RESUMO
BACKGROUND: The incidence and mortality of breast cancer are increasing annually. Breast cancer seriously threatens women's health and quality of life. We aimed to measure the clinical value of CPN1, a new serum marker of breast cancer and to evaluate the efficacy of CPN1 in combination with CA15-3. METHODS: Seventy samples of breast cancer with lymph node metastasis, seventy-three samples of nonmetastatic breast cancer and twenty-five samples of healthy human serum were collected. Serum CA15-3 concentration was determined by Roche Elecsys, and serum CPN1 concentration was determined by ELISA. RESULTS: In breast cancer patients, serum CPN1 concentration was positively correlated with tumour size, clinical stage and CA15-3 concentration (r = 0.376, P<0.0001). ROC curve analysis showed that the optimal critical concentration of CPN1 for breast cancer diagnosis was 32.8pg/ml. The optimal critical concentration of CPN1 in the diagnosis of metastatic breast cancer was 66.121pg/ml. CPN1 has a greater diagnostic ability for breast cancer (AUCCA15-3=0.702 vs. AUCCPN1=0.886, P<0.0001) and metastatic breast cancer (AUCCA15-3=0.629 vs. AUCCPN1=0.887, P<0.0001) than CA15-3, and the combined detection of CA15-3 and CPN1 can improve the diagnostic efficiency for breast cancer (AUCCA15-3+CPN1=0.916) and for distinguishing between metastatic and non-metastatic breast cancer (AUCCA15-3+CPN1=0.895). CONCLUSION: CPN1 can be used as a new tumour marker to diagnose and evaluate the invasion and metastasis of breast cancer. The combined detection of CPN1 and CA15-3 is more accurate and has a certain value in clinical application.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/sangue , Lisina Carboxipeptidase/sangue , Mucina-1/sangue , Adulto , Idoso , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Lisina Carboxipeptidase/genética , Lisina Carboxipeptidase/metabolismo , Pessoa de Meia-Idade , Adulto JovemRESUMO
Acute aortic dissection (AAD) is a catastrophic cardiovascular disease with high disability and mortality due to multiple fatal complications. However, the molecular changes of the serum proteome after AAD are not very clear. Here, we performed isobaric tags for relative and absolute quantitation- (iTRAQ-) based comparative proteomic analysis to investigate the proteome profile changes after AAD by collecting plasma samples from 20 AAD patients and 20 controls. Out of the 345 identified proteins, 266 were considered as high-quality quantified proteins (95%confident peptides ≥ 2), of which 25 proteins were accumulated and 12 were reduced in AAD samples. Gene ontology enrichment analysis showed that the 25 AAD-accumulated proteins were enriched in high-density lipoprotein particles for the cellular component category and protein homodimerization acidity for the molecular function category. Protein-protein interaction network analysis showed that serum amyloid A proteins (SAAs), complement component proteins, and carboxypeptidase N catalytic chain proteins (CPNs) possessed the key nodes of the network. The expression levels of six selected AAD-accumulated proteins, B2-GP1, CPN1, F9, LBP, SAA1, and SAA2, were validated by ELISA. Moreover, ROC analysis showed that the AUCs of B2-GP1 and CPN1 were 0.808 and 0.702, respectively. Our data provide insights into molecular change profiles in proteome levels after AAD and indicate that B2-GP1 and CPN1 are potential biomarkers for AAD.
Assuntos
Dissecção Aórtica/sangue , Proteoma/química , Proteínas de Fase Aguda , Adulto , Dissecção Aórtica/patologia , Biomarcadores/sangue , Proteínas de Transporte/sangue , Proteínas do Sistema Complemento/análise , Feminino , Humanos , Lisina Carboxipeptidase/sangue , Masculino , Glicoproteínas de Membrana/sangue , Pessoa de Meia-Idade , Proteína Amiloide A Sérica/análiseRESUMO
Essentials Two basic carboxypeptidases are present in plasma, B2 (CPB2) and N (CPN). Cpb2-/- and Cpn-/- mice were challenged in a hemolytic uremic syndrome (HUS) model vs. wild type. Cpb2-/- exacerbates HUS while Cpn-/- exacerbates cobra venom factor challenge vs. wild type mice. CPB2 and CPN have overlapping but non-redundant roles. SUMMARY: Background There are two basic carboxypeptidases in plasma. Carboxypeptidase B2 (CPB2) is activated from a circulating zymogen, proCPB2, and carboxypeptidase N (CPN) is constitutively active with both inactivating complement C3a and C5a. Aims To test the roles of CPB2 and CPN in complement-driven mouse models of cobra venom factor (CVF) challenge and hemolytic-uremic syndrome (HUS). Methods Cpb2-/- , Cpn-/- and wild-type (WT) mice were compared in an HUS model induced by Shiga toxin and lipopolysaccharide administration and following CVF administration. Results HUS was exacerbated in Cpb2-/- mice more than in Cpn-/- mice, compared with WT mice. Cpb2-/- mice developed the HUS clinical triad of microangiopathic hemolytic anemia, uremia and thrombocytopenia. Treatment with anti-C5 antibody improved survival of both Cpb2-/- and Cpn-/- mice. In contrast, when challenged acutely with CVF, the reverse phenotype was observed. Cpn-/- mice had markedly worse disease than Cpb2-/- mice, whereas the WT mice were resistant. Conclusions CPN and CPB2 play overlapping but non-redundant roles in regulating complement activation in vivo. The constitutively active CPN is key for inactivation of systemic C5a, whereas CPB2 functions as an on-demand supplementary anaphylatoxin inhibitor in inactivating excessive C5a formed locally.
Assuntos
Carboxipeptidase B2/sangue , Ativação do Complemento , Complemento C3/metabolismo , Complemento C5a/metabolismo , Síndrome Hemolítico-Urêmica/enzimologia , Lisina Carboxipeptidase/sangue , Animais , Carboxipeptidase B2/deficiência , Carboxipeptidase B2/genética , Ativação do Complemento/efeitos dos fármacos , Complemento C5a/antagonistas & inibidores , Complemento C5a/imunologia , Inativadores do Complemento/farmacologia , Modelos Animais de Doenças , Venenos Elapídicos/toxicidade , Endotoxinas , Genótipo , Síndrome Hemolítico-Urêmica/sangue , Síndrome Hemolítico-Urêmica/induzido quimicamente , Síndrome Hemolítico-Urêmica/tratamento farmacológico , Lisina Carboxipeptidase/deficiência , Lisina Carboxipeptidase/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteólise , Toxina Shiga IIRESUMO
BACKGROUND: Carboxypeptidase N (CPN) is important in regulating vasoactive peptide hormones, growth factors, and cytokines by specifically cleaving their C-terminal basic residues. We investigated whether circulating peptides specifically cleaved by CPN in the tumor microenvironment can be stage-specific indicators of breast cancer. METHODS: CPN activity was measured using an ex vivo peptide cleavage assay by incubating synthesized C3f peptide (His6-C3f_S1304-R1320-His6) in interstitial fluids of breast tumors and adjacent normal breast tissues in mice with orthotopic implantation of the human cell line MDA-MB-231. The nature and extent of peptide cleavage by CPN was investigated by fragment profiling using nanopore fractionation and mass spectrometry. The fragment profiles in interstitial fluid correlated with concentrations of CPN-catalyzed peptides in blood samples taken from the tumor-bearing mice, healthy women, and breast cancer patients. CPN expression in the same set of samples was further examined by immunohistochemistry and immunoblotting. RESULTS: We showed that generation of C3f_R1310-L1319 specifically correlated with the CPN expression level. In both the mouse and clinical patient samples, CPN was clearly increased in tumor tissues compared with normal breast tissue, whereas corresponding CPN abundance in blood remained constant. Concentrations of 6 CPN-catalyzed peptides predominantly increased in sera taken from the mice (n = 8) at 2 weeks after orthotopic implantation. Six homologous peptides displayed significantly higher expression in the patients' plasma as early as the first pathologic stage of breast cancer. CONCLUSIONS: Circulating CPN-catalyzed peptide concentrations reflect the CPN activity in tumors. These biomarkers show strong potential for the noninvasive and early diagnosis of breast cancer.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Detecção Precoce de Câncer , Lisina Carboxipeptidase/sangue , Peptídeos/sangue , Animais , Biomarcadores Tumorais/antagonistas & inibidores , Neoplasias da Mama/enzimologia , Modelos Animais de Doenças , Feminino , Humanos , Lisina Carboxipeptidase/metabolismo , Camundongos , Peptídeos/antagonistas & inibidores , Peptidomiméticos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Microambiente TumoralRESUMO
To date, several assays for procarboxypeptidase U (proCPU) determination exist, all having their own inherent disadvantages and advantages. A drawback of activity-based assays is the interference of the constitutively active carboxypeptidase N (CPN) in plasma. Recent screening of Bz-Xaa-Arg peptides with modified aromatic amino acids at the P1 position revealed a selective CPU substrate, N-benzoyl-ortho-cyano-phenylalanyl-arginine (Bz-o-cyano-Phe-Arg), which will allow straightforward determination of proCPU in plasma. Our assay shows an excellent linearity in the concentration range of 20-2600 U/L, with within- and between-run precision values of 2.7% and 4.6%, respectively. A good correlation with our high-performance liquid chromatography (HPLC)-assisted proCPU activity assay using hippuryl-l-arginine (HipArg) as substrate was found. Besides the major improvement regarding the selectivity, the assay is much easier to perform and far less time-consuming compared with the proCPU activity assay using HipArg as substrate.
Assuntos
Carboxipeptidase B2/sangue , Ensaios Enzimáticos/métodos , Calibragem , Cromatografia Líquida de Alta Pressão , Humanos , Lisina Carboxipeptidase/sangue , Padrões de Referência , Especificidade por SubstratoRESUMO
BACKGROUND: Aminopeptidase P (APP) plays an important role in the catabolism of kinins in human plasma, mostly for des-Arg(9)-bradykinin. Impaired degradation of this active bradykinin metabolite was found to be associated with a decreased APP activity in hypertensive patients who experienced angioedema while being treated with angiotensin I-converting enzyme inhibitors. The pathophysiology of hereditary angioedema is presently attributed only to a quantitative/qualitative C1 inhibitor (CI-INH) defect with increased bradykinin release. OBJECTIVES: In the context of androgen prophylaxis, increased CI-INH function cannot fully explain protection from angioedema attacks alone because of the limited reversion of the CI-INH defects. Therefore we hypothesized that androgen prophylaxis could enhance plasma APP activity. METHODS: Patients with hereditary angioedema were investigated for plasma metallopeptidase activities responsible for kinin catabolism (APP, angiotensin I-converting enzyme, and carboxypeptidase N) and for CI-INH function in treated and untreated patients. RESULTS: APP activity was asymmetrically distributed in untreated patients (n = 147): the mean value was significantly lower than the value in a reference healthy and unmedicated population (n = 116; P < or = .001). Prophylaxis with androgen induced a significant increase in APP activity (P < or = .001), whereas it did not affect the other metallopeptidase activities. In both patient groups, APP activity showed a significant inverse relationship to disease severity (P < or = .001). CONCLUSION: In addition to the effect on circulating CI-INH levels, the increase in APP levels brought on by androgens could contribute to a more effective control of the kinin accumulation considered to be responsible for the symptoms of angioedema.
Assuntos
Aminopeptidases/sangue , Androgênios/uso terapêutico , Angioedemas Hereditários/sangue , Angioedemas Hereditários/prevenção & controle , Metaloproteases/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Angioedemas Hereditários/fisiopatologia , Proteína Inibidora do Complemento C1/metabolismo , Humanos , Cininas/metabolismo , Lisina Carboxipeptidase/sangue , Metabolismo , Pessoa de Meia-Idade , Peptidil Dipeptidase A/sangue , Estudos Prospectivos , Índice de Gravidade de DoençaRESUMO
Human carboxypeptidase N (CPN) was discovered in the early 1960s as a plasma enzyme that inactivates bradykinin and was identified 8 years later as the major "anaphylatoxin inactivator" of blood. CPN plays an important role in protecting the body from excessive buildup of potentially deleterious peptides that normally act as local autocrine or paracrine hormones. This review summarizes the structure, enzymatic properties and function of this important human enzyme, including insights gained by the recent elucidation of the crystal structure of the CPN catalytic subunit and structural modeling of the non-catalytic regulatory 83 kDa subunit. We also discuss its physiological role in cleaving substrates such as kinins, anaphylatoxins, creatine kinase, plasminogen receptors, hemoglobin and stromal cell-derived factor-1alpha (SDF-1alpha).
Assuntos
Lisina Carboxipeptidase/sangue , Lisina Carboxipeptidase/química , Estrutura Terciária de Proteína , Motivos de Aminoácidos , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Lisina Carboxipeptidase/genética , Modelos Moleculares , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
The activity of peptidyl-dipeptidase A and carboxypeptidase N taking part in peptide metabolism in the serum of patients with Alzheimer disease were studied. The role of these enzymes in the metabolism of neuropeptides and beta-amyloid at the Alzheimer disease was discussed.
Assuntos
Doença de Alzheimer/sangue , Lisina Carboxipeptidase/sangue , Peptidil Dipeptidase A/sangue , Idoso , Doença de Alzheimer/enzimologia , Peptídeos beta-Amiloides/metabolismo , Estudos de Casos e Controles , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Carboxypeptidase N is a plasma zinc metallocarboxypeptidase which is constitutively expressed in the liver and was identified as the enzyme responsible for inactivating bradykinin and kallidin by removing the C-terminal arginine. Because CPN can cleave the C-terminal arginine of C3a, C4a and C5a it is often referred to as anaphylatoxin inactivator. Markedly reduced levels of circulating CPN are associated with recurrent angioedema and abnormal cutaneous polymorphonuclear cell infiltration. METHODS: In this paper we describe a fast kinetic coupled enzymatic assay for the sensitive measurement of carboxypeptidase N activities in serum samples. The assay makes use of the excellent CPN substrate Benzoyl-L-Alanyl-L-Arginine. RESULTS: This novel assay is very fast, easy to perform and combines good reliability and reproducibility with excellent correlation with the HPLC-assisted assay (r=0.927; n=140). CONCLUSION: The presented assay can be used for high throughput screening of this important regulator of inflammation in clinical plasma or serum samples.
Assuntos
Bioensaio , Inflamação/sangue , Lisina Carboxipeptidase/sangue , Adolescente , Adulto , Idoso , Anafilatoxinas/antagonistas & inibidores , Anafilatoxinas/metabolismo , Cromatografia Líquida de Alta Pressão , Dipeptídeos/metabolismo , Humanos , Inflamação/patologia , Cinética , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
The effect of a single atropine administration on the activities of angiotensin-converting enzyme and carboxypeptidase N taking part in peptides metabolism was studied. Changes in the enzymes activities after administration were evident during at least 72 h. The role of these enzymes in the metabolism of neuropeptides under the effect of atropine was discussed.
Assuntos
Atropina/farmacologia , Lisina Carboxipeptidase/sangue , Antagonistas Muscarínicos/farmacologia , Peptidil Dipeptidase A/sangue , Animais , Injeções Intraperitoneais , Masculino , Ratos , Fatores de TempoRESUMO
To shed light on the role of bradykinin in preeclampsia in addition to acute hypoxia, we measured the activity of kininase I, the enzyme responsible for its degradation, in umbilical plasma. Kininase I activity in umbilical arteries was compared with that in the umbilical veins. The relationship between kininase I and pH values was also evaluated in women with and without preeclampsia. Also, enzyme activity in supernatants of fetal hepatic cells (NFL/T) cultured under hypoxic or normoxic conditions were determined. Kininase I activity levels in fetal umbilical arteries and veins (n = 33) were similar (r = 0.77). Hypoxia caused suppression of kininase I activity in the supernatant cultures of NFL/T after one hour. However, after 8 and 24 hours, kininase I activity was significantly greater than under normoxic conditions (p < 0.05). Kininase I activity of fetal umbilical vein significantly decreased in the presence of acidemia in the uncomplicated group (n = 75, r = 0.42), whereas the activity negatively correlated with umbilical arterial pH in the preeclamptic group (n = 10, r = - 0.65). Kininase I activity levels in cases complicated with preeclampsia were significantly higher than without preeclampsia (49.2 +/- 9.1 vs. 66.2 +/- 11.3 nmol/ml/min). The present study indicates that kininase I acts as a regulatory factor in bradykinin degradation.
Assuntos
Sangue Fetal/enzimologia , Hipóxia Fetal/enzimologia , Lisina Carboxipeptidase/sangue , Bradicinina/metabolismo , Feminino , Humanos , Concentração de Íons de Hidrogênio , Cinética , Gravidez , Artérias Umbilicais , Veias UmbilicaisRESUMO
Angio-oedema is a rare but potentially life threatening side-effect of angiotensin-converting-enzyme (ACE) inhibitor treatment. Identification of individuals at risk of this adverse effect is not possible. Angio-oedema is associated with raised concentrations of bradykinin, which is mainly inactivated by ACE. We assessed the plasma activity of two other enzymes that catabolise bradykinin (aminopeptidase P and carboxypeptidase N) in 39 hypertensive patients with a history of angio-oedema during ACE inhibitor treatment and in 39 hypertensive patients who had never had ACE inhibitor associated side-effects. Patients with previous angio-oedema had a lower plasma activity of aminopeptidase P than did those who never presented with angio-oedema (p=0 003). Our data suggest that low plasma concentrations of aminopeptidase P could be a predisposing factor for development of angio-oedema in patients treated with ACE inhibitors.
Assuntos
Aminopeptidases/sangue , Angioedema/induzido quimicamente , Inibidores da Enzima Conversora de Angiotensina/efeitos adversos , Lisina Carboxipeptidase/sangue , Adulto , Idoso , Análise de Variância , Angioedema/sangue , Angioedema/enzimologia , Estudos de Casos e Controles , Humanos , Hipertensão/tratamento farmacológico , Pessoa de Meia-IdadeRESUMO
Pro-carboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), precursor of carboxypeptidase U and plasma carboxypeptidase B is present in plasma and following activation by thrombin/thrombomodulin and/or plasmin can remove arginine from the carboxyterminal of C3a and C5a. We have shown that this enzyme can remove terminal arginine from the C5a octapeptide much more efficiently than the classical anaphylatoxin inactivator, carboxypeptidase N (CPN). Since we have previously demonstrated that proCPR is significantly upregulated in the inflammatory state, this enzyme would appear to significantly contribute to the inactivation of C5a, the most potent of the complement derived anaphylatoxins.
Assuntos
Carboxipeptidase B2/farmacologia , Complemento C3a/antagonistas & inibidores , Complemento C5a/antagonistas & inibidores , Lisina Carboxipeptidase/farmacologia , Carboxipeptidase B2/sangue , Humanos , Hidrólise , Lisina Carboxipeptidase/sangue , Fatores de TempoRESUMO
BACKGROUND: Coagulation and inflammation are both important processes that contribute to glomerular injury. The present study was performed to evaluate the effects of recombinant human soluble thrombomodulin (RHS-TM) in a lethal model of thrombotic glomerulonephritis and to investigate the possible mechanisms. METHODS: Thrombotic glomerulonephritis was induced in rats by administration of lipopolysaccharide and rabbit anti-rat glomerular basement membrane antibody. One hour later, RHS-TM or heparin was administered, and the histological findings, renal functions, and coagulation parameters were evaluated. To evaluate the contribution of carboxypeptidase R (CPR) to the results obtained in rats treated with RHS-TM, plasma CPR levels were measured. Then, carboxypeptidase inhibitor (CPI), which prevents the function of CPR, was administered. RESULTS: Massive glomerular thrombosis and lung hemorrhage developed within five hours of disease induction, and all rats died within 24 hours. RHS-TM (3 mg/kg) prevented the progression of the disease and all rats survived. Heparin (250 U/kg/h) showed similar anti-thrombotic effect, but induced massive hemorrhage in the lungs or stomach. RHS-TM attenuated leukocyte/neutrophil infiltration in the glomerulus but heparin did not, suggesting that RHS-TM has anti-inflammatory properties. CPR levels in plasma were about threefold higher in rats treated with RHS-TM compared to those in rats treated with heparin. Furthermore, the inhibitory effect of RHS-TM on leukocyte/neutrophil infiltration was significantly diminished by injection of CPI. CONCLUSION: RHS-TM effectively attenuates the injuries of thrombotic glomerulonephritis in rats. The results indicate that RHS-TM, in addition to its anti-thrombotic action, may exert its anti-inflammatory properties by converting proCPR to CPR, which then inactivates anaphylatoxins. RHS-TM is a potential novel therapeutic tool for thrombotic glomerular injury and related disorders.
Assuntos
Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/etiologia , Trombomodulina/administração & dosagem , Trombose/complicações , Anafilatoxinas/metabolismo , Animais , Coagulação Sanguínea , Nitrogênio da Ureia Sanguínea , Carboxipeptidase B2/sangue , Complemento C5a/metabolismo , Creatinina/sangue , Modelos Animais de Doenças , Feminino , Fibrina/metabolismo , Glomerulonefrite/patologia , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Contagem de Leucócitos , Lisina Carboxipeptidase/sangue , Tempo de Tromboplastina Parcial , Contagem de Plaquetas , Tempo de Protrombina , Coelhos , Ratos , Ratos Wistar , Solubilidade , Trombina/metabolismo , Trombose/induzido quimicamente , Trombose/patologiaRESUMO
Because bradykinin (BK) appears to have cardioprotective effects ranging from improved hemodynamics to antiproliferative effects, inhibition of BK-degrading enzymes should potentiate such actions. The purpose of this study was to find out which enzymes are responsible for the degradation of BK in human plasma. Human plasma from healthy donors (n = 10) was incubated with BK in the presence or absence of specific enzyme inhibitors. At high (micromolar) concentrations, BK was mostly (>90%) degraded by carboxypeptidase N (CPN)-like activity. In contrast, at low (nanomolar) substrate concentrations, at which the velocity of the catalytic reaction is equivalent to that under physiological conditions, BK was mostly (>90%) converted into an inactive metabolite, BK-(1-7), by angiotensin-converting enzyme (ACE). BK-(1-7) was further converted by ACE into BK-(1-5), with accumulation of this active peptide. A minor fraction (<10%) of the BK was converted into another active metabolite, BK-(1-8), by CPN-like activity. The present study shows that the most critical step in plasma kinin metabolism, i.e., inactivation of BK, is mediated by ACE. Thus inhibition of plasma ACE activity would be cardioprotective by elevating the concentration of BK in the circulation.
Assuntos
Bradicinina/antagonistas & inibidores , Bradicinina/sangue , Lisina Carboxipeptidase/sangue , Peptidil Dipeptidase A/sangue , Ácido 3-Mercaptopropiônico/análogos & derivados , Ácido 3-Mercaptopropiônico/farmacologia , Adulto , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Proteínas Sanguíneas/farmacologia , Bradicinina/química , Captopril/farmacologia , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteases/farmacologia , TrítioRESUMO
Arginine carboxypeptidase (CPR) is a single-chain plasma protein generated during coagulation from a precursor (proCPR). proCPR is the same molecule as thrombin activable fibrinolysis inhibitor (TAFI), which retards fibrin clot lysis in vitro and most likely modulates fibrinolysis in vivo. In this study, the amount of CPR-total, which includes proCPR (TAFI) and CPR (activated TAFI), in hemophiliac patients was evaluated using a newly developed enzyme linked immunosorbent assay (ELISA). The amount of CPR-total in plasma or serum of most of the hemophiliac patients was in the range of healthy individuals. There was no significant difference in hemophiliac patients with or without HIV-1 infection. However, two out of the 74 hemophiliac patients showed a significantly high level. The upregulation of CPR-total might contribute to compensate for inefficient coagulation in some hemophiliac individuals.
Assuntos
Precursores Enzimáticos/sangue , Hemofilia A/enzimologia , Lisina Carboxipeptidase/sangue , Ativação Enzimática , Infecções por HIV/sangue , Infecções por HIV/enzimologia , Hemofilia A/sangue , HumanosRESUMO
There are two types of carboxypeptidases present in human blood, carboxypeptidase N (CPN) and arginine carboxypeptidase (CPR). CPR is generated during coagulation from a precursor (proCPR) which can be converted to the active form by trypsin in vitro. Since it is difficult to distinguish the two types of carboxypeptidases in human blood by the measurement of enzyme activity, we established a quantitative sandwich ELISA by which CPR can be quantitated. The amount of CPR in plasma, fresh serum and heated serum were essentially the same. Therefore the ELISA assay does not distinguish proCPR, activated CPR and inactivated CPR. With the ELISA method, CPR was quantitated in plasma from fifty patients with rheumatoid arthritis and eleven patients with severe hepatitis as well as healthy individuals. The amount of CPR in plasma obtained from patients with rheumatoid arthritis was not found to be lower than that of normal subjects. Furthermore, the patients who suffered severe hepatitis and had very low levels of CPR-total were fatal. This suggests that a decrease of CPR level might be a good indication of a patient's prognosis to death by hepatitis.
Assuntos
Precursores Enzimáticos/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Lisina Carboxipeptidase/sangue , Anticorpos Monoclonais/imunologia , Western Blotting , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/imunologia , Humanos , Lisina Carboxipeptidase/imunologia , Dodecilsulfato de SódioRESUMO
Arginine carboxypeptidase (CPR) is a novel carboxypeptidase which was first described by Campbell and Okada. CPR is generated from a stable precursor of CPR (proCPR) during coagulation or under other circumstances and is promptly inactivated at 37 C. Therefore, it is not easy to determine CPR in blood samples. Since proCPR can be separated from the other basic carboxypeptidase (carboxypeptidase N; CPN) by passing plasma through DEAE gel, we have established a method to determine the amount of proCPR after converting it to active CPR by trypsin treatment. We first separated the proCPR from CPN using a filter cup tube (FC tube) packed with DEAE Sephadex, and measured activity after conversion of the enzyme to its active form using trypsin. With this method, no significant decrease in proCPR was noted in the plasma of patients including those with rheumatoid arthritis (RA), although CPR activity in fresh sera has been reported to be decreased. This discrepancy suggests that proCPR is not depleted in most patient sera, but that the level of activity of the enzyme which converts proCPR into active CPR may be compromised in RA patients.