containing peptide regions of human ceruloplasmin. Tryptic digestion-derived copper peptides were fractionated by gel filtration chromatography, yielding two fractions, one with an apparent molecular weight of 11000 and the other 1000. The high molecular weight fraction (11K fraction) was found to contain 50% of the copper atoms of the ceruloplasmin molecule and behaved as a single copper-containing component by gel filtration chromatography and by isoelectric focusing. The low molecular weight fraction (1K fraction) was found to be a mixture of three or four copper peptides by isoelectric focusing. Acrylamide gel electrophoresis studies, amino acid composition analysis and terminal amino acid determinations showed the 11K fraction to be a complex composed of at least three peptides arising from different regions of the ceruloplasmin molecule. Two of the peptides of the 11K complex appear to be derived from the 19K fragment of the ceruloplasmin molecule (18); one peptide in the complex appears to correspond to the aspartic acid-rich portion, residues 7-30, and the other to the histidine-rich portion, residues 103-157. Most preparations of ceruloplasmin are reported to consist of three non-covalently linked fragments that have molecular weights of 67K, 50K and 19K. Dwulet and Putnam (20) proposed a model for the sequence structure of ceruloplasmin where the molecule exhibits a three-fold repeat pattern of two alternating structures, here termed X and Y.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
16.
We report the isolation and characterization of an apolipoprotein A-I mutant using a new technique for structural analysis of apolipoproteins based upon the combined techniques of protein isolation by isoelectric focusing in immobilized pH-gradients, reversed-phase HPLC of tryptic peptides, and subsequent molecular weight analysis of isolated peptides by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The particular advantages of the TOF-SIMS procedure in the characterization of proteolytic peptides are the detection limits in the picomole range, the accuracy of molecular weight determination (up to 3000 +/- 1 D), the speed of analysis, and the wide range of applications for involatile biomolecules. The described procedure for the analysis of apolipoproteins requires only 2 ml of serum as starting material. This method can be used to monitor for genetic polymorphisms and posttranslational modifications on a microscale basis. Applying these techniques, we characterized a new apolipoprotein A-I mutant with an amino acid exchange arginine177 by histidine. 相似文献
17.
Isolation of insulin degradation products from endosomes derived from intact rat liver 总被引:7,自引:0,他引:7
F G Hamel B I Posner J J Bergeron B H Frank W C Duckworth 《The Journal of biological chemistry》1988,263(14):6703-6708
Rats were injected with [125I]iodoinsulin labeled at either the A14 or B26 tyrosine, and the animals were killed and livers subcellularly fractionated to yield light (early or neutral) endosomes and heavy (late or acidic) endosomes. 125I-Labeled material was extracted from endosomes and analyzed by Sephadex G-50 filtration and high performance liquid chromatography (HPLC). Radiolabeled material in both types of endosomes is comprised of high molecular weight, insulin-sized, and low molecular weight components, with B chain-labeled small molecular weight material in two peaks, one corresponding to iodotyrosine and one to small peptides (Mr less than 1500). As compared with A chain label, however, less of the B chain material appears in the degradation components (both high and low molecular weight fractions) suggesting that a fragment of B chain containing the B26 residue is lost from the endosomes. Analysis on HPLC shows that significant amounts of the insulin-sized and high molecular weight material have proteolytic cleavage(s) in the B chain with an intact A chain. The B chain-derived labeled peptides elute from HPLC identically with products generated by insulin protease. These results therefore show substantial insulin degradation occurring in light endosomes prior to endosomal acidification and to receptor dissociation, suggesting receptor-bound insulin is a substrate for insulin protease. 相似文献
18.
19.
Masakazu Uramoto Masayuki Matsuoka J.G. Liehr James A. Mccloskey Kiyoshi Isono 《Bioscience, biotechnology, and biochemistry》2013,77(8):1901-1902
Eclosion hormone was purified 5,000-fold from the extracts of male adult heads of the silkworm, Bombyx mori. The fourteen-step purification procedure consisting of solvent extractions, fractional precipitations and chromatographies afforded a partially purified preparation of eclosion hormone, 1.8 μg of which showed activity in a Bombyx pharate adult. The hormone was inactivated by proteolytic enzymes. The molecular weight of eclosion hormone was estimated to be 8,400 ± 1,000 daltons by gel-filtration on Sephadex G-50. 相似文献
20.
The effects of fractionated bovine uterine flushings (BUF) on in vitro mouse embryo development were evaluated. Uterine flushings were nonsurgically collected from six cows and fractionated by ultrafiltration using 1,000 D and 30,000 D molecular weight exclusion limit membranes. Retentates were designated as 1,000 D (1 KR) and 30,000 D retentates (30 KR) and were evaluated for total protein, plasmin, plasmin inhibitor and support of mouse embryo development. Medium with bovine serum albumin (BSA) as a control was fractionated in a similar manner and frozen and thawed to assess any developmental limitations induced by the procedure. Consistent significant relationships, as determined by correlation-regression analysis, between the extent of embryo development and levels of protein, plasmin and plasmin inhibitor were not observed in either 1 KR BUF or 30 KR BUF. The numbers of embryos developing into blastocysts in 1 KR BUF on Days 9, 12, 15, 18 and 21 of the collection period were reduced when compared with the 30 KR BUF (P<0.05). Fewer blastocysts hatched in 1 KR BUF than in 30 KR BUF on Days 12, 15, 18 and 21 of the collection period (P<0.05). Embryo development in medium with 1 KR BSA tended to be superior to development in unfractionated media and in 30 KR BSA at comparable protein levels. No detrimental effects of freezing and thawing culture media on embryo development were observed (P>0.10). These data suggest that luteal phase BUF contain an inhibitor of embryo development, and commercial BSA preparations may possess a small molecular weight contaminant which reduces in vitro embryo survivability. 相似文献