RESUMO
High molecular weight zinc ion-dependent acid p-nitrophenylphosphatase (HMW-ZnAPase) was purified from bovine liver to homogeneity as judged by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The partial sequence of the purified enzyme electroblotted on PVDF membrane reveals a 95% sequence homology with human and bovine liver fructose-1,6-bisphosphate aldolase isozyme B (FALD B). FALD B was isolated from bovine liver using an affinity elution from phosphocellulose column. FALD B from bovine liver shows a native and subunit molecular weight that is indistinguishable from that of HMW-ZnAPase. In addition, an affinity purified antiserum raised in rabbits against purified HMW-ZnAPase cross-reacts with bovine liver FALD B and rabbit muscle isozymes. Despite these similarities, HMW-ZnAPase does not show FALD activity and bovine liver FALD does not display any zinc ion-p-nitrophenylphosphatase activity. These results suggested the existence of structural and immunological similarities between bovine liver HMW-ZnAPase and FALD B. Differences in some amino acid residues in enzyme activity indicate that they may be involved in different biochemical functions.
Assuntos
4-Nitrofenilfosfatase/química , Frutose-Bifosfato Aldolase/química , Fígado/enzimologia , Zinco/química , Sequência de Aminoácidos , Animais , Western Blotting , Bovinos , Eletroforese em Gel de Poliacrilamida , Humanos , Íons , Isoenzimas/química , Dados de Sequência Molecular , Peso MolecularRESUMO
The major acid phosphatase form (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was purified from the soluble extract of barley roots. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr approximately 38,000 in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme was Mr 77,600 and 79,000 as determined, respectively, by gel filtration on a Sephadex G-100 column and by density gradient ultracentrifugation. The isoelectric point was about 6.28. The enzyme is competitively inhibited by molybdate (Ki = 9 x 10(-7) M). NaF, Ag(+), Hg(2+), Pb(2+) and Zn(2+) are also inhibitors, while other cations showed no effect. The enzyme hydrolyzes a wide variety of natural and synthetic phosphate esters. In particular, the enzyme seems to be active on ATP, o-phosphotyrosine, o-phosphoserine and glucose 1-phosphate. The pH dependence studies between pH 4-8 using p-nitrophenylphosphate as substrate and diethylpyrocarbonate inactivation indicate the presence of essential histidine residue at the active site.
Assuntos
Fosfatase Ácida/isolamento & purificação , Grão Comestível/enzimologia , Hordeum/enzimologia , Fosfatase Ácida/antagonistas & inibidores , Fosfatase Ácida/metabolismo , Cátions/farmacologia , Cromatografia em Gel , Cromatografia por Troca Iônica , Citoplasma/enzimologia , Temperatura Alta , Concentração de Íons de Hidrogênio , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Cinética , Peso Molecular , Especificidade por SubstratoRESUMO
The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes were studied. The Cu2+ exposure of mussels caused severe perturbations in haemocyte actin and fibronectin organization with respect to non-exposed organisms. Cytoskeletal actin was analysed by indirect immunofluorescence, using an antitotal actin monoclonal antibody, and by rhodamine-conjugated phalloidin. The majority of haemocytes from Cu(2+)-exposed mussels displayed a round morphology, with short and blunt filopodia; they lacked the polarized phenotype which was typical in control samples. The cytoskeleton alteration, more evident after phalloidin staining, resulted in the disappearance of filamentous actin. The actin cortical meshwork also appeared disorganized. The cytoskeletal morphology studied by transmission electron microscopy after negative staining of Triton X-100-treated haemocytes confirmed these observations. The structural organization of actin when analysed by Western blotting showed a larger number of Triton-soluble actin pools in treated mussel haemocytes. Fibronectin was studied by indirect immunofluorescence using a polyclonal antiserum directed against mussel fibronectin. In treated mussels, fibronectin appeared to be strongly disorganized and its levels decreased in both haemocytes and haemolymph. The mechanism(s) of the copper-induced alterations on actin and fibronectin organization in mussel immunocytes is discussed.
Assuntos
Actinas/efeitos dos fármacos , Cobre/farmacologia , Fibronectinas/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Actinas/metabolismo , Animais , Bivalves/efeitos dos fármacos , Citoesqueleto/química , Citoesqueleto/efeitos dos fármacos , Fibronectinas/metabolismo , Hemócitos/químicaRESUMO
The effect of phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) on head regeneration in decapitated planarians (Dugesia lugubris s.1.) has been studied. TPA-treatment soon after head amputation dramatically inhibited the regenerative process. Ultrastructural analysis revealed that the migration of fixed parenchymal cells (FPCs) to the wound area was strongly activated by TPA. FPCs interacted with various types of cells inducing lysis and phagocyting cell debris. The resulting fluid was removed through diaphanous protrusions appearing at the level of the wound zone. Moreover the close association of FPCs with neoblastlike cell clusters in the parenchyma indicated their possible role in the modulation of neoblast migration.
RESUMO
Fibronectin (FN)-like protein has been localized by immunoelectron microscopy in the extracellular matrix (ECM) of planaria Dugesia lugubris s.l. The immunolabeling was present in both intercellular spaces of epidermal cells and the basement membrane, however the amount and distribution of gold particles seemed to be substantially different. FN-like material increased markedly during the passage of migrating cells through the basement membrane from the parenchyma to the epidermis. Gold particles were often found at cell-matrix contacts. Our result suggest that FN-like molecules detected in planarian ECM may be involved not only in cell adhesion but also in promoting cell migration and in regulating the epidermal cell turnover.
RESUMO
Actin expression in some Platyhelminthe species was demonstrated by western-blotting and immunocytochemical analysis using two distinct anti-actin antibodies: the anti-total actin that reacts against all actin isoforms of higher vertebrates and the anti-alpha SM-1 that recognizes the alpha-smooth muscle (alpha SM) isotype of endothermic vertebrates (Skalli et al., 1986). Western-blotting experiments showed that all species tested, including some free-living Platyhelminthes (Tricladida and Rhabdocoela) and the parasitic Fasciola hepatica, were stained by anti-total actin antibody while only Dugesidae and Dendrocoelidae showed a positive immunoreactivity against anti-alpha SM-1. These results were confirmed by cytochemical immunolocalization using both avidin biotin conjugated peroxidase reaction on paraffin sections, and immunogold staining on Lowicryl 4KM embedded specimens. Our findings may contribute to the understanding of Platyhelminthes phylogeny.
Assuntos
Actinas/biossíntese , Platelmintos/metabolismo , Animais , Western Blotting , Imuno-Histoquímica , Microscopia EletrônicaAssuntos
Actinas/metabolismo , Actinas/genética , Actinas/imunologia , Animais , Peixes , Expressão GênicaRESUMO
Three clones containing satellite DNA sequences were selected from a randomly sheared genomic DNA library of Picea abies (clones PAF1, PAG004P22F (2F), and PAG004E03C (3C)). PAF1 contained 7 repeats that were 37-55 bp in length and had 68.9%-91.9% nucleotide sequence similarity. Two 2F repeats were 305-306 bp in length and had 83% sequence similarity. Two 3C repeats were 193-226 bp in length and had a sequence similarity of 78.6%. The copy number per 1C DNA of PAF1, 2F, and 3C repeats was 2.7 x 10(6), 2.9 x 10(5), and 2.9 x 10(4), respectively. In situ hybridization showed centromeric localization of these sequences in two chromosome pairs with PAF1, all pairs but one with 2F, and three pairs with 3C. Moreover, PAF1 sequences hybridized at secondary constrictions in six pairs, while 2F-related sequences were found at these chromosome regions only in four pairs. These hybridization patterns allow all chromosome pairs to be distinguished. PAF1-related repeats were contained in the intergenic spacer (IGS) of ribosomal cistrons in all six nucleolar organizers of the complement, while sequences related to 2F were found on only one side of the rDNA arrays in four pairs, showing structural diversity between rDNA regions of different chromosomes.
Assuntos
Cromossomos de Plantas , DNA Satélite/genética , Picea/genética , Sequência de Bases , Southern Blotting , Centrômero , Hibridização in Situ Fluorescente , Cariotipagem , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido NucleicoRESUMO
1. Zn2+-dependent acid p-nitrophenylphosphatase from chicken liver was purified to homogeneity. 2. The purified enzyme moves as a single electrophoretic band at pH 8.3 in 7.5% acrylamide and was coincident with the enzyme activity. 3. Gel filtration on Sephadex G-200 gave an apparent molecular weight of 110,000 with two apparent identical subunits of 54,000-56,000 as determined by sodium dodecyl sulphate gel electrophoresis. 4. The maximum of enzyme activity was obtained in the presence of 3-5 mM ZnCl2 at pH 6-6.2, however, higher concentrations of metal are inhibitory. The enzyme hydrolyses p-nitrophenylphosphate, o-carboxyphenylphosphate and phenylphosphate, was insensitive to NaF and was inhibited by phosphate and ATP. The Km for p-nitrophenylphosphate was 0.28 x 10(-3)M at pH 6 in 50 mM sodium acetate/100 mM NaCl. 5. Phosphate is a competitive inhibitor (Ki = 0.5 x 10(-3)M) whereas ATP seems to be a non-competitive inhibitor (Ki = 0.35 x 10(-3)M). The isoelectric point determined by isoelectric focusing on polyacrylamide gel is 7.5. 6. Cell fractionation studies indicate that the Zn2+-dependent acid p-nitrophenylphosphatase of chicken liver is a soluble enzyme form.
Assuntos
4-Nitrofenilfosfatase/isolamento & purificação , Fígado/enzimologia , Monoéster Fosfórico Hidrolases/isolamento & purificação , Frações Subcelulares/enzimologia , Zinco/metabolismo , 4-Nitrofenilfosfatase/metabolismo , Animais , Galinhas , Cinética , Fígado/ultraestrutura , Peso Molecular , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Fosfatos/metabolismoRESUMO
The presence of a Zn2+-dependent acid p-nitrophenyl phosphatase (EC 3.1.3.2) in bovine liver was described. The enzyme was purified to apparent homogeneity and migrates as a single band during electrophoresis on polyacrylamide gel. The enzyme requires Zn2+ ions for catalytic activity, other bivalent cations have little or no effect. The enzyme, of Mr 118,000, optimum pH 6-6.2 and pI 7.4-7.5, was inhibited by EDTA, tartrate, adenine and ATP, but not by fluoride. The common phosphate esters are poor substrates for the enzyme, which hydrolyses preferentially p-nitrophenyl phosphate and o-carboxyphenyl phosphate. The Zn2+-dependent acid p-nitrophenyl phosphatase of bovine liver was different from the high-Mr acid phosphatases previously detected in mammalian tissues.
Assuntos
4-Nitrofenilfosfatase/metabolismo , Fígado/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , 4-Nitrofenilfosfatase/antagonistas & inibidores , 4-Nitrofenilfosfatase/isolamento & purificação , Animais , Bovinos , Cromatografia por Troca Iônica , Cobalto/farmacologia , Eletroforese em Gel de Poliacrilamida , Manganês/farmacologia , Especificidade por Substrato , Zinco/farmacologiaRESUMO
Two acid phosphatase forms were isolated from chicken liver by gel filtration on Sephadex G-100. These enzymes, termed I and II, have similar Km- and Vmax-values, but differ in molecular weight, optimum pH, sensitivity to various inhibitors and substrate specificity. The results were compared with the numerous literature reports of mammalian acid phosphatases.
Assuntos
Fosfatase Ácida/isolamento & purificação , Isoenzimas/isolamento & purificação , Fígado/enzimologia , Animais , Galinhas , Cinética , Peso MolecularRESUMO
The hemolysate of the pike, Esox lucius, contains multiple molecular forms of hemoglobin molecules. Analytical gel electrofocusing, disc-gel electrophoresis and gel filtration chromatography indicate that the hemoglobin heterogeneity may be due to different charge isomers with mol. wt values similar to that of the human. Ion exchange chromatography on CM-cellulose permits to separate a hemoglobin component in a pure form. Globin chains analysis of this fraction and their isolation was also reported.
Assuntos
Hemoglobinas/isolamento & purificação , Salmonidae/sangue , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Globinas/isolamento & purificaçãoRESUMO
The subcellular localization of high and low molecular weight acid phosphatases in chicken liver was studied. The high molecular weight acid phosphatase is mainly associated with the particulate fraction, particularly with the mitochondrial-lysosomal fraction, whereas the low molecular weight form seems to be a soluble cytoplasmic enzyme. Biochemical properties including optimal pH, molecular weight determination and the effect of some modifier substances indicate that mitochondria-lysosomes and microsomes contain the same high molecular weight acid phosphatase form.
Assuntos
Fosfatase Ácida/isolamento & purificação , Isoenzimas/isolamento & purificação , Fígado/enzimologia , Fosfatase Ácida/metabolismo , Animais , Galinhas , Isoenzimas/metabolismo , Lisossomos/enzimologia , Microssomos Hepáticos/enzimologia , Mitocôndrias Hepáticas/enzimologia , Peso Molecular , Frações Subcelulares/enzimologiaRESUMO
Human liver contains high molecular weight-type Zn2+-dependent acid p-nitrophenylphosphatase (HMW-ZnAP). The enzyme was purified 1000-fold by a new procedure, including preparative isoelectrofocusing. The HMW-ZnAP was homogeneous in non-denaturing disk-gel electrophoresis with an MW of about 93 kDa determined by Sephadex G-100 chromatography. A single polypeptide chain of 43 kDa was detected on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), suggesting a homodimeric structure. The isoelectric point (pI) was 7.2-7.4. Human liver HMW-ZnAP requires Zn2+-ions for activity; other divalent cations are ineffective or act as inhibitors. It dephosphorylated p-nitrophenylphosphate (pNPP) (Km = 0.24 mM), o-carboxyl phenylphosphate (oCPP) (Km = 0.92 mM) and phenylphosphate (PhP) (Km = 1.42 mM). Other substrates including [32P]-labelled casein or phosvitin, adenyl nucleotides and myo-inositol-1-phosphate, were not dephosphorylated. Human liver HMW-ZnAP obeys Michaelis-Menten kinetics with pNPP as substrate; the enzyme was competitively inhibited by inorganic phosphate (Ki = 0.55 mM), and by oCPP (Ki = 0.65 mM) and PhP (K = 1.16 mM). Adenosine monophosphate (AMP), adenosine diphosphate (ADP) and ATP displayed mixed-type inhibition. The enzyme was also inhibited by some modifiers such as EDTA, oxalate, p-chloromercurybenzoate, tartrate, imidazole, cyanide, cysteine, histidine and diethylpyrocarbonate, but not by fluoride or okadaic acid. Human liver HMW-ZnAP is sensitive to temperatures higher than 40 degrees C. The pH-dependence of the steady-state kinetic parameters indicates the existence of an essential ionizable group with a pKa of 7.25-7.50, similar to that of histidine. However, diethylpyrocarbonate inactivation experiments suggest that other amino acid residues may also be involved in enzyme catalysis.
Assuntos
4-Nitrofenilfosfatase/isolamento & purificação , Fígado/enzimologia , Zinco/metabolismo , 4-Nitrofenilfosfatase/análise , 4-Nitrofenilfosfatase/antagonistas & inibidores , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Focalização Isoelétrica , Cinética , Peso Molecular , Especificidade por Substrato , TemperaturaRESUMO
Low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase is largely expressed in chick brain tissue during development. The enzyme was purified from brain extract prepared from 19-day-old chick embryos and from adult chickens using ammonium sulfate fractionation, gel filtration on Sephadex G-75 and two DEAE-Cellulose ion-exchange chromatography steps. The purified enzymes from embryo and adult chick brains show identical molecular weight values (about 18-20 kDa) and biochemical and structural properties such as substrate specificity, sensitivity to inhibitors, and number of free reactive sulphydryl groups. These data suggest that they are the same enzyme protein. Although the total acid phosphatase activity does not change appreciably during development, the activity associated with the low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase markedly increases after birth and reaches the adult values within the first week of life. Taken together, our results suggest an involvement of the low molecular weight acid phosphatase/phosphotyrosyl protein phosphatase in postnatal development and maturation of chick brain tissue. The variations in tyrosine phosphorylation profile of chick brain polypeptides analyzed by Western blotting at the same developmental stages are also reported.
Assuntos
Fosfatase Ácida/metabolismo , Encéfalo/enzimologia , Proteínas Tirosina Fosfatases/metabolismo , Fosfatase Ácida/química , Animais , Western Blotting , Encéfalo/embriologia , Embrião de Galinha , Embrião não Mamífero/enzimologia , Desenvolvimento Embrionário , Peso Molecular , Proteínas Tirosina Fosfatases/químicaRESUMO
A new minor haemoglobin, L, was isolated from the haemolysates of chicken embryos more than 7 days old. Electrophoresis in denaturing conditions and tryptic peptide maps of the globins show that the beta-like globin of HbL is identical to that of the minor haemoglobin H(beta H) while the alpha-like globin is very similar to that of the adult haemoglobin D (alpha D). HbL completes the description of the map of the minor chicken haemoglobins during embryonic development. In early embryos two minor haemoglobins, M and E, are produced which have the same beta-like globin (epsilon) and differ in their alpha-like globins (alpha D and alpha A, respectively). The same two alpha-like globins will make up the minor haemoglobins of the late embryo, L and H, which differ from HbM and HbE on account of their beta-like globin (beta H). The native tetramers L and M are hard to distinguish from each other. However the constituent epsilon globin can be easily separated from beta H by electrophoresis on polyacrylamide gel in formic acid. With this method we found that the switch of the minor haemoglobins in the blood of chicken embryos starts at the 7th incubation day. The two red cell populations, primitive and definitive, present in the blood of 7-day-old embryos were separated on an albumin gradient and their minor haemoglobins analysed. The haemoglobin couple M/E was found in the primitive erythroid cells whereas the L/H couple was found in the definitive ones. The disappearance of the early haemoglobin couple and its substitution by the late one during embryonic development correlates with the replacement of erythroid lines in the blood.
Assuntos
Eritrócitos/análise , Hemoglobinas/isolamento & purificação , Animais , Embrião de Galinha , Eletroforese em Gel de Poliacrilamida , Focalização Isoelétrica , Mapeamento de PeptídeosRESUMO
1. Four acid phosphatases (AcPase I, II, III and IV) were found in the liver of the frog Rana esculenta. 2. AcPases I, II, III, and IV were associated with the microsomal, mitochondrial-lysosomal, nuclear and soluble fractions respectively and showed apparent molecular weights of about 240,000, 110,000, 38,000 and 17,000. 3. All the enzymes show acid pH optima, and similar Km values for p-nitrophenylphosphate. 4. AcPases I, II, and III hydrolyze most of the common phosphate esters whereas AcPase IV hydrolyzes effectively only p-nitrophenylphosphate, phenylphosphate and flavine mononucleotide. 5. AcPases I and II are inhibited by NaF and tartrate. AcPases III and IV are tartrate resistant. 6. Temperature inhibits AcPases I, II, IV, whereas it activates AcPase III.
Assuntos
Fosfatase Ácida/isolamento & purificação , Isoenzimas/isolamento & purificação , Fígado/enzimologia , Rana esculenta/metabolismo , Animais , Cromatografia em Gel , Hidrólise , Cinética , Fígado/análise , Frações Subcelulares/enzimologia , TemperaturaRESUMO
Three haemoglobins were isolated by ion-exchange chromatography from the haemolysates of embryonic duck erythrocytes up to 8 days of development. The component globins were characterized both by electrophoresis in dissociating conditions and by finger-printing analysis. The major haemoglobin fraction E1 appears to be an embryonic tetramer since its constituent globins are different from all the others synthesized during embryonic and adult life. The two minor fractions E2 and E3 show alpha-type subunits that are very similar to those of the two adult haemoglobins A1 and A2 respectively. They are present all through embryonic life, as demonstrated by chromatographic analysis. For these reasons they have been considered foetal. The two haemoglobins typical of the adult animal are found in the red cells of the embryo from 8 days of incubation. Their relative amounts change continuously during embryonic development and reach the adult value after hatching.
Assuntos
Patos/embriologia , Hemoglobinas/análise , Animais , Cromatografia por Troca Iônica , Patos/sangue , Eletroforese em Gel de Poliacrilamida , Eritrócitos/análise , Hemoglobina A/análise , Hemoglobina E/análise , TripsinaRESUMO
A protein kinase, type NII, has been purified from wheat germ chromatin. The enzyme, which uses both ATP and GTP as phosphoryl donors, catalyzes the phosphorylation of casein, phosvitin and E. coli RNA polymerase, but not of histone proteins. Polypeptide bands at 46 kDa, 37 kDa and 25 kDa were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autophosphorylation of the 25 kDa subunit was observed following incubation of the purified kinase with (gamma-32P)ATP and (gamma-32P)GTP.
Assuntos
Cromatina/química , Proteínas Quinases/isolamento & purificação , Triticum/enzimologia , Caseínas/metabolismo , Núcleo Celular/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Cinética , Fosforilação , Fosvitina/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Triticum/ultraestruturaRESUMO
3 molecular forms (P1, P2 and P3) of acid phosphatase (E.C. 3.1.3.2) have been detected in chicken liver homogenate. The different intracellular localization of these molecules has been demonstrated by cellular fractionation and electrophoretic analysis. P1 and P2 phosphatases are both present in the particulate fraction. P3 is present in a pure form in the soluble fraction. The difference between the enzyme molecules present in the particulate fraction and that in the soluble one is confirmed by the different activation-inhibition effect of various ions and substances on the enzymatic activity of subcellular fractions.