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1.
A sensitive enzyme-linked immunosorbent assay (ELISA) for quantitation of human epidermal growth factor (EGF) was employed to study EGF in urine and blood. The EGF/creatinine ratio in urine was significantly higher for women (range and (median); 0.20-0.83 (0.50) nmol EGF/mmol creatinine) than for men (0.17-0.63 (0.30) nmol EGF/mmol creatinine). We were not able to demonstrate EGF in plasma (median plasma EGF < 0.01 nmol/l) whereas serum contained a range and (median) of 0.02-0.31 (0.12) nmol EGF/l. The amount of EGF in serum showed a weak correlation to the platelet count (r = 0.327). EGF was partly purified by affinity chromatography from urine (urine EGF) and from activated platelets in platelet rich plasma (blood EGF). Both blood and urine contained a high molecular weight form of EGF (HMW EGF) as well as 6 kDa EGF. HMW EGF from blood was similar to HMW EGF from urine concerning behaviour upon gel filtration, pI and apparent affinity constant for binding to the EGF receptor. However, HMW EGF constituted approx. 40% of blood EGF but only 10% of urinary EGF. The 6 kDa EGF from both blood and urine contained two isopeptides with pI around 4.40 and 4.15 but in various proportions. The apparent affinity constant for binding to the EGF receptor for blood 6 kDa EGF was 1.8 x 10(10) l/mol compared to 1.0 x 10(10) l/mol for urinary 6 kDa EGF and 0.8 x 10(10) l/mol for HMW EGF from both blood and urine. The present study suggests that the processing of the EGF precursor differs in the blood and in the kidneys and that 6 kDa EGF from blood and urine binds to the EGF receptor with a higher apparent affinity constant than does HMW EGF.  相似文献   

2.
Angiotensin I-converting enzyme (ACE) activity was analyzed in human urine collected from mild hypertensive untreated patients. DEAE-cellulose chromatography using linear gradient elution revealed two forms of angiotensin I-converting enzyme, eluted in the conductivity of 0.75 and 1.25 mS. The fractions of each conductivity were pooled and submitted to direct gel filtration in an AcA-34 column, and the apparent molecular weights of urinary ACEs were estimated as 90 kDa (for ACE eluted in 0.75 mS) and 65 kDa (for ACE eluted in 1.25 mS). Both enzymes have a K(i) of the order of 10(-7) M for the specific inhibitors studied, and are able to hydrolyze luteinizing hormone-releasing hormone and N-acetyl-Ser-Asp-Lys-Pro as described for N-domain ACE. By Western blot analysis, both peaks were recognized by ACE-specific antibody Y4, confirming the molecular weight already described. A plate precipitation assay using monoclonal antibodies to the N-domain of ACE showed that both forms of ACE binds with all monoclonal antibodies to the active N-domain ACE, suggesting that these forms of human urine ACEs resemble the N-fragment of ACE. The HP2 ACE (65 kDa) is similar to low molecular weight (LMW) ACE from normal subjects, and the HP2 ACE (90 kDa) is different from high molecular weight (190 kDa) and LMW (65 kDa) normal ACEs. The 90 kDa ACE could have an important role in development of hypertension. It will be fundamental to elucidate the molecular mechanism responsible for the genesis of this isoform.  相似文献   

3.
Vitronectin is a cell-adhesive glycoprotein in serum and plasma, also termed serum spreading factor and complement S-protein. It consists of a mixture of a polypeptide of molecular weight 75 kilodalton (kDa) and its nicked product of 65 kDa plus 10 kDa. By a quantitative immunoblotting assay, human blood samples could be classified into three distinct vitronectin types; type I (58% of the population) was 75 kDa rich and 65 kDa poor, type II (35% of the population) contained approximately equal amounts of 75 kDa and 65 kDa, and type III (5% of the population) was 75 kDa poor and 65 kDa rich. The vitronectin type did not correlate with age, sex, or ABO blood type.  相似文献   

4.
A low molecular weight (LMW) kininogen was isolated from pooled human serum by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Sephadex G-150, and Sephadex G-100. It was shown to be homogeneous by ultracentrifugation, polyacrylamide gel electrophoresis, and immunoelectrophoresis. The sedimentation coefficient, S020,W, of purified LMW kininogen was 3.85 s, and its molecular weight was determined to be 78,000 by Sephadex G-100 gel-filtration. The LMW kininogen contained 79.3% protein, 8.0% hexose, 3.9% hexosamine, and 4.9% sialic acid. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antigenic determinant of LMW kininogen was not related to the sialic acid and kinin moieties in the kininogen molecule, but could not be distinguished from that of high molecular weight (HMW) kininogen. In the quantitative single radial immunodiffusion test, a sialic acid-free LMW kininogen reacted to a greater extent with the antiserum than the native LMW kininogen. The kininogen level in human serum was estimated by single radial immunodiffusion. The antiserum cross-reacted with monkey serum, but not with sera from dogs, rats, and mice, horses, pigs, guinea pigs, oxen, and rabbits.  相似文献   

5.
Summary A factor with a molecular weight of less than 1 kDa in the mucosa of the bovine small intestine (low molecular weight factor or LMW factor) stimulated DNA synthesis in rat hepatocytes in primary culture. This factor only showed its activity when it was added with a larger factor with a molecular weight of 30 kDa that was also found in the same tissue (high molecular weight factor or HMW factor). The LMW factor probably acts to enhance the action of a hepatotrophic growth factor, since EGF and HGF can substitute for the HMW factor. The action of the LMW factor was not due to the actions of low molecular weight substances such as norepinephrine, estradiol, triiodothyronine, and putrescine, which enhance the action of EGF or HGF, since substantial amounts of these substances were not found in the extract. When intraperitoneally administered into rats, after two-thirds hepatectomy, the LMW factor enhanced hepatocyte proliferation without the administration of the HMW factor. In the regenerating liver, a hepatotrophic growth factor(s), which acts synergistically with the LMW factor, might be properly provided, but the supply of the LMW factor might be below the level that maximally stimulates hepatocyte proliferation.  相似文献   

6.
Renalase is a recently discovered secretory protein, which is suggested to play a role (which still remains elusive) in regulation of blood pressure. Earlier it was purified from urine of healthy volunteers by means of ammonium sulfate fractionation and subsequent affinity chromatography (Xu et al. (2005), J. Clin. Invest., 115, 1275). The resultant purified preparation of renalase contained 2 proteins with molecular masses of 35 and 67?C75 kDa. The authors believed that the latter represents a dimerization (aggregation) product of the 35 kDa protein. In this study we have detected relanase in urinary samples of 2 of 6 volunteers only after immunoaffinity enrichment of urinary samples subjected to ammonium sulfate precipitation. Electrophoresis of the purified preparation also demonstrated the presence of 2 proteins with molecular masses of 35 and 66 kDa, respectively. Mass spectrometry analysis of these proteins identified 35 and 66 kDa proteins as renalase and serum albumin, respectively. Thus, our results do not support suggestion on formation of renalase dimers and they indicate that urinary renalase excretion significantly varies in humans.  相似文献   

7.
Epidermal growth factor in blood   总被引:3,自引:0,他引:3  
The presence of receptors for epidermal growth factor (EGF) in a wide variety of human tissues and also some tumours indicates an as yet undefined role for EGF and it is therefore necessary to know precise concentrations in blood and other fluids. We have investigated the occurrence of EGF in the circulation and found that in platelet rich plasma, EGF levels were 51 +/- 5 pmol/l (mean +/- S.E.M., n = 6) while in platelet poor plasma levels were 2.9 +/- 0.9 pmol/1. In contrast, serum EGF was 37 +/- 7 pmol/l if separated at 30 min and rose to 117 +/- 5 pmol/l if separated at 270 min. Gel chromatography showed that all residual EGF immunoreactivity in platelet poor plasma resided in the high molecular weight form thought to be non biologically active. In serum, delay in separation resulted in an increase in the proportion of EGF immunoreactivity co-eluting with EGF standard. These results suggest that EGF in the circulation is associated with platelets and that the process of blood coagulation leads to release of free EGF.  相似文献   

8.
Cell extract and spent culture supernatant proteins from Streptococcus pyogenes Manfredo strain (type M5) were each separated to give 22 narrow range molecular weight fractions by blot-elution from SDS-polyacrylamide gels. Eluted samples and unfractionated proteins were screened for T cell stimulatory activity using human peripheral blood mononuclear cells (PBMC) from healthy adults in proliferation assays. Responses were measured in 4- and 7d cultures. Responses to a wide range of cell extract proteins were revealed by fractionation, the degree of response to each fraction varying between donors. Unfractionated culture supernatant proteins elicited proliferative responses by PBMC from all individuals examined. Responses to culture supernatant fractions containing 25–33 kDa proteins could be attributed to known superantigens. Furthermore, samples from culture supernatants containing higher molecular weight fractions (>45 kDa) elicited responses in 50% of donors in 7d cultures, suggesting that these fractions contained common recall antigens. The efficacy of using electroeluted samples to identify T lymphocyte stimulatory proteins was confirmed by demonstrating that a known superantigen of S. pyogenes Manfredo strain, streptococcal pyrogenic exotoxin C (SPEC), could be fractionated successfully using this method and its activity recovered. Our results show that human T cell responses to group A streptococci involve a remarkably wide range of both cell-associated and released streptococcal proteins.  相似文献   

9.
The heterogeneity of serum lipoproteins (excluding very low density (VLDL) and intermediate density (IDL) lipoproteins) and that of lipoproteins secreted by HepG2 cells has been studied by immunoblot analysis of the apolipoprotein composition of the particles separated by polyacrylamide gradient gel electrophoresis (GGE) under nondenaturing conditions. The reactions of antibodies to apoA-I, apoA-II, apoE, apoB, apoD, and apoA-IV have revealed discrete bands of particles which differ widely in size and apolipoprotein composition. GGE of native serum lipoproteins demonstrated that apoA-II is present in lipoproteins of limited size heterogeneity (apparent molecular mass 345,000 to 305,000) and that apoB is present in low density lipoproteins (LDL) and absent from all smaller or denser lipoproteins. In contrast, serum apoA-I, E, D, and A-IV are present in very heterogeneous particles. Serum apoA-I is present mainly in particles of 305 to 130 kDa where it is associated with apoA-II, and in decreasing order of immunoreactivity in particles of 130-90 kDa, 56 kDa, 815-345 kDa, and finally within the size range of LDL, all regions where there is little detectable apoA-II. Serum apoE is present in three defined fractions, one within the size range of LDL, one containing heterogeneous particles between 640 and 345 kDa, and one defined fraction at 96 kDa. Serum apoD is also present in three defined fractions, one comigrating with LDL, one containing heterogeneous particles between 390 and 150 kDa, and one band on the migration front. Most of serum apoA-IV is contained in a band comigrating with albumin. GGE of centrifugally prepared LDL shows the presence of apoB, apoE, and apoD, but not that of apoA-I. However, the particles containing apoA-I, which, in serum, migrated within the LDL size range and as bands of 815 to 345 kDa, were recovered upon centrifugation in the d greater than 1.21 g/ml fraction. GGE of high density lipoproteins (HDL) indicated that most of apoA-I, A-II, and A-IV were present in lipoproteins of the same apparent molecular mass (390-152 kDa). ApoD tended to be associated with large HDL, and this was also significant for HDL apoE, which is present in lipoproteins ranging from 640 to 275 kDa. GGE of very high density lipoproteins (VHDL) presented some striking features, one of which was the occurrence of apolipoproteins in very discrete bands of different molecular mass. ApoA-II was bimodally distributed at 250-175 kDa and 175-136 kDa, the latter fraction also containing apoA-I.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

10.
F2-isoprostane excretion rate and diurnal variation in human urine   总被引:6,自引:0,他引:6  
8-Iso-PGF2alpha is formed in vivo by non-enzymatic free radical catalysed oxidation of arachidonic acid. Urinary measurement of this compound has previously been shown to reflect the oxidative stress of the body in human and animal studies. To investigate the normal excretion rate and a possible diurnal variation of 8-iso-PGF2alpha excretion in humans urinary samples were collected from ten healthy volunteers of both sexes at different times during a day and as a 24-h urine sample. The samples were analyzed by a newly developed radioimmunoassay with a specific antibody against free 8-iso-PGF2alpha. There was no diurnal variation in the urinary levels of 8-iso-PGF2alpha during the day in this study. Neither was there any statistically significant difference between the 8-iso-PGF2alpha levels at any time of the day or in the morning urine samples compared to the 24-h urine samples. In conclusion, all urine samples collected at any time of the day, preferably a morning urine sample (representative urine from 6-8 hours), can thus be used to obtain a reliable and adequate value of the amount of the 8-iso-PGF2alpha excretion in urine in healthy individuals.  相似文献   

11.
To investigate the molecular features of hEGF in the circulatory system, we analyzed hEGF-like immunoreactivity (hEGF-LI) in human serum and plasma by our two-site enzyme immunoassay (EIA), using an aliquot of each fraction obtained by gel filtration of human serum or plasma on Sephadex G-100. The results demonstrated that the majority of the hEGF-LI in the plasma (HMW X hEGF-LI) emerged in the void volume, while a small amount of hEGF-LI (LMW X hEGF-LI) eluted at a position similar with that of standard hEGF. After reduction of HMW X hEGF-LI with 2-mercaptoethanol, hEGF-LI emerged at the same elution position as that of standard hEGF, suggesting that the predominant form of hEGF may circulate as a complex with some macromolecule(s) in human blood. The biochemical properties of the plasma component(s) with high molecular weight which form the complex with hEGF and the physiological significance of this circulating complex still remain to be clarified.  相似文献   

12.
By use of immunoblot analysis, we demonstrate the presence of a pro-Epidermal growth factor (EGF) with an approximate molecular weight of 165 kDa in adult mouse urine. In addition, urine contains four high molecular weight EGFs with approximate molecular weights of 116, 97, 66 and 56 kDa. The 165 kDa pro-EGF as well as the 66 and 56 kDa EGFs also are detectable in mouse kidney extract. Neither urine nor kidney contain the mature EGF of 6 kDa. The 165 kDa pro-EGF is the major product synthesized in renal tissue and secreted in urine. The finding of high molecular weight EGFs in urine suggests that part of pro-EGF secreted into urine undergoes partial proteolysis distal to its site of synthesis.  相似文献   

13.
Two forms of epidermal growth factor (EGF) have been purified to homogeneity from rat urine by immunoaffinity chromatography and gel filtration. For one of the purified peptides the molecular mass has been determined to be 5891 by mass spectrometry. This peptide consists of 51 amino acid residues. The sequence of the first 48 amino acid residues is identical to the previously published sequence for submandibular rat EGF. The C-terminal three residues (49-51) are Trp-Trp-Lys. The other purified peptide has a molecular mass of 45 kDa as determined by SDS-polyacrylamide gel electrophoresis. The N-terminal sequence is Asn-Tyr-Lys-Asp-(Cys)-Gly-Pro-Gly-Gly-(Cys)-Gly-Ser-His-Ala. Both the high and the low molecular mass form of urinary rat EGF are able to bind to the human placenta receptor for EGF.  相似文献   

14.
Topography of human placental receptors for epidermal growth factor   总被引:1,自引:0,他引:1  
These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.  相似文献   

15.
Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.  相似文献   

16.
Human fibroblast (WS-1) cells in culture synthesized and secreted an epidermal growth factor which cross-reacted with human epidermal growth factor (hEGF) purified from human urine. hEGF secreted by the cells (designated as WS-1 EGF or fibroblast EGF) and hEGF isolated from urine (designated as urine EGF) were immunologically indistinguishable. The molecular weight of fibroblast EGF estimated by gel filtration was identical with that of hEGF from urine. On chromatofocusing chromatography, fibroblast EGF was eluted mainly at pH 4.26 as a sharp symmetric peak with a minor peak at pH 4.62, like urine EGF. These results suggested that EGF synthesized and secreted by human fibroblast cells is an identical molecule to that of hEGF in human urine.  相似文献   

17.
《Cytokine》2015,71(2):97-103
Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3 kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.  相似文献   

18.
Human collagen type IX was isolated from the media of organ cultures of fetal or infant hyaline cartilage. It consisted of three distinct, disulfide-bonded polypeptides of 115, 84, and 72 kDa, respectively. Digestion with chondroitinase ABC reduced the apparent molecular mass of the 115-kDa chain to about 65 kDa demonstrating that also human collagen type IX is a proteoglycan. In the electron microscope, the molecule had a rigid rod-like structure with characteristic kinks and with a globular domain at one end. Digestion of human collagen type IX with pepsin leads to somewhat heterogeneous fragments. Affinity-purified antibodies to the mixture of fragments specifically reacted with the fragment HMW without cross-reaction with chicken HMW. LMW of both species were recognized to the same low extent. Mechanically generated fibril fragments from human fetal cartilage were heterogeneous in diameter. Significantly, they could be immunostained for collagen type IX in a D-periodic pattern and regardless of the fibril diameter. Some fibrils were poorly labeled, again independently of the diameter. Therefore, the role of collagen type IX in cartilage probably is not to control directly the lateral growth during fibrillogenesis but rather to stabilize the fibril network.  相似文献   

19.
Two acid alpha-glucosidase components of different molecular sizes (80 and 71 kDa) were separated from human placenta by DEAE-cellulose chromatography. Their catalytic properties were similar, and they showed almost the same molecular structure with regard to immunological properties and carboxy-terminal sequences, although the amino acid composition, the total hexose content, and the circular dichroism spectra were different. The pulse-chase labeling acid alpha-glucosidase with [3H]leucine revealed a processing pathway from a 110 kDa precursor to a 95 kDa intermediate form, then finally to 80 and 71 kDa mature forms. However, the sequence of appearance was different between the two mature enzymes. The 80 kDa component appeared first after chase for 5 h, and then the 71 kDa component followed. Their amounts became equal at 2 to 4 days. When ammonium chloride or leupeptin was added to the culture medium after chase for 5 h, the 71 kDa component failed to appear and the 80 kDa component was not converted to 71 kDa. It is concluded that probably only a part of the 80 kDa component is processed to form the 71 kDa component, although another possibility that cannot be excluded is that these two components are converted independently from the common intermediate 95 kDa protein.  相似文献   

20.
The heterogeneity of dipeptidyl peptidase IV (EC 3.4.14.5) was investigated in normal human serum. Thin-layer analytical isoelectric focusing revealed the presence of multiple molecular forms of the enzyme, their isoelectric points being in the pH range of 3.30-4.25. The maximum of enzyme activity appeared around pH 3.50. After treatment with neuraminidase the pI shifted to 4.70-5.40 with two maxima at pH 5.00 and 5.15. The Triton X-100 solubilized as well as the papain-treated-Triton X-100 solubilized enzyme from the whole human adult jejunal biopsy were also found to be heterogeneous. They focused--both before and after neuraminidase treatment--at pH values different from those of the enzyme of normal human serum. There was almost no pI shift after neuraminidase treatment of the intestinal enzyme from adult enterobiopsy. Electrophoresis in continuous polyacrylamide gradient gels as well as gel chromatography on Bio-Gel A-1.5m revealed two molecular forms of dipeptidyl peptidase IV in normal human serum. The estimated relative molecular mass of the major enzyme form was 250 000 in both the separation techniques used. On the other hand, the apparent relative molecular mass of the minor enzyme form was 450 000 as assessed by gradient gel electrophoresis, and 550 000, when estimated by gel chromatography. The Km values for glycyl-L-proline-4-nitroanilide as substrate with the major and minor forms of the serum enzyme were 1.60 +/- 0.39 X 10(-4) mol/l and 1.60 +/- 0.13 X 10(-4) mol/l, respectively. Our results indicate that the dipeptidyl peptidase IV in normal human serum is a heterogeneous enzyme as far as its charge and molecular size are concerned.  相似文献   

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