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In the past, almost all studies on naphthalene cataract were based on in vivo experiments. Such studies are laborious and time-consuming and are complicated by systemic toxicity arising from the metabolites of naphthalene. In order to study the direct effects of naphthalene metabolites on the lens, we established an in vitro 'naphthalene cataract' model system by exposing rat lens to naphthalene dihydrodiol (2.5 x 10(5) M) containing medium for 48 hr. Under these conditions, we analysed several biochemical parameters including the glutathione level, protein mixed disulfides, protein patterns on SDS-gels, active transport, NA+/K(+)-ATPase activities and the measurement of naphthalene metabolites in the cultured lenses. The results showed that both the morphological and biochemical changes were very similar to those observed in lenses of rats fed naphthalene (1 g kg-1 day-1). Furthermore, ALO1576 completely blocked the in vitro changes as it did in vivo. Therefore, this model system can be used as a new tool to investigate the mechanism of naphthalene cataract formation. Other naphthalene metabolites such as 1-naphthol, 2-naphthol, 1,2-dihydroxynaphthalene and 1,2-naphthoquinone were also studied in vitro and the results showed that the effects of these naphthalene metabolites were very different from those observed in naphthalene cataracts in vivo.
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PMID:Establishment of a naphthalene cataract model in vitro. 154 43

Chaen et al. (1986. J. Biol. Chem. 261:13632-13636) showed that treatment of relaxed single muscle fibers with para-phenylenedimaleimide (pPDM) results in inhibition of a fiber's ability to generate active force and a diminished ATPase activity. They postulated that the inhibition of force production was due to pPDM's ability to prevent crossbridges from participating in the normal ATP hydrolysis cycle. We find that the crossbridges produced by pPDM treatment of relaxed muscle cannot bind strongly to the actin filaments in rigor, but do bind weakly to the actin filaments in the presence and also absence of ATP. After pPDM treatment, fiber stiffness, as measured using ramp stretches of varying duration, is ATP-insensitive and identical to that of untreated relaxed fibers (both at high [165 mM] and low [40 mM] ionic strength). These results suggest that the pPDM-treated crossbridges, in both the presence and absence of ATP, are locked in a state that resembles the weakly-binding myosin ATP state of normal crossbridges. Their resemblance to the ATP-crossbridges of relaxed untreated fibers is quite strong; both bind to actin about equally tightly and have similar attachment and detachment rate constants. We also found that crossbridges are locked in a weakly-binding state after treatment with N-phenylmaleimide (NPM). In muscle fibers, this method of producing weakly-binding crossbridges appears preferable to pPDM treatment because, unlike treatment with pPDM, it does not increase the fiber's resting tension and stiffness and it does not disrupt the titin band seen on SDS-PAGE.
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PMID:Formation of ATP-insensitive weakly-binding crossbridges in single rabbit psoas fibers by treatment with phenylmaleimide or para-phenylenedimaleimide. 154 25

Digital-imaging fluorescence microscopy with fura-2 allows the determination of intracellular calcium concentration ([Ca2+]i) in single cells. At a cell density of 10(5) cells/petri dish 44% of the chick embryo heart cells had a high [Ca2+]i of 99.4 +/- 7.1 nM and 56% of the cells a low [Ca2+]i of 27.8 +/- 4.4 nM (mean +/- SE). This laboratory previously reported that high-[Ca2+]i and low-[Ca2+]i cells from chick embryo hearts differ in their sensitivity to cardiac glycosides, as shown by measuring the increase in [Ca2+]i to reach a new steady state [Ahlemeyer, B., Weintraut, H., Seibold, G. & Schoner, W. (1991) in The sodium pump: recent developments (Kaplan, J. H. & De Weer, P., eds) pp. 653-656, Rockefeller University Press, New York]. This time we used N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) which binds irreversibly to amino groups of the Na+/K(+)-ATPase, and sheep anti-digoxigenin Fab fragments coupled with fluorescein isothiocyanate to identify different cardiac glycoside-binding sites. Half-maximal labelling of high-[Ca2+]i cells was obtained at 0.36 nM HDMA, and at 12.0 nM with the low-[Ca2+]i cells. Specific labelling of the cells by HDMA was 91% and 80% in high-[Ca2+]i and low-[Ca2+]i cells, respectively, as revealed by competition experiments with a 1000-fold excess of ouabain. HDMA half-maximally elevated the [Ca2+]i of high-[Ca2+]i cells at a concentration of 50 pM and that of low-[Ca2+]i cells at 8.0 nM. Concentrations higher than 0.1 microM produced signs of intoxication. When the labelled cells were subjected to a SDS/PAGE, a 100-kDa band was found to contain HDMA. The electrophoretic mobility of a protein labelled at 10 nM HDMA was slightly higher than that of a protein labelled at 1.0 microM. The data suggest that different isoforms of the alpha-subunit of Na+/K(+)-ATPase may exist in low-[Ca2+]i and high-[Ca2+]i cells of chick embryo heart.
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PMID:Chick heart cells with high intracellular calcium concentration have a higher affinity for cardiac glycosides than those with low intracellular calcium concentration, as revealed by affinity labelling with a digoxigenin derivative. 155 87

A method has been developed for the purification of bovine adrenal kinesin combining ion exchange chromatography on phosphocellulose and Mono-Q (FPLC), affinity binding to microtubules in the presence of tripolyphosphate and gel filtration on Superose 6 (FPLC). From 100 g of tissue this procedure yields 200 micrograms of a remarkably pure kinesin as assayed by SDS-PAGE and electron microscopy of rotary shadowed specimens. The enzyme has a Ca++ ATPase of 0.4 mumol/min per mg and a Mg++ ATPase of 0.03 mumol/min per mg in the absence of microtubules. The addition of microtubules (5 microM) activates the Mg++ ATPase activity by almost 70-fold to a value of 1.9 mumol/min per mg. This purification procedure results in a fairly large amount of a remarkably pure adrenal kinesin with high specific activity which is an important improvement over the method previously available.
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PMID:An improved method for the purification of kinesin from bovine adrenal medulla. 156 Jan 82

Partially purified (Na+,K+)-ATPase (E.C. 3.6.1.3.) was investigated in the epileptic cortex of audiogenic DBA/2 mice and in the primary and secondary foci of cats with acute or chronic freeze lesions. No differences in specific activities measured at 3 mM K+ were observed between epileptic and control cortex, except an increase of enzymic activities in the primary focus of acutely lesioned cats. The (Na+,K+)-ATPase catalytic subunits were resolved by SDS-gel electrophoresis and their phosphorylation levels were measured in presence of K+ ions and phenytoin. K+ was more effective in inducing maximal dephosphorylation of (Na+,K+)-ATPase in C57/BL, with identical affinity in the two strains. Phenytoin decreased the net phosphorylation level of (Na+,K+)-ATPase by about 50% in C57/BL mice, but only by 20% in DBA/2 mice. Both K+ and phenytoin dephosphorylating influences were decreased in primary and secondary foci of acutely lesioned cats. Those changes were limited to the alpha(-) subunit. In chronic cats, the dephosphorylating step of the (Na+,K+)-ATPase catalytic subunit recovered a normal affinity to K+, but its sensitivity to phenytoin remained decreased. Those differences in K+ and phenytoin influences on brain (Na+,K+)-ATPases between control and epileptic cortex might be responsible for the ictal transformation and seizure spread. In cats, the alteration of the alpha(-) isoform could mainly affect the glial cells.
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PMID:Phosphorylation of brain (Na+,K+)-ATPase alpha catalytic subunits in normal and epileptic cerebral cortex: I. The audiogenic mice and the cat with a freeze lesion. 165 58

We examined the activity and phosphorylation level of (Na+,K+)-ATPase (E.C. 3.6.1.3) partially purified from normal and epileptic human cortices. Control patients (n = 11) were operated on for a non-epileptogenic deep brain lesion, while epileptic patients (n = 10) were operated on for temporal or frontal originating partial seizures, resistant to medications or secondary to evolutive brain tumors. No differences in the specific activity of microsomal (Na+,K+)-ATPase were observed between the two groups of patients. After partial purification of the enzyme followed by SDS-polyacrylamide gel electrophoresis, (Na+,K+)-ATPase catalytic subunit had a decreased affinity for K+ in human epileptic cortex and lost its sensitivity to phenytoin dephosphorylation. Indirect evidence suggests that those abnormalities of (Na+,K+)-ATPase in human epileptic cortex hold preferentially true for the alpha(-) enzymatic subunit. Those results indicate that, in human epileptic cortex, (Na+,K+)-ATPase and most probably its glial subtype is altered in its K+ regulation and phenytoin sensitivity and could be responsible for ictal transformation and seizure spread.
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PMID:Phosphorylation of brain (Na+,K+)-ATPase alpha catalytic subunits in normal and epileptic cerebral cortex: II. Partial seizures in human epilepsy. 165 59

The Mg(2+)-ATPase present in rabbit skeletal-muscle transverse tubules is an integral membrane enzyme which has been solubilized and purified previously in this laboratory [Kirley (1988) J. Biol. Chem. 263, 12682-12689]. The present study indicates that, in addition to the approx. 100 kDa protein (distinct from the sarcoplasmic-reticulum Ca(2+)-ATPase) seen previously to co-purify with the Mg(2+)-ATPase activity, there are also proteins having molecular masses of 160, 70 and 43 kDa. The 70 and 43 kDa glycosylated proteins (50 and 31 kDa after deglycosylation) are difficult to detect by SDS/PAGE before deglycosylation, owing to the broadness of the bands. Additional purification procedures, cross-linking studies and chemical and enzymic deglycosylation studies were undertaken to determine the structure and relationship of these proteins. Both the 97 and 160 kDa proteins were demonstrated to be N-glycosylated at multiple sites, the 97 kDa protein being reduced to a peptide core of 84 kDa and the 160 kDa protein to a peptide core of 131 kDa after deglycosylation. Although the Mg(2+)-ATPase activity is resistant to a number of chemical modification reagents, cross-linking inactivates the enzyme at low concentrations. This inactivation is accompanied by cross-linking of two 97 kDa molecules to one another, suggesting that the 97 kDa protein is involved in ATP hydrolysis. The existence of several proteins along with the inhibition of ATPase activity by cross-linking is consistent with the interpretation of the susceptibility of this enzyme to inactivation by most detergents as being due to the disruption of a protein complex of associated subunits by the inactivating detergents. The 160 kDa glycoprotein can be partially resolved from the Mg(2+)-ATPase activity, and is identified by its N-terminal amino acid sequence as angiotensin-converting enzyme.
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PMID:The Mg(2+)-ATPase of rabbit skeletal-muscle transverse tubule is a highly glycosylated multiple-subunit enzyme. 165 80

Yeast transformants harboring the CBS1 gene under the control of the strong ADC1 promoter on a high copy number plasmid express the mitochondrial CBS1 protein at artificially high levels. Over-expressed protein is imported into mitochondria and correctly processed to yield the mature mitochondrial 23.5 kDa form, but differs in its solubility properties from CBS1 in wild-type mitochondria. It forms insoluble protein aggregates, which are refractory to solubilization with 1% Taurodeoxycholate. We exploited this observation to separate CBS1 from the bulk of mitochondrial proteins and to isolate CBS1 after SDS gel electrophoresis. Determination of the amino-terminal amino acids of the purified protein reveals that the mature CBS1 protein starts with Ile30, at the characteristic distance of +2 amino acids from an arginine residue (Arg28). The cleavage site shows a remarkable homology to that of subunit 9 of the F0F1 ATPase from Neurospora crassa.
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PMID:Over-expression, purification and determination of the proteolytic processing site of the yeast mitochondrial CBS1 protein. 165 14

Organotin compounds have been shown to interfere with cardiovascular system. We have studied the in vitro and in vivo effects of tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT) on the cardiac SR Ca2+ pump, as well as on protein phosphorylation of SR proteins, in order to understand the relative potency of these tin compounds. All the three tin compounds inhibited cardiac SR 45Ca uptake and Ca(2+)-ATPase in vitro in a concentration-dependent manner. The order of potency for Ca(2+)-ATPase as determined by IC50, is TBT (2 microM) greater than TET (63 microM) greater than TMT (280 microM). For 45Ca uptake, it followed the same order i.e., TBT (0.35 microM) greater than TET (10 microM) greater than TMT (440 microM). In agreement with the in vitro results, both SR Ca(2+)-ATPase and 45Ca uptake were significantly inhibited in rats treated with these tin compounds, indicating that these tin compounds inhibit cardiac SR Ca2+ transport. cAMP significantly elevated (70-80%) the 32P-binding to SR proteins in vitro in the absence of any organotin. In the presence of organotins, cAMP-stimulated 32P-binding to proteins was significantly reduced, but the decrease was concentration dependent only at lower concentrations. The order of potency is TBT greater than TET greater than TMT. In agreement with in vitro studies, cAMP-dependent 32P bound to proteins was significantly reduced in rats treated with TBT, TET and TMT. SDS-polyacrylamide gel electrophoresis of the cardiac SR revealed at least 30 Coomassie blue stainable bands ranging from 9 to 120 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of Ca2+ transport associated with cAMP-dependent protein phosphorylation in rat cardiac sarcoplasmic reticulum by triorganotins. 165 51

Three isoforms of the alpha subunit of Na,K-ATPase, alpha 1, alpha 2, and alpha 3 have been characterized at the DNA, mRNA and protein levels. In admixtures, isoforms migrate as doublets (i.e. alpha 1 and another band originally designated alpha +, comprising alpha 2 + alpha 3) when analyzed by SDS-PAGE. As deduced from cDNA sequences their masses range from 111.7 to 112.6 kDa. With conventional protein standards, however, SDS-PAGE yields nominal masses of 85-105 kDa. In this system, the presence of a doublet that reacted with a polyclonal anti-Na,K-ATPase antibody in the kidney was interpreted as indicating two molecular or conformational species of the kidney alpha sub-unit (Siegel, G.J. and Desmond, T.J. (1989) J. Biol. Chem. 264, 4751-4754). We report that Na,K-ATPase purified from dog, guinea pig and rat kidney medulla or from rat brain, can yield two distinct bands when analyzed by SDS-PAGE or STS-PAGE, migrating between 85 and 105 kDa. An additional band migrating at 117 and 120 kDa appears often in enzyme purified from rat and guinea pig kidney medulla. The apparent molecular weights and relative intensities of these bands vary with temperature and duration of incubation during sample preparation. N-terminal sequencing and monospecific antibody probes revealed that the two distinct bands obtained from the kidney enzyme consist only of the alpha 1 isoform. The band appearing at 117-120 kDa also contains only the alpha 1 N-terminal sequence. In contrast, as reported earlier (Sweadner, K.J. (1979) J. Biol. Chem. 254, 6060-6067), the doublet seen in brain preparations consists of alpha 1 and alpha 2 or (alpha 2 + alpha 3). We conclude that monospecific antibody probes or N-terminal sequencing must be used to identify Na,K-ATPase isoforms by SDS- or STS-PAGE. In addition, gel conditions that may affect the mobilities of the isoforms are discussed.
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PMID:Anomalous mobilities of Na,K-ATPase alpha subunit isoforms in SDS-PAGE: identification by N-terminal sequencing. 166 Nov 52


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