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1.
Immunoreactive dynorphin (I-dynorphin) was measured by radioimmunoassay in human cerebrospinal fluid (CSF). I-dynorphin concentration in CSF was 30 +/- 2 pg/ml. Sephadex G-25 column chromatography showed the main peak eluted at the position of dynorphin-(1-17). HPLC elution profile of this major peak from gel filtration showed a large peak corresponding to the position of dynorphin-(1-17) and small peaks corresponding to the positions of dynorphin-(1-13), dynorphin-(1-12) and other unknown peptides.  相似文献   

2.
Multiple forms of immunoreactive dynorphin (I-dynorphin) in rat anterior pituitary (AP), intermediate-posterior pituitary (IP) and medial basal hypothalamus (MBH) were examined. Three I-dynorphin peaks were observed on gel filtration. The first peak (big form) was eluted near the position of beta-LPH, and was predominant in AP. The second peak (middle form) was eluted near the position of ACTH. The third peak (small form) was eluted at the position of dynorphin (1-13), ane was predominant in IP and MBH. The heterogeneity of this small form was examined by ion exchange chromatography and reverse phase high performance liquified chromatography (HPLC). I-dynorphin peaks were observed at the positions of dynorphin(1-17), (1-13), (1-11), (1-10) and other peptides. These results strongly suggest (i) the presence of dynorphin(1-17), (1-13), (1-11) and (1-10) in rat IP, (ii) dynorphin(1-11) and (1-10) as the major components in this small form, (iii) the difference of I-dynorphin processing in AP, IP and MBH.  相似文献   

3.
The possible existence in rat brain tissues of shorter peptides related to VIP has been examined. VIP and PHI both contain paired basic amino acid residues at which posttranslational cleavage of these peptides might occur. Antiserum to VIP(22-28) was raised in rabbits. The antiserum was carboxy-terminus directed, showing cross-reactivity with all tested peptides containing the VIP carboxy-terminus sequences. Chromatographic analysis of rat brain extracts demonstrated that recovered VIP(22-28) immunoreactivity [VIP(22-28)-ir] was heterogeneous, consisting of a major fraction [60-70% of total VIP(22-28)-ir] which eluted as authentic VIP(1-28) on gel filtration and on reversed phase high performance liquid chromatography (HPLC) columns. A second fraction (30-35% of total VIP(22-28)-ir] eluted from gel filtration columns in the position of VIP(22-28). HPLC analysis of this fraction from extracts of rat cortex, hippocampus, and midbrain indicated that it was heterogeneous. One component corresponded to authentic VIP(22-28). The other two components have not been identified; one appears to be a VIP fragment intermediate in size between VIP(1-28) and VIP(22-28).  相似文献   

4.
Two main forms of immunoreactive insulin have been identified in cultures of foetal mouse brain using HPLC and gel filtration. The major component which resembled proinsulin was converted by trypsin to the minor form which was similar to authentic pancreatic insulin in chromatographic behaviour. Both components showed immunological properties comparable to insulin and proinsulin including sensitivity of the former to reduction and alkylation.  相似文献   

5.
A combination of several chromatographic and assay systems was used to characterize the opioid peptides in rat stomach extracts. Partial purification of opioid material in acetic acid extracts of the corpus plus antrum regions of the rat stomach was carried out by gel filtration chromatography on Sephadex G-50, followed by adsorption onto Amberlite XAD-2 resin. A single peak in opioid activity was determined by both radioreceptor assay (RRA) and bioassay. By high performance liquid chromatography, this peak was resolved into five distinct components, characterized by RRA and (or) radioimmunoassay, with retention times corresponding to methionine enkephalin (met-enk), leucine enkephalin, met-enk-arg6-gly7-leu8, met-enk-arg6-phe7, and dynorphin 1-13. Closer examination of the dynorphin component revealed the presence of dynorphins 1-17, 1-13, and 1-8. Trypsin digestion of the partially purified (Sephadex G-50 and Amberlite XAD-2 chromatographed) extract resulted in an overall increase in opioid activity, suggesting the presence of larger, possibly precursor forms.  相似文献   

6.
Specimens from human porcine mucosal and muscular tissue from the entire gastrointestinal tract were extracted in acid ethanol, subjected to chromatography and analysed for somatostatin-like immunoreactivity by region-specific radioimmunoassays. The concentration of somatostatin-like immunoreactivity from man and pig ranged from 1.13 +/- 0.37 to 101.15 +/- 33.93 pmol/g wet weight, and from 7.64 to 159.48 +/- 23.79 pmol/g wet weight, respectively. In both species the highest concentrations were found in the jejunum. The immunoreactivity in intestinal mucosal extracts was distributed among four major peaks, two of which were identified by HPLC as somatostatin 1-28 and somatostatin 1-14, respectively. A peak of approx. 10 kDa was resolved by ion exchange plus HPLC into three components, two containing at least part of the somatostatin 1-14 sequence as well as (part of) the somatostatin 1-28(1-14) sequence (but differing in charge), the third containing only the 1-28(1-14) sequence. These peptides probably represent uncleaved and partially cleaved prosomatostatin. The fourth component to be identified by gel filtration was slightly larger than somatostatin 1-14. Extracts from the antrum, the pancreas and from muscular tissues contained almost exclusively somatostatin 1-14, and very little somatostatin 1-28, indicating that the somatostatin precursor is processed differently at these sites. Furthermore, extracts of porcine gastric antrum, analysed for somatostatin 1-28(1-14) immunoreactivity, showed two immunoreactive forms in the mucosa and three major forms in the muscular layers.  相似文献   

7.
I Yoshida  T Koyama  K Ogura 《Biochemistry》1987,26(21):6840-6845
Hexaprenyl diphosphate synthase from Micrococcus luteus BP-26, which has been known to be dissociated into two essential components, designated as components A and B, during hydroxyapatite chromatography [Fujii, H., Koyama, T., & Ogura, K. (1982) J. Biol. Chem. 257, 14610-14612], was also resolved similarly by Sephadex G-100 or DEAE ion-exchange chromatography. Each component takes various self-aggregated forms. The apparent molecular mass of component B estimated by gel filtration on Superose 12 varied depending on its concentration, ranging from approximately 18 to 49 kilodaltons (kDa). On the other hand, the apparent molecular mass of component A varied depending on not only its concentration but also the ionic strength of the medium, ranging from approximately 13 to 24 kDa. When a mixture of components A and B preincubated in the presence of Mg2+ but in the absence of substrate was subjected to Superose 12 gel filtration, they were eluted at positions identical with those observed when they were chromatographed individually. In contrast, when a mixture of components A and B incubated in the presence of Mg2+ and substrates was filtrated on Superose 12, the elution positions were markedly changed, showing that an approximately 24-kDa aggregate of component A (designated as A) and an approximately 27-kDa aggregate of component B (designated as B) were the major species. Evidence was also obtained to show that farnesyl diphosphate (FPP) binds to the components to form an aggregate, A-B-FPP-Mg2+, which probably represents an intermediary state of enzyme catalysis.  相似文献   

8.
Immunochemical study on PHI/PHM with use of synthetic peptides   总被引:2,自引:0,他引:2  
We have synthesized PHI and PHM (human PHI) as well as their fragments, PHI (1-6), PHI (1-15), PHI (14-19), PHI (14-27), PHI (20-27), PHM (1-15) and PHM (13-27), by the solution or solid-phase method for peptide synthesis. Using the highly purified synthetic peptides as immunogens or haptenic immunogens, five kinds of PHI/PHM specific antisera were produced. The major antibody-recognition sites of the five antisera were located respectively in the PHI C-terminal (R8201), in the PHI N-terminal (R8403), in the PHM C-terminal (R8502), and in the PHM whole molecule (R8702 and R8703). Radioimmunoassays (RIAs) with antisera R8201, R8403 and R8502, respectively, showed a wide distribution of immunoreactive (IR) PHI/PHM in porcine and human gastrointestinal and brain tissues. The concentrations of IR-PHI in the porcine gastrointestinal tissues, however, differed between the R8201 and R8403 RIAs employed for measurement. By using these two different PHI RIAs, the IR-PHI in the porcine brain tissue extract was shown to be almost a single component coeluting with synthetic PHI in gel filtration. The IR-PHI in the extract of porcine lower intestine on the other hand, contained, besides a PHI-like component, unidentified component(s) eluting immediately after synthetic PHI in gel filtration; this crossreacted with the PHI C-terminal specific R8201 antiserum but not with the N-terminal specific R8403 antiserum, suggesting the presence of the C-terminal-related fragment(s) of PHI in the tissues.  相似文献   

9.
The peptides that represent the major components with alpha-endorphin- and gamma-endorphin-like immunoreactivity in the rat neurointermediate lobe were purified to homogeneity and chemically characterized. Rat neurointermediate lobes were extracted by boiling and homogenization in acetic acid. Peptide purification was based on gel filtration, followed by two high-pressure liquid chromatography steps. Pools containing peptides with the size and immunochemical properties of alpha- and gamma-endorphins were resolved by reverse-phase high-pressure liquid chromatography into multiple immunoreactive components. The major forms were finally purified by paired-ion high-pressure liquid chromatography. The amino acid compositions of these peptides fitted the beta-endorphin sequences 1-16 and 1-17. Tryptic mapping, aminopeptidase M digestion, chromatographic characterization, and immunoreactivity to an antiserum recognizing the N alpha-acetylated terminus of endorphins showed that these peptides were indistinguishable from N alpha-acetyl-alpha-endorphin (N alpha-acetyl-beta-endorphin 1-16), and N alpha-acetyl-gamma-endorphin (N alpha-acetyl-beta-endorphin 1-17). The NH2-terminal residue of the peptides was identified by mass spectrometry as N alpha-acetyltyrosine, substantiating the identity of the peptides. The results demonstrate the existence of N alpha-acetylated alpha- and gamma-endorphin as endogenous peptides in the neurointermediate lobe of the rat pituitary gland. In view of their occurrence and biological properties they should be considered significant members of the pro-opiomelanocortin family.  相似文献   

10.
The distribution of five major products of proenkephalin B [dynorphin1-17, dynorphin B, dynorphin1-8, alpha-neo-endorphin and beta-neo-endorphin] was studied in regions of rat brain and pituitary. The distribution pattern of immunoreactive (ir) dynorphin B (= rimorphin) was found to be similar to that of ir-dynorphin1-17, with the highest concentrations being present in the posterior pituitary and the hypothalamus. HPLC and gel filtration showed the tridecapeptide dynorphin B to be the predominant immunoreactive species recognized by dynorphin B antibodies in all brain areas and in the posterior pituitary. In addition, two putative common precursor forms of dynorphin B and dynorphin1-17 with apparent molecular weights of 3,200 and 6,000 were detected in brain and the posterior pituitary. The 3,200 dalton species coeluted with dynorphin1-32 on HPLC. In contrast with all other tissues, anterior pituitary ir-dynorphin B and ir-dynorphin1-17 consisted exclusively of the 6,000 dalton species. Concentrations of dynorphin1-8 were several times higher than those of dynorphin1-17 in striatum, thalamus, and midbrain while posterior pituitary, hypothalamus, pons/medulla, and cortex contained roughly equal concentrations of these two opioid peptides. No dynorphin1-8 was detected in the anterior pituitary. Concentrations of beta-neo-endorphin were similar to those of alpha-neo-endorphin in the posterior pituitary. In contrast, in all brain tissues alpha-neo-endorphin was found to be the predominant peptide, with tissue levels in striatum and thalamus almost 20 times higher than those of beta-neo-endorphin. These findings indicate that differential proteolytic processing of proenkephalin B occurs within different regions of brain and pituitary. Moreover, evidence is provided that, in addition to the paired basic amino acids -Lys-Arg- as the "typical" cleavage site for peptide hormone precursors, other cleavage signals also seem to exist for the processing of proenkephalin B.  相似文献   

11.
Two novel N-terminal fragment pairs of porcine pro-gamma-melanocyte-stimulating hormone (MSH)-(1-103), viz. pro-gamma-MSH-(1-30)/(2-30) and pro-gamma-MSH-(1-67)/(2-67) were characterized. A third pair of peptides of still larger size was also detected. The two characterized peptide pairs terminate at sites different from the dibasic sequences typical of prohormone cleavage. This suggests either a different processing event or proteolysis during purification; in both cases cleavages are selective since the two peptides end at distinct positions. Unlike most previously described pro-gamma-MSH forms, which begin with Trp at position 1 in pro-opiomelanocortin (position -105 in relation to the start of ACTH), the novel peptide pairs show N-terminal heterogeneity with one of the components beginning with Trp as in other forms, and the second component (present in relative amounts of 10-70%) beginning with Cys at position 2 (corresponding to position -104).  相似文献   

12.
The procedure of high performance ion-exchange chromatography has been used to fractionate subfragment 1 of myosin (SF1) into its isoenzymic forms. In contrast to conventional ion-exchange procedures which yield two fractions corresponding to SF1(A1) and SF1(A2), the high performance liquid chromatography (HPLC) procedure resolves SF1 into four discrete fractions. The first pair that is eluted appears to be A1-containing isoenzymes while the latter pair corresponds to the A2 forms based on their polypeptide compositions by gel electrophoresis in the presence of sodium dodecyl sulfate. By gel electrophoresis under nondenaturing conditions it is not possible to differentiate between the two fractions corresponding to each isoenzyme. Although very minor differences between the fractions can be seen by the presence of extraneous peptides, these are present in far below stoichiometric amounts and, therefore, make it very unlikely that the superior fractionation by the HPLC procedure is based on their presence. An examination of the heavy chain heterogeneity in each of these fractions by peptide mapping revealed that the extra separation was based on this factor. Thus the HPLC procedure is capable of providing separation of SF1 into heavy chain-based isozymes as well as the light chain forms. ATPase measurements of these fractions reveal only minor differences in the Ca2+- and EDTA-activated ATPase.  相似文献   

13.
The reactive sulfhydryl group (SHD) (Kawakita et al. (1980) J. Biochem. 87, 609-617) which is essential for the decomposition of the E-P intermediate of Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum has been identified. One sample of sarcoplasmic reticulum membranes was reacted for 3 min with 0.4 mM N-[3H]ethylmaleimide at pH 7.0 at 30 degrees C to a labeling density of 1 mol/mol ATPase without loss of the Ca2+-transporting activity. Another sample of the membranes was treated similarly with non-radioactive N-ethylmaleimide and then labeled with 0.4 mM N-ethyl[14C]maleimide for 17 min. An extensive loss of the Ca2+-transporting activity occurred during the period of this radio-labeling, thus substantiating the 14C-labeling of SHD. The labeled membranes were digested by thermolysin, and the labeled peptides were fractionated by gel filtration and reversed-phase HPLC. Two major radioactive peptides were present in both 3H- and 14C-labeled thermolytic digests, and each of the major components of 14C-labeled peptides had a counterpart in the major components of 3H-labeled peptides which behaved identically on HPLC. The major 14C-labeled peptides were purified and found to be identical with the two SHN peptides, TL-I and TL-II (Saito-Nakatsuka et al. (1987) J. Biochem. 101, 365-376), and 0.5 mol/mol ATPase each of Cys344 and Cys364 was assigned as SHD. It seems that the Ca2+-transport system retains its activity while either of the two Cys residues is unoccupied, but loses it when both of them are modified with N-ethylmaleimide.  相似文献   

14.
15.
The protein composition of the cytolytic factor (NKCF) secreted by natural killer cells was analyzed. Natural killer cells were isolated from spleens of nude rats and purified in the Percole gradient. SDS-PAAG analysis of NKCF revealed the presence of major proteins with an approximate molecular weight of 62-67 kDa. Biochemical properties of these proteins such as fractionation on a TSK-3000 SW gel filtration column, electrophoretical mobility in reduced conditions, enhanced cytolytical activity in the presence of Ca2+ demonstrate the similarity with the pore-forming protein--perforin. Apart from the major component, proteins with m. w. 42 kDa, 31-28 kDa, 17 kDa and 12-14 kDa were also found. It is possible that the Ca2+-independent cytolytical activity is bound to one of these minor protein components of NKCF.  相似文献   

16.
Pro-opiocortin peptides in rat cerebrospinal fluid   总被引:1,自引:0,他引:1  
Cerebrospinal fluid (CSF) taken from rats implanted with chronic cisternal cannulae was subjected to gel filtration chromatography on Sephadex G-50. Fractions were monitored using radioimmunoassays for N-terminal pro-opiocortin (N-POC), gamma 3-melanotropin (gamma 3-MSH), C-terminal adrenocorticotropin (C-ACTH), alpha-endorphin, beta-endorphin, gamma-lipotropin (gamma-LPH) and alpha-MSH. Two peaks which corresponded in elution position to rat N-POC (1-74) and gamma 3-MSH were detected. The major C-ACTH-immunoreactive (IR) peak was found to correspond to 14k ACTH. While no alpha-endorphin immunoreactivity was detected in rat CSF, three beta-endorphin-IR peaks were identified in positions expected for beta-LPH, beta-endorphin (1-31) and beta-endorphin (1-27), as well as a major peak of activity with the elution characteristics and cross-reactivity of rat gamma-LPH. HPLC of the alpha-MSH-IR material in rat CSF revealed the presence of a major peak of immunoreactivity whose retention time did not correspond to the known oxidised and reduced forms of alpha-MSH and its desacetylated and diacetylated derivatives. The identity of this peak is unknown.  相似文献   

17.
Little is known of the identity of gastrin/cholecystokinin (CCK)-like peptides in protochordates. These animals are at a level of organization corresponding to that from which the vertebrate line arose; in order to shed light on the origins of gastrin/CCK-like peptides, we have studied by immunochemical methods these peptides in a protochordate, Ciona intestinalis. In radioimmunoassay, boiling water extracts of the neural ganglion reacted with C-terminal specific gastrin/CCK antibodies, but not N-terminal or intact G17 specific antibodies. Of particular importance was the fact that a gastrin antibody which reacts weakly with CCK8 showed full activity with the Ciona material, suggesting that it resembles the C-terminus of gastrin. A single major peak was found by gel filtration and HPLC. In immunohistochemistry, nerve cell bodies were found in the cortical regions of the ganglion, and abundant fibres ramified in the central neuropile. We conclude that peptides of the gastrin/CCK series occur in nervous tissue in protochordates, and that while they are distinguishable from known forms of both gastrin and CCK, they resemble C-terminal fragments of the mammalian gastrins.  相似文献   

18.
We have developed a reverse-phase HPLC method to purify 125I-labeled products resulting from the chloramine-T-based iodination of glucagon and have used the products [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon) to study the receptor binding of glucagon and the cell-mediated metabolism of the hormone by isolated canine hepatocytes. The extent of binding of the three labeled glucagons to cell receptors differed at steady state (8.5, 11.9, and 12.6% of the three peptides, respectively, becoming cell-associated), but each of the labeled glucagons approached steady state binding at the same rate. Further, unlabeled glucagon competed for the binding of each of the labeled peptides in parallel under steady state conditions, and each of the peptides showed potent activity in inhibiting [14C]fructose incorporation into glycogen. Gel filtration of the acetic acid-extracted, cell-associated products of radiolabeled glucagon binding revealed 10-20% of the material as a shoulder on the descending limb of the peak of hormone for each of the three labeled peptides. Trypsin digestion of the lower molecular weight peptide derived from [(125I)iodoTyr13]glucagon resulted in a fragment containing residues 13 to 17 as the only detectable radiolabeled product. On the other hand, trypsin digestion of the analogous peptide derived from [(125I)iodoTyr10]glucagon revealed, in addition to the radiolabeled fragment containing residues 1 to 12, a major fragment identified by radiosequence analysis to contain residues 4 to 12 and a minor fragment identified to contain residues 7 to 12. We conclude that (a) notwithstanding apparent differences in affinities exhibited by [(125I)iodoTyr13]glucagon, [(125I)iodoTyr10,13]glucagon, and [(125I)iodoTyr10]glucagon for binding to canine hepatocytes, the interactions of all three peptides with the glucagon receptor are functionally equivalent, and (b) the cell-mediated metabolism of receptor-bound glucagon involves the formation of hormone-derived peptides in which the biologically important NH2-terminal region of the hormone has been modified by limited proteolytic cleavage.  相似文献   

19.
The C-terminal half of the bovine myelin basic protein, peptide 89-169, was treated with BNPS-skatole [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine], and the products were isolated by repeated gel filtration through Sephadex G-50. They consisted of uncleaved peptide 89-169 in which approximately 30% of the tyrosine had been monobrominated and the tryptophan converted to oxindolealanine, peptide 116-169 modified by partial bromination (30%) of the tyrosine, and two chromatographic forms of peptide 89-115. The major form contained the lactone of dioxindolealanine at the C terminus; the minor form contained the uncyclized oxidation product. Each form of peptide 89-115 was resolved into several components by electrophoresis in polyacrylamide gels (10%, w/w) containing 1 M acetic acid and 8 M urea. The presence of three of these components could be explained by partial deamidation of Asn-91 and Gln-102. Studies on the oxidation of tryptophan-containing model peptides by BNPS-skatole indicated that the reaction can also include partial bromination of the dioxindole and its lactone and partial cleavage at the amino peptide bond of the tryptophan.  相似文献   

20.
Antibodies directed against the Met-enkephalin-related hexapeptide, Met-enk Arg6, have been used in radioimmunoassays in the characterization of material in rat brain, and bovine striatum, colon, and adrenal medulla. Met-enk Lys6 reacted 0.27 relative to Met-enk Arg6, but Leu-enk Arg6 and C-terminal extensions or deletions of Met-enk Arg6 showed less than 0.02 immunoreactivity. In rat brain, the concentration of Met-enk Arg6-like immunoreactivity was less than 20 pmol X g-1 in all regions, but after trypsinization of tissue extracts there were up to 80-fold increases in immunoreactivity as a result of cleavage of C-terminally extended forms. The tryptic product eluted as Met-enk Arg6 on gel filtration. In control extracts of rat brain there were at least three immunoreactive forms of Met-enk Arg6; one eluted in the position of the hexapeptide standard on gel filtration and HPLC while the others had properties of N-terminally extended forms. In bovine striatum and colon the hexapeptide-like material predominated; but in bovine adrenal extracts, there were relatively low concentrations of the hexapeptide and, instead, the dominant immunoreactive forms corresponded to two components that were probably N-terminally extended variants. Trypsin again produced marked increases in immunoreactivity. HPLC studies indicated that Met-enk Arg6Phe7- and Met-enk Arg6Gly7Leu8-like immunoreactive peptides were important substrates in bovine brain for the production of hexapeptide immunoreactivity after trypsin. The differences in the patterns of immunoreactive forms in bovine adrenal, colon, and brain are consistent with tissue variations in the pathways of posttranslational processing of the precursor molecules.  相似文献   

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