Protein synthesis in mouse embryo fibroblastic (NIH-L1) cells was stimulated by addition of fibronectin (FN) to the culture medium as a soluble factor. This stimulatory activity of FN appeared only when the cells were cultured over their saturated density. A hexapeptide (GRGDSP) containing the RGDS cell attachment site of FN could not prevent the stimulation of protein synthesis. Moreover, activity of the stimulatory FN was not affected by inclusion of cytochalasin B, which is known to release FN from cell surface by disorganizing actin cytoarchitecture. These results suggest that the signaling of change in protein synthesis rate by FN may be distinct from signaling involving cell attachment and spreading.
Mouse thymocytes were fractionated into heavy (subtype I, 79% of total cell number), medium (subtype II, 18%) and light (subtype III, 3%) ones by Percoll density centrifugation and they were identified as immature (subtype I and II) and mature (subtype III) thymocytes based on their proliferative response to mitogens. Whereas the nuclear activity of poly (ADP-ribose) polymerase (EC 2.4.2.30) in the subtype III was only one half that of denser subtypes, it increased two-fold upon mitogen stimulation. The sensitivity of three thymocyte subtypes to the dexamethasone cytotoxicity, as judged by the extent of the DNA cleavage, depletion of NAD and cell viability, was highest in the subtype I and lowest in the subtype III. The possible involvement of poly ADP-ribosylation in the apoptotic (programmed) cell death during intrathymic development of immature to mature thymocytes is discussed.
The effects of arachidonic acid and arachidonoyl CoA on the catabolism of prostaglandin E2 in a 105000 x g supernatant fraction from rabbit kidney cortex were examined. Arachidonic acid reduced the 15-hydroxy prostaglandin dehydrogenase activity by 50% at 20 microM, while arachidonoyl CoA showed weak inhibition for the enzyme activity (15% at 20 microM). However, arachidonoyl CoA strongly inhibited the prostaglandin delta 13 reductase activity, the concentration required for 50% inhibition being about 3 microM. The dehydrogenase inhibition by arachidonic acid was non-competitive with regard to NAD+ and prostaglandin E2, respectively. Arachidonoyl CoA was also a non-competitive inhibitor for the reductase with regard to NADH and 15-keto prostaglandin E2, respectively. These results suggest that arachidonic acid and arachidonoyl CoA can be important modulating factors in prostaglandin catabolism by the kidney cortex.
The degradation of big-endothelin (big-ET) in the soluble fraction of porcine lung was investigated. The degradation in the presence of p-chloromercuribenzoate (PCMB), pepstatin A, and EDTA resulted in the accumulation of two newly-formed fragments, big-ET (23-39) and big-ET (1-22), the latter called endothelin-valine (ET-Val). The generation of the two fragments was inhibited by diisopropylfluorophosphate (DFP). The enzyme responsible, called ET-Val-generating endopeptidase, was isolated from porcine lung by a procedure including chromatographies on columns of DEAE-cellulose, hydroxylapatite, Mono Q, p-mercuribenzoate-Sepharose, and Superose 6. The molecular weight of the enzyme was 140,000 and the pH optimum of the activity was 7.0. The activity was strongly inhibited by DFP, but scarcely inhibited by PCMB, EDTA, and pepstatin A. Thus, the isolated enzyme was classified as a serine protease cleaving big-ET at the Val22-Asn23 bond.
We measured the binding of a drug oxyphenylbutazone to the N-terminal peptic fragment of human serum albumin in 0.1 M Tris buffer, pH 8.0 (Kass = 2.4 10(5) M-1) and in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate and buffer in isooctane (Kass. = 2.7 10(5) M-1). In the absence of any measured change in conformation of the fragment in reverse micelles, the peptide affinity for the drug is not decreased, in contrast to what is observed in intact albumin (HSA) under similar conditions. The interaction and the subsequent unfolding of HSA at the membrane-mimetic interface, constitutes thus a drug release-facilitating mechanism.
Bovine brain adenosine deaminase cytoplasmatic form was purified about 450 fold by salt fractionation, column chromatography on DEAE-cellulose, octyl-sepharose 4B and affinity chromatography on CH-sepharose 4B 9-(p-aminobenzyl)adenine. The purified enzyme was homogeneous on disc gel electrophoresis; the enzyme had a molecular mass of about 65 kDa with an isoelectric point at pH 4.87. The Km values for adenosine and 2'-deoxyadenosine were 4 x 10(-5) and 5.2 x 10(-5) M, respectively. The enzyme showed a great stability to temperature with a half life of 15 hours at 53 degrees C significantly different compared to that known for other mammalian forms of this enzyme. Aza and deaza analogs of adenosine and erythro-9-(2-hydroxy-3-nonyl) adenine were good inhibitors of the bovine brain enzyme with little difference with respect to those reported for the adenosine deaminases purified from other sources. Kinetic constants for the association and dissociation of coformycin and 2'-deoxycoformycin with the bovine brain adenosine deaminase are reported.
A poly(U), poly(C) specific RNase of apparent MW 34 kDa has recently been purified from 6 day old larvae of the insect Ceratitis capitata. Two monoclonal antibodies were obtained by immunizing mice with this protein. Immunoblot analysis of the RNase revealed that both antibodies recognize the 34 kDa protein. Furthermore, immunoprecipitation experiments show that both antibodies were capable of precipitating the ribonuclease without affecting its catalytic activity.
A specific protein exhibiting immunological cross-reactivity with chicken riboflavin carrier protein has been purified to homogeneity from human amniotic fluid by use of ion-exchange and affinity chromatography. The protein is similar to its avian counterpart in terms of molecular size, distribution of 125I-labelled tryptic peptides during finger printing, and preferential binding to riboflavin. Immunologically, they are homologous since most of the monoclonal antibodies raised against the avian protein cross-react with the purified human vitamin carrier.
The aim of this study has been to compare the effects of chronic ethanol administration on the lipid composition and fluidity of chick-liver microsomes and mitochondria. Cholesterol levels were increased only in microsomal membranes whilst the fatty-acid profiles were considerably altered in the major phospholipids in both membranes. By using fluorescence polarization we found that ethanol produces a disorganisation of acyl chains in the core of microsomes and mitochondria and consequently causes higher fluidity. In addition, opposite changes were induced by ethanol in the polar region of the microsomes, inducing a more rigid area in the membrane.
In this study we have examined the effect of hCG-induced desensitization on RNA synthesis in rat Leydig cells. In vitro [3H]-uridine incorporation into RNA decreased after a high, single dose of hCG (100 IU). This effect was maximal after the second and third day of treatment. When Leydig cells were incubated in vitro for 30 min or more, a marked decrease of total and poly(A)+ RNA synthesis was observed. This was not due to reduced cell permeability to the radioactive nucleotide, indicating a truly decreased RNA synthesis during desensitization. The magnitude of this inhibitory effect (73-80%) suggests that desensitization may involve other biological functions of Leydig cell.
In the present study adrenergic and muscarinic cholinergic receptors have been investigated in human lung parenchyma in cancer and previous airways diseases (chronic pneumonia, tuberculosis). It has been found that the number of muscarinic sites significantly increases in cancer and does not change both in chronic pneumonia and tuberculosis lung parenchyma in comparison with the normal tissue; the number of beta-adrenergic sites decreases in cancer, chronic pneumonia, as well as severe tuberculosis lung parenchyma. The important role of beta-adrenergic and muscarinic receptors in the formation of human lung adenocarcinoma is discussed.
The interferon antagonist sarcolectin, the protease inhibitor aprotinin and calcyclin whose expression is regulated by growth stimulation in quiescent fibroblasts display sialic acid-dependent binding to fetuin, visualized by solid-phase assays using biotinylated fetuin. The potential functional importance of this property prompted its comparative characterization with an array of neutral or negatively charged sugars, sulfated polysaccharides and sialoglycoproteins as inhibitors of binding of biotinylated fetuin to the immobilized proteins. The results revealed that this activity of sarcolectin and calcyclin is nearly unaffected by charge-free carbohydrates in contrast to aprotinin, calcyclin exhibits a notable affinity for Neu5Gc and together with aprotinin for phosphorylated sugars, and that sarcolectin's binding is affected to the highest extent by sulfated sugars relative to aprotinin and calcyclin.
A protease from house dust mite(Dermatophagoides farinae) having high specificity towards a substrate of blood coagulation factor XIIa catalyzes the activation of kallikrein-kinin system in plasma (Takahashi et al., 1990). To prevent the formation of kinin by the mite-protease, inhibition of the protease with its inhibitors was tested in vitro and in vivo. Its kinetic studies revealed that Ki values are 3.9 x 10(-10) M for aprotinin, 3.0 x 10(-9) M for soybean trypsin inhibitor (Kunitz) and 2.5 x 10(-8) M for gabexate mesylate. Enhancement of blood permeability in guinea pigs caused by the protease was markedly suppressed by these inhibitors.
The pseudo-first-order rate constant of rabbit muscle creatine kinase (CK), in the direction of ATP synthesis (kf), was determined by saturation-transfer 31P NMR. When pH was varied between 6.0 and 7.4, kf increased linearly at both 20 degrees C and 37 degrees c. The corresponding flux is very small between pH 6.0 and 6.5, in contrast to previous studies. Up to 50 h exposure of the CK enzyme to high concentrations of inorganic phosphate (Pi), a known inhibitor in certain situations, had negligible effect on enzymatic flux in the physiological pH range. Thus under in vivo conditions, such as in stroke, where pH falls as low as 6.2 and Pi rises to high levels, the rate of the CK reaction may be severely reduced due to pH but not due to high Pi concentrations.
Early biosynthesis of short-life ATP was observed in plasma membranes of target cells stimulated by insulin or other polypeptide growth factors in the presence of all components of aerobic phosphorylation and cytochrome c. The effect is always mediated by the binding of insulin or growth factors to specific receptors. Erythrocyte plasma membranes are a convenient model to study the phenomenon. Insulin-stimulated synthesis of the plasma membrane "signal" ATP in an amount of 1-10 nM is potentized by ionophores carbonyl cyanide p-trifluorometoxyphenylhydrazone and monensin and inhibited by amiloride and ouabain. It is supposed that the plasma membrane "signal" ATP readily generated in response to a growth or mitogenic factor is an "amplifier" or "coupling agent" in the transduction of a signal to growth, proliferation, and mitogenesis. Biosynthesis of the plasma membrane "signal" ATP seems to be associated with partial reversion of Na+, K+ -ATPase with the participation of the plasma membrane redox chain as a proton generator.
The purpose of the present study was to purify and kinetically characterize N-acetyl-beta-D-hexosaminidases A and B (EC 3.2.1.52) from the caput, corpus and caudal regions of the adult rat epididymis. The molecular mass of the purified native enzyme was approximately 250,000 and approximately 223,000 daltons for the A and B isozymes, with a subunit molecular mass of approximately 63,000 and approximately 56,000 daltons, as determined by size exclusion chromatography and gel electrophoresis under reducing conditions. The apparent Michaelis-Menten constant and maximum velocity values were 0.60, 1.55 and 0.68 mM and 0.54, 3.20 and 2.30 microM/min./mg protein for the enzyme purified from the caput, corpus and caudal regions, respectively. These values were determined by using p-nitrophenyl-N-acetyl-beta-D-glucosaminide as the substrate. These data suggest that the enzyme may be more active in the corpus region of the epididymis than in the caput and caudal regions.
Parietal cell secretion can be stimulated by both histaminergic and cholinergic agonists. We have recently found that inhibition of calmodulin-dependent protein kinase II (CaMK II) activity can abolish cholinergic but not histaminergic stimulation of parietal cell secretion (Am. J. Physiol. 262:G118-122). We have investigated the presence of calmodulin-binding proteins and CaMK II in isolated rabbit parietal cells. Calmodulin-binding proteins with apparent molecular masses of 50, 60, 85, 100, and 240 kDa were observed. The major calmodulin-binding species was a 50 kDa band which was enriched in 50,000 g. microsomal membranes. The 50 kDa calmodulin binding comigrated with immunoreactivity for CaMK II. Partial purification of the microsomal CaMK II demonstrated a 250 kDa oligomer. The results demonstrate that CaMK II is the major calmodulin-binding protein in parietal cells and is associated primarily with light microsomal membranes.
Tumor promoter (phorbol-12-myristate-13-acetate:PMA) stimulates the production of prostaglandin E2 (PGE2) in a dose- and time-dependent manner in cloned rat thymic epithelial cells, TEA3A1. This stimulation of PGE2 production by PMA was blocked by pretreatment of cells with protein kinase C (PKC) inhibitor staurosporin and was abolished in PKC down modulated cells. PMA treatment significantly stimulated the release of arachidonic acid from the cells, but had no effect on arachidonic acid incorporation and on cyclooxygenase enzymatic activity of the cells. These results indicate that PMA stimulates PGE2 production through PKC mediated activation of phospholipase A2 (PLA2) in thymic epithelial cells.
Renal cortex homogenates from aged (greater than 5 y) rabbits showed decreased specific activities of brush border membrane enzymes compared to those from control young (6 m) rabbits but the specific enzyme activities of basolateral membrane, endoplasmic reticulum and mitochondria did not differ between the two groups. The stimulatory effects of parathyroid hormone (PTH) on the Ca(2+)-pump enzyme [(Ca(2+)+Mg2+)-ATPase] activity in kidney cortex homogenates were markedly less in aged rabbits, but the effect of cAMP on this enzyme activity was similar. Moreover, the production of cAMP induced by PTH was markedly less in the renal cortex homogenates from aged rabbits. From these results, we have proposed the following mechanism; aging--decrease in the response of cAMP to PTH in renal cortex--decrease in the stimulatory effect of PTH via cAMP on the Ca(2+)-pump enzyme--decreased reabsorption of Ca2+ from ureter--increased urinary Ca2+ secretion. This pathway may contribute to the worsening of senile osteoporosis.
A polysaccharide-peptidoglycan complex containing different phosphorylated sugars from Micrococcus lysodeikticus cell wall has been isolated and purified. The peptidoglycan contained muramic acid 6-phosphate and N-acetylglucosamine 6-phosphate as phosphorylated sugars in addition to other sugar residues. Mild acid hydrolysis of the peptidoglycan and subsequent reduction of the released polysaccharide showed therein the presence of glucose and N-acetyl-glucosamine in the linkage of the external polysaccharide residues to the peptidoglycan through phosphodiester linkage. These data suggest the presence of polysaccharide chains linked to a peptidoglycan core through two phosphorylated sugars via two different terminal carbohydrate residues of the external polysaccharide chains in a same polymer.
