1. The effects of okadaic acid (OA) and phorbol-12-myristate-13-acetate (PMA) on protein phosphorylation were studied in human term placentas. 2. When samples treated with tumour promoters were compared with untreated samples, the phosphorylation of a 135 kDa protein was significantly decreased; OA also produced a decrease in phosphorylation of a 24 kDa protein. 3. Both substances produced an alteration in the proportions of bands of masses 170, 65 and 24 kDa, relative to total phosphorylation; PMA treatment also affected the band of mass 135 kDa. 4. Placental cell extracts were also subjected to Western blotting with a protein kinase C (PKC) antibody, reportedly specific for the alpha- and beta-isoforms. 5. Two immunoreactive proteins were detected; an 80 kDa band, presumably corresponding to the alpha- or beta-PKC, and a 64 kDa protein, which could be a degradation production of the 80 kDa protein or it could correspond to another form of the enzyme. The expression of PKC did not change on treatment with PMA.
1. High-Km aldose reductase purified from dog kidney inner medulla was easily converted into aldose reductase by incubation in the neutral buffer solution. 2. High-Km aldose reductase was found to be in multiple forms, and was separated into three kinds of species designated as a-, b- and c-forms by HPLC. 3. The a-form observed as a single peak by HPLC was assumed to be present in three forms (a1-, a2- and a3-forms), one was aldose reductase (a1-form) and the others were the precursors of aldose reductase (a2- and a3-form). 4. The b-form was rapidly converted into the a3-form, followed slowly by the a2-form and finally into the a1-form. 5. The c-form was either directly converted into the a1-form, or indirectly into the a2-form followed by the a1-form. 6. Four kinds of species (a2-, a3-, b- and c-forms) of high-Km aldose reductase were finally converted into aldose reductase (a1-form).
1. The sialidase activity of human thymocyte was examined by a fluorogenic assay. 2. These studies revealed that human thymocyte sialidase activity is essentially acid-active and membrane-bound since 59.6% and 33% of the total activity was recovered in the lysosome-enriched and microsomal fractions, respectively. 3. A weak activity was also detected in the cytosolic fraction. 4. However, the acidic optimum pH of this soluble sialidase was at variance with the general concept of mammalian soluble sialidases which are known to be optimally active at more neutral pH. 5. This acidic soluble sialidase seems to be a general characteristic of the human T-cell lineage since examination of mature circulating T-cells revealed that they contain a soluble sialidase activity similar to that observed in thymocytes. 6. Analysis of mature and immature thymocyte subpopulation obtained by differential PNA agglutination indicated that this enzymatic system was not altered during the course of thymic maturation. 7. These results suggest that unlike in T-cell activation where changes in the level of sialidase activity were shown to influence the extent of cell surface sialylation and thereby the cell physiology, this enzymatic system seems not to be involved in the fluctuation of cell surface sialic acid content observed during thymic maturation.
1. Treatment of isolated rat liver mitochondria with methyl methacrylate (MM) produced membrane disruption as evidenced by the release of citrate synthase, and changes in the ultrastructure of mitochondria.2. At concentration 0.1%, MM uncoupled oxidative phosphorylation as evidenced by stimulation of state 4 respiration supported either by pyruvate plus malate or succinate (+rotenone) and ATP-ase activity in intact mitochondria.3. At concentration 1% MM stimulated ATP-ase activity in intact mitochondria and succinate (+ rotenone) oxidation at state 4 and was without effect on this substrate oxidation at state 3.4. MM inhibited pyruvate plus malate oxidation either at state 3 or in the presence of uncoupling agents.5. MM inhibited the NADH oxidase of electron transport particles at a concentration which failed to inhibit either succinic oxidase or the NADH-ferricyanide reductase activity.6. The data presented suggest that in the isolated mitochondria MM inhibits NADH oxidation in the vicinity of the rotenone sensitive site of complex I.7. The general conclusion is that MM may block an electron transport and to uncouple oxidative phosphorylation in rat liver mitochondria. The overall in vitro effect would be to prevent ATP synthesis which could result in cell death under in vivo conditions.
In conclusion, it appears that in general an increase in the fatigue resistance of a muscle is accompanied by an increase in its oxidative capacity. Fatigue resistance of a muscle seems to be partly determined by its oxidative capacity. On the single motor unit (Burke et al, 1973; Hamm et al, 1988; Kugelberg and Lindegren 1979; Larsson et al, 1991) and single fibre level (Nemeth et al, 1981) the relation between fatigue resistance and oxidative capacity seems to be valid. However, this does not appear necessarily to be the case on the level of the whole muscle. Kugelberg and Lindegren (1979) suggested, that the endurance of each link in the chain of events leading to contraction is under aerobic conditions matched to the contractile capacity of the fibre expressed by its oxidative enzyme activity. Therefore, it might be that several tests for endurance capacity are more strenuous than the aerobic capacity of the muscle. Indeed, several studies suggest that the Burke test (Burke et al, 1973) or other fatiguing protocols might primarily test for other endurance-related properties as the excitation-contraction coupling (Kernell et al, 1987; Mayne et al, 1991b). Another explanation for the discrepancy in changes in oxidative capacity and fatigue resistance might be, that the mechanical responses of the motor units (which have different biochemical and contractile properties) during the fatigue test do not summate linearly during whole muscle contraction as was found by Gardiner and Olha (1987).(ABSTRACT TRUNCATED AT 250 WORDS)
1. The effect of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE) on the formation of thromboxane (TX) B2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid in washed rabbit platelets was examined. 2. 13-HPODE inhibited TXB2 and HHT formation without affecting 12-HETE production. 3. 13-Hydroxy-9,11-octadecadienoic acid which was produced rapidly from 13-HPODE, did not suppress the formation of TXB2 and HHT, indicating the requirement of the hydroperoxy moiety for the inhibitory effect of 13-HPODE on TXB2 and HHT formation. 4. Experiments utilizing mannitol and dimethyl sulfoxide (hydroxy radical scavengers) revealed that the action of 13-HPODE is not due to hydroxy radicals which are expected to be formed from 13-HPODE. 5. These results suggest that 13-HPODE is a selective inhibitor of platelet cyclo-oxygenase and may have functional effects within platelets.
This review describes the protein kinases that are involved in long-term potentiation (LTP). The following items are described.1. Ca2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC) may play pivotal roles in the different phases of the expression of LTP. This involvement has been indicated mainly by using specific inhibitors of these kinases. The involvement of the CaMKII alpha-subunit was confirmed in mutant mice which were deficient in the gene for the subunit.2. Involvement of persistently active protein kinases in the maintenance of LTP has been proposed and, since then, several studies have focused upon the persistent kinase. Both PKC and CaMKII are possible sources of the persistent kinase activities.3. Protein kinases other than CaMKII or PKC (ex. protein kinase A, tyrosine kinases, mitogen-activated kinase) also play roles in the expression of LTP.4. Finally, the importance of postsynaptic density as a device/where complex chemical reactions related to neuronal signal transduction occur is discussed.
Attachment of mammalian embryos to the uterine wall involves the coordinated development of both the embryo and the uterine epithelium to an attachment-competent state. This coordination is achieved directly or indirectly through the actions of ovarian steroids. Acquisition of attachment competence is proposed to reflect two processes. The first is the loss of non-adhesive glycoproteins at the cell surface of embryos, e.g. zona pellucida subunits, as well as uterine epithelial cells, e.g. mucin glycoproteins. The second process is the functional expression of complementary adhesion-promoting molecules at these cell surfaces. A series of studies indicates that heparan sulfate proteoglycans and their corresponding binding sites can play an important role in the initial stage of embryo attachment to the uterine surface.
1. In the presence of polyethylene glycol, the self-association of human spectrin is enhanced in a manner that depends approx. exponentially on the mass concentration of polyethylene glycol. 2. For a given mass concentration, the enhancement is independent of the molecular weight of the polyethylene glycol. 3. These data are consistent with the operation of excluded volume effects, and support the contention that the association of spectrin is likely to be increased in the presence of the high concentration of hemoglobin within the erythrocyte in vivo.
1. Adenosine 5'-phosphoramidate hydrolase of 29 kDa was isolated from rat liver cytosol. 2. It consisted of two subunits of 14 kDa. 3. It hydrolyzed nucleoside 5'-monophosphoramidates into nucleoside 5'-monophosphates and ammonia, while it did not hydrolyze adenylyl phosphoramidate, adenylyl imidodiphosphate and N-phosphorylated compounds like phosphocreatine, N omega-phosphoarginine, 6-phospholysine and 3-phosphohistidine. 4. Divalent cations and cyclic AMP had no effect on the hydrolytic activity.
A computer comparison of protein sequences revealed similarity between the 30.4 kDa subunit of complex I from the fungus Neurospora crassa and the ORF5 subunit of formate hydrogenlyase from Escherichia coli. The ORF5 protein was previously known to be homologous to the 49 kDa component of the mitochondrial enzyme. We show that the 30.4 kDa corresponds to the N-terminal part while the 49 kDa subunit corresponds to the C-terminal portion of the bacterial protein. Thus, this bacterial protein represents a fusion of the two mitochondrial polypeptides suggesting that the two complex I genes arose from a single ancestor. Our results indicate that the 30.4 kDa and 49 kDa subunits are part of a structural and functional unit in complex I.
1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity.2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5-20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein.3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein.4. The rat testis AcPase IV is a metal activated enzyme in which Mg2+ is the metal activating agent with a K-a = 0.88 x 10(-3) M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg2+ ions, is 0.23 x 10(-3) M.5. The enzyme preferentially hydrolizes p-nitrophenylphosphate, phenylphosphate and ATP.
1. We have studied fatty acid oxidation and ketone bodies production in liver mitochondria from rats exposed to cold for 10 days and in rats adapted to cold for 40 days. We have examined State 3 respiration and ketone bodies production with palmitoylcarnitine as substrate in isolated mitochondria, together with mitochondrial protein mass and hepatocyte oxygen consumption. 2. Mitochondrial protein mass increases both in cold exposed and in cold adapted rats. 3. State 3 respiration and hepatocyte respiration increase only in cold exposed rats. 4. Ketone bodies production increases in cold adapted rats. 5. Taken together, our results suggest that the increased hepatic mitochondrial protein mass and the increased fatty acid oxidation subserve two different roles: an increase in ATP production in cold exposed rats and an increase in ketone bodies production in cold adapted rats.
1. Exogenous and endogenous. tyrosine protein phosphorylation activities were examined in soluble and particulate fractions from various normal tissues by using poly-[Glu(80)Na, Tyr(20)] and a monodonal antibody specific for phosphotyrosine. 2. Phosphorylation of the exogenous substrate by the particulate forms of TPKs was 2- to 10-fold higher than by soluble forms. The activities of particulate and soluble enzymes decreased in the following order: spleen > (thymus = kidney) > testes greater than or equal to (pancreas = liver = brain) > heart. 3. The level of endogenous phosphorylation in the tissues decreased respectively in the following order: thymus > brain greater than or equal to (pancreas = liver) > spleen > testes > kidney > heart for the particulate fractions, and spleen > thymus > brain > pancreas greater than or equal to liver > testes > kidney > heart for the soluble fractions. 4. A large number of phosphotyrosine-containing proteins were detected. In addition, several phosphotyrosine-containing proteins of similar molecular weight were found in different tissues and fractions.
1. The effect of URO I on the activity of ALA-D, PBGase, deaminase and URO-D, both in aerobiosis and anaerobiosis, was studied. 2. Photoinactivation of the enzymes was much lower in an anaerobic than in an aerobic atmosphere. 3. Dark inactivation in the absence of oxygen was lower than its presence. 4. Preincubation in the presence of ALA or PBG protected the enzymic activity of ALA-D, PBGase and deaminase against URO I-inactivation both under u.v. light and in the dark. 5. Photoinactivating action of URO I would be mediated by reactive oxygen species generated by the excited porphyrin after its absorption of light. Dark inactivation, in aerobiosis, can also be partly mediated by amino acid oxidation, although to a lesser extent than that observed under u.v. light.
1. A modification of the Nelson and Mason (1970) method for tyrosinase (EC 1.14.18.1) extraction, purification and separation from Agaricus bisporus is proposed.2. Modifications primarily concern: (a) enzyme recovery by performing pellet re-extractions, (b) preparative isoelectric focusing purification replacing a gel-permeation, (c) use of polyvinyipyrrolidone to remove contaminant ''brown proteins'', instead of basic Pb acetate.3. With this procedure, it is possible to enhance the amount of tyrosinase recovered, thus reducing the quantity of mushrooms required (90% less). The enzyme obtained displays a better degree of purification (between 81- and 92-fold), and the total extraction time is lower than that of the original technique.
