Mary Lou Caspers

Separator isoelectric focusing: An improved method of protein analysis and purification

Abstract Addition of small amphoteric substances or separators to the carrier ampholytes used in isoelectric focusing improves the separation of protein components. The technique has been applied to partially oxidized human hemoglobin, goat anti-bovine pancreatic oxidized ribonuclease, human γ-globulin, and barley β-amylase. Each of these proteins is a complex mixture of species which can be resolved selectively and reproducibly by this method. Superior separation is provided by this technique when compared to focusing in commercially available narrow-range ampholytes. Addition of separators usually results in the flattening of the pH profile of the gel. Inspection of the protein patterns indicates that no new protein bands arise, although different patterns of existing components are obtained by choice and concentration of separator as well as by concentration of carrier ampholytes. This technique, thus, offers the advantage of making conventional isoelectric focusing more versatile.

Expression of γ-glutamyltranspeptidase in a transformed rat cerebral endothelial cell line

Brain capillary endothelium in vivo contains high levels of gamma-glutamyltranspeptidase activity. In addition, the presence of this enzyme has been used as a marker of neoplastic cells. Normal rat cerebral endothelial cells in culture exhibit a specific activity for gamma-glutamyltranspeptidase of 2 units/10(6) cells. In vitro transformation of these cells is achieved by the use of an avian retrovirus, Schmidt-Ruppin RSV-strain D. The resultant cell line, designated RCE-T1, demonstrates a significant increase in gamma-glutamyltranspeptidase activity up to 20 units/10(6) cells in early passage levels (9-26) after which enzyme activity declines and returns to normal levels by passage 80. This variation in enzyme activity correlates with histochemical staining for this enzyme. Furthermore, the enzyme activity increases linearly over a 1000-fold range of cell concentrations. Various culture modifications do not influence this pattern of enzyme expression. These parameters include trypsin dissociation, cell freezing, degree of confluency and culture maintenance with serum or with conditioned medium obtained from passage levels exhibiting high or low enzyme activity. RCE-T1 cells will provide a unique model system to study the distribution and regulation of this enzyme during differentiation and viral carcinogenesis.

Natural pH gradients formed by amino acids: Ampholyte distribution, time course, use in electrofocusing of protein, relation to pH gradients in isotachophoresis, and separator effects

Abstract Natural pH gradients can be formed from as few as seven amino acids when a polyacrylamide gel, containing a mixture of the 0.01 m amino acids, is placed in an electric field between 0.01 m anolyte and catholyte solutions of the isoelectric “leading” and “trailing” amino acids in the mixture. The amino acids seem to distribute at or near the anodic and cathodic pH gradient terminals, as well as in a position along the pH gradient approximating their isoelectric point (pI). Only the two amino acids at the terminals in contact with identical electrolytes at their pIs are isoelectric and distribute in single peaks. The electrofocusing at or near its pI of a protein in a pH gradient formed by amino acids was demonstrated. Stacking of the seven amino acids (0.015 mmol each) in a multiphasic buffer system operative at pH 10.4 gives rise to a spatially extended pH gradient within the stack suitable for “cascade stacking.” After transposition of the extended stack between isoelectric “leading” (anolyte) and “trailing” (catholyte) constituents, this pH gradient was maintained. Amino acid electrofocusing provides a tool for the comprehensive physical-chemical analysis of pH gradients and electrofocusing by measurement of the positions, peak widths, and peak shapes of defined carrier ampholytes as a function of time.

Inhibition by lead of human erythrocyte (Na+ + K+)-adenosine triphosphatase associated with binding of 210Pb to membrane fragments

Fragmented human erythrocyte membranes were exposed to PbCl2 for 10–40 min at 23°C prior to (Na+ + K+)-ATPase assay. Inhibition increased with exposure time. Enzyme activity in 5 μg membrane protein was inhibited 50% after a 10-min exposure to 1.0 nmol PbCl2 (25 μM final concentration) and was inhibited 100% after 40 min. When membranes at various concentrations were exposed to PbCl2 for 40 min, inhibition was linear with the ratio of PbCl2 to protein. Inhibition of 100% was obtained at 0.2 nmol PbCl2/μg protein. A graph of activity vs. [protein] in the presence of PbCl2 intercepted the abscissa to the right of the origin, indicating that lead acts as an irreversible or very slowly reversible inhibitor. Addition of 1 mM 2,3-dimercaptopropanol, 1,3-dithiothreitol, dl-penicillamine or EDTA after 40 min exposure to 100 μM PbCl2 restored 45, 64, 81 and 92% of the (Na+ + K+)-ATPase, respectively. These chelators, excluding EDTA, prevented inhibition when added before PbCl2. Two washings of the membrane fragments with water or 10 mM imidazole-HCl (pH 7.4) did not restore activity. 210Pb bound tightly to membrane fragments and beginning of saturation was observed at 0.19 nmol Pb2+ bound/μg protein. This corresponded to 200 μM final concentration of PbCl2 in the ATPase assay. At 0.2 nmol PbCl2/μg protein (100% inhibition of ATPase), from 0.10–0.17 nmol of lead was bound per μg protein. Under the same conditions, 1 mM dl-penicillamine removed 80% of the bound lead which correlated with its restoration of ATPase activity. Pb2+ does not appear to denature the enzyme. The irreversible kinetics may be related to sequestration of Pb2+ within vesicles that interfere with the accessibility of chelators to Pb2+ binding sites.

Aluminum-induced alterations in [3H]Ouabain binding and ATP hydrolysis catalyzed by the rat brain synaptosomal (Na++K+)-ATPase

The (Na(+)+K+)-ATPase is responsible for maintenance of the ionic milieu of cells. The objective of this study is to investigate the effect of aluminum, an ion implicated in several neurological disorders, on ATP hydrolysis catalyzed by the rat brain synaptosomal (Na(+)+K+)-ATPase and on the binding of [3H]ouabain to this enzyme. AlCl3 (25-100 microM) inhibits the phosphatase activity of the (Na(+)+K+)-ATPase in a dose-dependent manner. AlCl3 appears to act as a reversible, noncompetitive inhibitor of (Na(+)+K+)-ATPase activity by decreasing the maximum velocity of the enzyme without significantly affecting the apparent dissociation constant with respect to ATP. AlCl3 may affect Mg2+ sites on the (Na(+)+K+)-ATPase but does not appear to interact with Na+ or K+ sites on the enzyme. In contrast to this inhibitory effect on the phosphatase function of the enzyme, AlCl3 (1-100 microM) stimulates the binding of [3H]ouabain to the (Na(+)+K+)-ATPase. This effect is due to an increase in the maximum [3H]ouabain binding capacity of the enzyme with no change in the [3H]ouabain binding affinity. These data support the hypothesis that AlCl3 may stabilize the phosphorylated form of the synaptosomal (Na(+)+K+)-ATPase which increases [3H]ouabain binding while inhibiting the phosphatase activity of the enzyme.

Autoradiographic visualization and characterization of [3H]ouabain binding to the Na+, K+-ATPase of rat brain and pineal

Ouabain binds to the catalytic subunit of Na+,K+-ATPase and specific [3H]ouabain binding can be used as a measure of the number of active enzyme molecules present in a given tissue. Specific [3H]ouabain binding can be demonstrated in frozen, cryostat sections from rat brain and pineal and these sites have the characteristics of Na+,K+-ATPase. Incubations carried out in the absence of ATP or the presence of excess unlabeled ouabain reduces specific binding by greater than or equal to 98%. The addition of K+ or omission of Mg2+ also result in a decrease in specific binding. Strophanthidin, digoxin and digoxigenin displace [3H]ouabain binding with IC50 values of 0.73, 0.48 and 1.4 microM, respectively. Scatchard analyses of specific [3H]ouabain binding in brain sections shows a single class of non-interacting binding sites with an apparent affinity (Kd) of 339 nM and a maximal binding capacity (Bmax) of 34.9 pmol/mg protein. [3H]Ouabain binding is unevenly distributed throughout the brain with the olfactory nuclei, superior colliculus, dentate gyrus, pontine nuclei and pineal gland having a relatively high density of binding sites. The outer layers (1-3) of the cerebral cortex show more labeling than the inner layers (4-6) and most other brain areas have intermediate levels of [3H]ouabain binding sites, whereas white matter has virtually no specific binding. Computer-assisted densitometry was used to measure changes in specific [3H]ouabain binding after kainic acid injection into the caudate nucleus. An initial increase in [3H]ouabain binding was observed at 1 and 24 h after lesioning and a decrease in [3H]ouabain binding was evident by 9 days after lesioning.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of aluminum-induced inhibition of δ-aminolevulinic acid dehydratase in vitro

Abstract Anemia, one consequence of aluminum toxicity, may be due to inhibition of enzymes in the heme biosynthetic pathway. In this study, the in vitro effect of aluminum on rat liver and erythrocyte δ-aminolevulinic acid dehydratase (8-ALA dehydratase), an enzyme that is sensitive to a number of metal ions, was investigated. The presence of 1–10 μM AlCl 3 caused a concentration-dependent inhibition of liver δ-ALA dehydratase activity. The K i for AlCl 3 -induced inhibition of δ-ALA dehydratase was 4.1 μM, and 10 μM AlCl 3 virtually abolished δ-ALA dehydratase activity (99% inhibition). Erythrocyte δ-ALA dehydratase was also inhibited by similar concentrations of AlCl 3 and displayed a K i of 1.1 μM. AlCl 3 (5 μM) decreased the V max by 50% but did not change the K m , suggestive of reversible, noncompetitive inhibition. Sodium citrate (50 μM) when added with AlCl 3 completely restored δ-ALA dehydratase activity to basal levels. Thus, disruption of δ-ALA dehydratase occurred at low micromolar levels of AlClP 3 in vitro , which may help to explain abnormalities in the heme pathway in cases of aluminum poisoning.

Carrier ampholyte distribution in isoelectric focusing

Abstract The spatial distributions of individual components of carrier ampholyte mixtures obtained by isoelectric focusing in cylindrical (6 mm × 75 mm) polyacrylamide gels (PAG) and in a thin-layer Sephadex gel (TLSG) were determined by ion-exchange chromatography of the eluates of gel slices. A ca. 1-mm slice of PAG separating the A and B forms of β-lactoglobulin (isoelectric points of 5.21 and 5.34, respectively) was found to contain at least 12–14 ampholyte components out of a total of at least sixty in the ampholyte mixture (LKB Ampholine, pH 3.5–10, Lot No. 17). Fifteen to twenty components were found in each 2-mm slice of a sequence of six slices of PAG subsequent to isoelectric focusing of sperm whale myoglobin with the same ampholyte mixture. An ampholyte mixture prepared by the copolymerization of triethylenetetramine with acrylic acid was focused in TLSG on 20 cm × 5 cm plates and the eluates obtained from ca. 5-mm slices were analyzed by ion-exchange chromatography. The results were compared with the caramelization pattern obtained by the Felgenhauer technique. The spatial distributions of individual ampholyte components were broad with half-band widths of ca. 1 cm. The effects of sample load, ampholyte concentration, and duration of focusing on spatial distribution were investigated in the PAG focusing of [ 14 C]histidine. The narrowest and most symmetrical distributions were obtained with small sample load and high ampholyte concentration.

Control of [3H]ouabain binding to cerebromicrovascular (Na+ + K+)-ATPase by metal ions and proteins

The (Na+ + K+)-ATPase is localized to the cerebral endothelium, i.e. the blood-brain barrier, and is important for the maintenance of the brain electrolyte environment. Data from the present study indicate that Pb2+ inhibits the binding of [3H]ouabain to the cerebral microvascular (Na+ + K+)-ATPase in a time- and dose-dependent manner. Pb2(+)-induced inhibition developed slowly with a maximum obtained after 40 min. Inhibition of [3H]ouabain binding to the enzyme was 48% at 10 microM Pb2+ and appeared maximal (89%) at 100 microM Pb2+ when compared to [3H]ouabain binding in untreated microvessels at 40 min. In contrast, 100 microM Al3+ caused a 55% increase in [3H]ouabain binding to the (Na+ + K+)-ATPase, relative to untreated microvessels at 40 min. Insulin or bovine serum albumin stimulated [3H]ouabain binding to the enzyme when added at similar concentrations. However, the addition of both insulin and bovine serum albumin did not result in an additive effect. These results show that insulin exerts a nonspecific effect on [3H]ouabain binding to the (Na+ + K+)-ATPase similar to that evoked by bovine serum albumin. However, the metal ions Pb2+ and Al3+ provoke selective alterations in the cerebromicrovascular (Na+ + K+)-ATPase with Pb2+ inhibiting and Al3+ stimulating [3H]ouabain binding.

Control of the Na+,K(+)-ATPase under normal and pathological conditions.

The Na+,K(+)-ATPase is an important enzyme in determining the ionic milieu of the cerebromicrovasculature and neurons. The effect of hypertension or aging on this enzyme, as well as its susceptibility to regulation by fatty acids or aluminum, is the focus of this study. A significant increase (34%) in the apparent affinity constant (KD) but no change in the maximum binding capacity (Bmax) for [3H]ouabain binding to the cerebromicrovascular Na+,K(+)-ATPase occurs after induction of acute hypertension. In addition, long chain unsaturated fatty acids stimulate the binding of [3H]ouabain to the enzyme in microvessels from normotensive and hypertensive rats. The synaptosomal Na+,K(+)-ATPase is sensitive to aluminum. AlCl3 (1-100 microM) inhibits the K(+)-dependent-p-nitrophenylphosphatase (K(+)-NPPase) activity of the Na+,K(+)-ATPase in a dose-dependent manner. AlCl3 (100 microM) decreases the Vmax by 14% but does not alter the KM, suggestive of non-competitive inhibition. The enzyme from aged brain displays a greater Vmax, but shows the same susceptibility to AlCl3 as the enzyme from younger brain. In summary, disruption of the Na+,K(+)-ATPase may underlie, at least in part, abnormalities of nerve and vascular cell function in disorders where elevated concentrations of fatty acids or metal ions are involved.