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J R Sachs

Publications and source records attributed to J R Sachs.

At least 19 recordsLinked to original sources
PubMed2001

Structure of Na+,K+-ATPase at 11-A resolution: comparison with Ca2+-ATPase in E1 and E2 states.

Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the crystals had p1 symmetry. A large number of helical symmetries were observed, so a three-dimensional structure was calculated by averaging both Fourier-Bessel coefficients and real-space structures of data from the different symmetries. The resulting structure clearly reveals cytoplasmic, transmembrane, and extracellular regions of the molecule with densities separately attributable to alpha and beta subunits. The overall shape bears a remarkable resemblance to the E2 structure of rabbit sarcoplasmic reticulum Ca2+-ATPase. After aligning these two structures, atomic coordinates for Ca2+-ATPase were fit to Na+,K+-ATPase, and several flexible surface loops, which fit the map poorly, were associated with sequences that differ in the two pumps. Nevertheless, cytoplasmic domains were very similarly arranged, suggesting that the E2-to-E1 conformational change postulated for Ca2+-ATPase probably applies to Na+,K+-ATPase as well as other P-type ATPases.

Adenosine Triphosphatases↗
PubMed2000

Ligands presumed to label high affinity and low affinity ATP binding sites do not interact in an (alpha beta)2 diprotomer in duck nasal gland Na+,K+-ATPase, nor Do the sites coexist in native enzyme.

The interaction of ligands deemed to be ATP analogues with renal Na(+),K(+)-ATPase suggests that two ATP binding sites coexist on each functional unit. Previous studies in which fluorescein 5-isothiocyanate (FITC) was used to label the high affinity ATP site and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was used to probe the low affinity site suggested that the two sites coexist on the same alphabeta protomer. Other studies in which FITC labeled the high affinity site and erythrosin-5-isothiocyanate (ErITC) labeled the low affinity site led to the conclusion that the high and low affinity sites exist on separate interacting protomers in a functional diprotomer. We report here that at 100% inhibition of ATPase activity by FITC, each alphabeta protomer of duck nasal gland enzyme has a single bound FITC. Both TNP-ADP and ErITC interact with FITC-bound protomers, which unambiguously demonstrates that putative high and low affinity ATP sites coexist on the same protomer. In unlabeled nasal gland enzyme, TNP-ADP and ErITC inhibit both ATPase activity and p-nitrophenyl phosphatase activity, functions attributed to the putative high and low affinity ATP site, respectively, by interacting with a single site with characteristics of the high affinity ATP binding site. In FITC-labeled enzyme, TNP-ADP and ErITC inhibit p- nitrophenyl phosphatase activity but at much higher concentrations than with the unmodified enzyme. Low affinity sites do not exist on the unmodified enzyme but can be detected only after the high affinity site is modified by FITC.

Adenosine Diphosphate↗
PubMed2000

Alphabeta protomers of Na+,K+-ATPase from microsomes of duck salt gland are mostly monomeric: formation of higher oligomers does not modify molecular activity.

The distance that separates alphabeta protomers of the Na(+), K(+)-ATPase in microsomes and in purified membranes prepared from duck nasal salt glands was estimated by measuring fluorescence resonance energy transfer between anthroylouabain bound to a population of alphabeta protomers and either N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain or 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain bound to the rest. Energy transfer between probes bound in the microsomal preparation was less than in the purified membranes. The efficiency of energy transfer between anthroylouabain and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain was 29.2% in the microsomes compared with 62.6% in the purified preparation. Similar results were obtained with 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain as acceptor. We calculate that either the protomer bound probes were on the average 13 A farther apart in the microsomes than in the purified membranes, or that 53% of the protomers are monomeric in the microsome preparation. Microsomes prepared in the presence of phalloidin (a toxin that binds to F actin and stabilizes the actin-based cytoskeleton) showed less quench than those prepared in its absence. The data support the hypothesis that protomers are kept apart by their association with the cytoskeleton. The turnover rate while hydrolyzing ATP is the same in the microsomal and purified preparations; higher oligomer formation has no significant effect on the enzyme reaction mechanism.

4-Nitrophenylphosphatase↗
PubMed1999

Role of polyamine structure in inhibition of K+-Cl- cotransport in human red cell ghosts.

1. K+-Cl- cotransport in human red cell ghosts is inhibited by divalent inorganic cations, soluble polycations and amphipathic organic cations. These findings suggest a common mechanism of inhibition, namely, binding of the cations to negative charges at the surface of a hydrophobic structure. 2. We have characterized the inhibitory capacity of a number of polyamines in order to obtain information about the nature of the charges with which they interact. Neomycin inhibited swelling-stimulated cotransport. The diquaternary amines dimethonium and decamethonium were relatively ineffective inhibitors. These compounds are thought to shield negative charges, but not bind to them. 3. Comparison of a homologous series of polyamines indicated that primary amines were better inhibitors than secondary amines, that inhibition increased with the charge of the polyamine, and that inhibition increased as the distance separating the amines increased. 4. The results indicate that the negative charges to which polycations bind are multiple and mobile. Since they must be associated with a hydrophobic environment, it is likely that they are negatively charged phospholipids located in the inner leaflet of the bilayer membrane. 5. Heating red cells or ghosts to 49 C denatures spectrin. Heating markedly increased K+ uptake in swollen ghosts but not in shrunken ghosts. The increase in uptake was reversed when swollen ghosts were shrunk even though denaturation of spectrin was not reversed. Polyamines, which inhibited swelling-activated K+ uptake in control ghosts, similarly inhibited the increased uptake in heated ghosts. 6. We speculate that spectrin, which is closely associated with the inner bilayer leaflet, shields negative charges in a volume-dependent manner and so regulates volume-sensitive K+ transport.

Amines↗
PubMed1999

Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites.

The phosphorylation capacity of Na+,K+-ATPase preparations in common use is much less than expected on the basis of the molecular weight of the enzyme deduced from cDNA sequences. This has led to the popularity of half-of-the-sites or flip-flop models for the enzyme reaction mechanism. We have prepared Na+,K+-ATPase from nasal salt glands of salt-adapted ducks which has a phosphorylation capacity and specific activity near the theoretical maxima. Preparations with specific activities of >60 micromol (mg of protein)-1 min-1 at 37 degrees C had phosphorylation capacities of >60 nmol/mg of protein, and the rate of turnover of the enzyme was 9690 min-1, within the range reported for the enzyme from other sources. The fraction of the maximal specific activity of the enzyme compared well with the fraction of the protein on SDS-PAGE which was alpha and beta chains, especially at the highest specific activity which indicates that all of the alphabeta protomers are active. The gels of the most reactive preparations contained only alpha and beta chains, but less active preparations contained a number of extraneous proteins. The major contaminant was actin. The preparation did not contain any protein which migrated in the molecular weight range of the gamma subunit. The subunit composition of the enzyme was alpha1 and beta1 only. This is the first report of a pure, homogeneous, fully active preparation of the protein. Reaction models which incorporate a half-of-the-sites or flip-flop mechanism do not apply to this enzyme.

Amino Acid Sequence↗
PubMed1995

Electromechanical spectroscopy of cartilage using a surface probe with applied mechanical displacement.

This study focuses on an approach for the nondestructive assessment of cartilage degeneration in vivo by quantitation of bulk material properties based on measurements made at the top surface of the tissue. A model of an electromechanical coupled poroelastic medium [Sachs and Grodzinsky (1989) Physicochem. Hydrodyn. 11, 585-614] is used to interpret the behavior of a diagnostic probe configuration suitable for such surface measurements of cartilage electromechanical and poroelastic properties via arthroscopy. The response of a planar layer of tissue to a periodic displacement imposed at the articular surface is described. This displacement produces a periodic electric streaming potential and mechanical stress in the bulk and at the surface of the tissue layer with the same frequency and wavelength as the imposed displacement. Using boundary conditions and parameter values relevant to cartilage, the results show that surface measurements of the stress and potential can be used to determine bulk material properties including tissue thickness, moduli, hydraulic permeability, and electrokinetic coupling coefficients. The relation between the temporal frequency and spatial wavelength of the surface excitation and the amplitude, phase, and penetration depth of the stress and potential is investigated numerically and asymptotically. Good agreement has been found between the long wave limit (with parameter values taken from the literature) and compression-induced streaming potential data from previous experiments in uniaxial confined compression. The results show that use of independently imposed temporal frequency and spatial wavelength may enable detection and imaging of focal regions of cartilage degeneration via nondestructive surface spectroscopy.

Biomechanical Phenomena↗
PubMed1994

The role of (alpha beta) protomer interaction in determining functional characteristics of red cell Na,K-ATPase.

We have examined the possibility that interaction of (alpha beta) protomers within a diprotomer is responsible for some anomalous characteristics of red cell Na,K-ATPase by examining their response to two inhibitors, FITC and H2DIDS, which bind covalently, and to ouabain, which debinds slowly from red cell pumps. The phenomena we examined were: (1) the biphasic curve relating Na,K-ATPase activity to ATP concentration, and (2) protection of Na pumps against vanadate inhibition by external Na. If interaction of (alpha beta) protomers within a diprotomer were responsible for these phenomena, random inactivation of (alpha beta) protomers should have resulted in a high proportion of (alpha beta) promtomers with an inhibited protomer as a partner, and therefore should have significantly altered the consequences of subunit interaction. With each inhibitor, 60-70% inhibition of ATPase activity did not alter the functional characteristics of the residual activity. We conclude that interaction of functional (alpha beta) protomers does not explain the phenomena which we investigated. This is consistent with our previous observation that Na,K pumps of red cell membranes exist as monomeric (alpha beta) protomers (Martin, D.W. and Sachs, V.R. (1992) J. Biol. Chem. 267, 23922-23929).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Soluble polycations and cationic amphiphiles inhibit volume-sensitive K-Cl cotransport in human red cell ghosts.

We have measured the effect of soluble polycations (spermine and methylglyoxal) and cationic amphiphiles (sphingosine and tetracaine) on K-Cl cotransport in shrunken and swollen red cell ghosts. All substances inhibited cotransport, and for each agent, the concentration at which inhibition was half-maximal was about the same for swollen and shrunken ghosts. Acetylspermine was a much less effective inhibitor than spermine, which demonstrates that inhibition depends on the cationic groups of spermine. Spermine was a more effective inhibitor in ATP-free ghosts than in ghosts containing ATP, which eliminates the possibility that inhibition of cotransport activity results from inhibition of protein kinase activity. Inhibition by spermine is as effective in K-free ghosts as in high-K ghosts; spermine does not inhibit cotransport by reducing the effective K concentration at the inner membrane surface. We conclude that regulation of K-Cl cotransport involves negative charges (phosphatidylserine or phosphatidylinositides) at the inner membrane surface and suggest a model that accounts for our findings.

Carrier Proteins↗
PubMed1993

The role of ATP in swelling-stimulated K-Cl cotransport in human red cell ghosts. Phosphorylation-dephosphorylation events are not in the signal transduction pathway.

Volume-sensitive K-Cl cotransport occurs in red blood cells of many species. In intact cells, activation of K-Cl cotransport by swelling requires dephosphorylation of some cell protein, but maximal activity requires the presence of intracellular ATP. We have examined the relation between K-Cl cotransport activity and ATP in ghosts prepared from human red blood cells. K-Cl cotransport activity in swollen ghosts increased by ATP, and the increase requires Mg so that it almost certainly results from the phosphorylation of some membrane component. However, even in ATP-free ghosts residual volume-sensitive K-Cl cotransport can be demonstrated. This residual cotransport in ATP-free ghosts is greater in the presence of vanadate, a tyrosyl phosphatase inhibitor, and in ghosts that contain ATP cotransport is reduced by genistein, a tyrosyl kinase inhibitor. Okadaic acid, an inhibitor of serine and threonine phosphatases, inhibits K-Cl cotransport in ghosts as it does in intact cells. Experiments in which ghosts were preexposed to okadaic acid showed that the protein dephosphorylation that permits K-Cl cotransport can proceed to completion before the ghosts are swollen and K transport measured and therefore dephosphorylation is not a response to ghost swelling. In experiments with ATP-free ghosts we found that phosphorylation is not necessary to increase the cotransport rate when shrunken ghosts are swollen, nor is rephosphorylation necessary to decrease the cotransport rate when swollen ghosts are shrunken. Cotransport is greater in swollen than in shrunken ghosts even when the swollen and shrunken ghosts have the same concentration of cytoplasmic solutes. We conclude that, although phosphorylation and dephosphorylation modify the activity of the cotransporter in swollen and in shrunken ghosts, neither of these processes nor any other known messenger is involved in signal transduction between the cell volume sensor and the cotransporter as originally proposed by Jennings and Al-Rohil (Jennings, M. L., and N. Al-Rohil. 1990. Journal of General Physiology. 95: 1021-1040).

Adenosine Triphosphate↗
PubMed1992

Cross-linking of the erythrocyte (Na+,K+)-ATPase. Chemical cross-linkers induce alpha-subunit-band 3 heterodimers and do not induce alpha-subunit homodimers.

Earlier studies (Periyasamy, S. M., Huang, W.-H., and Askari, A. (1983) J. Biol. Chem. 258, 9878-9885) suggested that Cu2+ and o-phenanthroline induced the formation of cross-linked homodimers between alpha-subunits of the erythrocyte (Na+,K+)-ATPase. This was interpreted as indicating that alpha-subunits existed in close proximity in native erythrocyte membranes. The alpha-subunit and band 3 monomers have similar molecular weights (M(r) approximately 100,000) and exist in the membrane in molar ratios of approximately 1:3000 alpha-subunit:band 3. We explored the possibility that alpha-subunit and band 3 could be induced to form heterodimeric structures in the presence of cross-linking reagents. Using methods similar to those employed in the above-cited reference we demonstrated that cross-linked dimers containing phosphorylated alpha-subunits had proteolytic sensitivity that was inconsistent with the formation of alpha-subunit homodimers and fully consistent with heterodimer formation between alpha-subunit and band 3. The data also indicated that alpha-subunit-band 3 heterodimer formation is dependent on the conformational state of the (Na+,K+)-ATPase. Using the appropriate reagents we obtained cross-linked products which were consistent with heterodimer formation between alpha- and beta-subunits of the (Na+,K+)-ATPase. Our data argue against a close association between pairs of (Na+,K+)-ATPase alpha-subunits in the human red cell membrane.

Copper↗
PubMed1991

Perialveolar interstitial resistance and compliance in isolated rat lung.

We have developed a method to characterize fluid transport through the perialveolar interstitium using micropuncture techniques. In 10 experiments we established isolated perfused rat lung preparations. The lungs were initially isogravimetric at 10 cmH2O arterial pressure, 2 cmH2O venous pressure, and 5 cmH2O alveolar pressure. Perialveolar interstitial pressure was determined by micropuncture at alveolar junctions by use of the servo-null technique. Simultaneously a second micropipette was placed in an alveolar junction 20-40 microns away, and a bolus of albumin solution (3.5 g/100 ml) was injected. The resulting pressure transient was recorded for injection durations of 1 and 4 s in nonedematous lungs. The measurements were repeated after gross edema formation induced by elevated perfusion pressure. We model the interstitium as a homogeneous linearly poroelastic material and assume the initial pressure distribution due to the injection to be Gaussian. The pressure decay is inversely proportional to time, with time constant T, where T is a measure of the ratio of interstitial tissue stiffness to interstitial resistance to fluid flow. A linear regression was performed on the reciprocal of the pressure for the decaying portion of the transients to determine T. Comparing pressure transients in nonedematous and edematous lungs, we found that T was 4.0 +/- 1.4 and 1.4 +/- 0.6 s, respectively. We have shown that fluid transport through the pulmonary interstitium on a local level is sensitive to changes in interstitial stiffness and resistance. These results are consistent with the decreased stiffness and resistance in the perialveolar interstitium that accompany increased hydration.

Airway Resistance↗
PubMed1991

Disseminated Pneumocystis carinii infection with hepatic involvement in a patient with the acquired immune deficiency syndrome.

Extrapulmonary infection with Pneumocystis carinii (P. carinii) in AIDS patients is uncommon, and is often described only at postmortem examination. Although most antemortem cases involve spread to the bone marrow or spleen, P. carinii involvement of other organs has only recently been described. Despite the frequency of liver enzyme abnormalities in AIDS patients with a history of P. carinii pneumonia, P. carinii has been observed only rarely in the liver. We present a well-documented case of P. carinii involving the liver in an AIDS patient with P. carinii pneumonia and progressive liver enzyme abnormalities. We suggest that P. carinii infection should be considered in the differential diagnosis of AIDS-related liver disease.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Mucinous cystadenoma: pitfalls of differential diagnosis.

Cystic neoplasms of the pancreas often are difficult to differentiate from pseudocysts. It has been proposed that a history of clinical pancreatitis, elevated serum pancreatic enzymes, elevated cyst fluid amylase, and a communication with the pancreatic duct suggest the diagnosis of a pseudocyst. We report the case of a young woman who presented with a cystic mass in the pancreas and was thought to have a pseudocyst because of the above; at surgery, a mucinous cystadenoma was documented. The pitfalls of differentiating neoplastic cysts of the pancreas from pseudocysts are discussed.

Amylases↗
PubMed1988

Volume-sensitive K influx in human red cell ghosts.

K influx into resealed human red cell ghosts increases when the ghosts are swollen. The influx demonstrates properties similar to volume-sensitive K fluxes present in other cells. The influx is, for the most part, insensitive to the nature of the major intracellular cation and therefore is not a K-K exchange. The influx is much greater when the major anion is Cl than when the major anion is NO3; Cl stimulates the flux and, at constant Cl, NO3 inhibits it. Increase in the influx rate is rapid when shrunken ghosts are swollen or when NO3 is replaced by Cl. The volume-sensitive K influx requires intracellular MgATP at low concentrations, and ATP cannot be replaced by nonhydrolyzable ATP analogues. The volume-sensitive influx is inhibited by Mg2+ and by high concentrations of vanadate, but is stimulated by low concentrations of vanadate. It is not modified by cAMP, the removal of Ca2+ by EGTA, substances that activate protein kinase C, or by inhibition of phosphatidylinositol kinase. The influx is inhibited by neomycin and by trifluoperazine.

1-Phosphatidylinositol 4-Kinase↗
PubMed1988

Phosphate inhibition of the human red cell sodium pump: simultaneous binding of adenosine triphosphate and phosphate.

1. The Na+-K+ exchange carried out by the Na+ pump of human red cell ghosts and the Na+ + K+-dependent adenosine triphosphatase (Na+,K+-ATPase) activity of human red cell membranes are inhibited by MgPO4 rather than by free phosphate; similarly, the substrate for the K+-K+ exchange carried out by the pump is MgPO4 rather than free phosphate. 2. Inhibition of the Na+, K+-ATPase activity by MgPO4 is only partially competitive (mixed type) with ATP, and MgPO4 inhibition of the Na+-K+ exchange measured in Na+-free solutions and in K+-free ghosts which contain ATP at relatively high concentration is partially uncompetitive (mixed type) with external K+. 3. When measurements were made in K+-free ghosts and Na+-free solutions, or when Na+,K+-ATPase activity was measured at high ATP concentrations, inhibition by MgPO4 was non-competitive with cell Na+. This observation is not consistent with the Albers-Post reaction mechanism of the Na+ pump, and suggests the presence of an alternative reaction pathway in which ATP combines with the enzyme before phosphate is released. 4. MgPO4 monotonically inhibited the uncoupled Na+ efflux which occurs in solutions free of both Na+ and K+. The uncoupled efflux seemed to be more sensitive to MgPO4 inhibition than the Na+-K+ exchange. 5. Trinitrophenyladenosine-5'-tetraphosphate stimulated the K+-K+ exchange in the presence of MgPO4, and the characteristics of stimulation by TNP adenosine tetraphosphate were little different from the characteristics of stimulation by trinitrophenyladenosine-5'-triphosphate or -5'-diphosphate. The nucleotide binding site at which K+-K+ exchange is stimulated must be able to accommodate a nucleotide with a linear array of four phosphate groups.

Adenosine Triphosphate↗