The Na-K-2Cl cotransporter is in a permanently activated state in cytoplasts from Ehrlich ascites tumor cells

Brief incubation of Ehrlich ascites tumor cells with cytochalasin B causes the formation of blebs in the surface membrane. Gentle homogenization removes the blebs as intact cytoplasts which contain neither mitochondrian or nucleus, nor other cytoplasmic membranous organelles. The Na-K-2Cl cotransporter is present in the cytoplasts in a permanently activated state, whereas the Na-K-2Cl transport system in unperturbed intact cells is silent. Pretreatment of intact cells with cytochalasin B for l min stimulates the bumetanide-inhibitable K⁺ influx fivefold. The influx into purified cytoplasts when expressed per g protein is three- to fourfold higher than the influx into cytochalasin B-treated intact cells. Thus, the membrane vesicles are enriched with the cotransporter, and the cotransporter is present in an activated state. The K influx into cytoplasts is inhibited about 40% by Na-free, Cl-free or bumetanide-containing media and to a similar extent by Fab fragments prepared from antiserum against purified proteins of the cotransporter. The K _I for bumetanide was 0.19±0.06 m for the cytoplasts as compared to 0.67±0.11 m for the intact cells. SDS gel electrophoresis of membrane proteins from the cytoplast membranes compared to the membranes of intact cells shows a reduced number of bands and a majority of bands showing reduced staining, whereas a few bands are stained more intensely. Particularly notable is a band at 80 kD, which is similar to the molecular weight previously reported for the main membrane protein isolated from intact cells using a bumetanide-Sepharose affinity column. An immunoblot of the cytoplast preparation using antibodies against the purified bumetanide binding proteins showed strong immunodetection of the 80 kD protein.We are grateful to Marianne Schiødt, Birgit Blytmann Jørgensen, Thomas Krarup and Beverley Dyer for expert assistance. This work was supported by grants from the Danish Natural Science Research Council (11-6835 to E.K.H.) and the National Institutes of Health (DK 33640 to P.B.D.) and by a Carlsberg Foundation research fellowship (to F.J.).     

Genetic mapping of 14 short tandem repeat polymorphisms on human chromosome 22

We have constructed a linkage map of 14 short tandem repeat polymorphisms (11 with heterozygosity > 70%) on the long arm of human chromosome 22 using 23 non-CEPH pedigrees. Twelve of the markers could be positioned uniquely with a likelihood of at least 1,000:1, and distributed at an average distance of 6.62 cM (range 1.5–16.1 cM). The sex-combined map covers a total of 79.6 cM, the female map 93.2 cM and the male map 64.6 cM. Based on comparisons between physical maps and other genetic maps, we estimate that our map covers 70%–80% of the chromosome. The map integrates markers from previous genetic maps and uniquely positions one marker (D22S307). Data from physical mapping on the location of four genetic markers correlates well with our linkage map, and provides information on an additional marker (D22S315). This map will facilitate high resolution mapping of additional polymorphic loci and disease genes on chromosome 22, and act as a reference for building and verifying physical maps.     

Substrate slope and orientation in a land hermit crab,Coenobita clypeatus (Decapoda,Coenobitidae)

Summary Coenobita clypeatus were tested on an underwater inclined plane in order to determine their behavioral threshold for perceiving slope, and to investigate the relative importance of input from the statocyst organ and from the weight of the body and gastropod shell on upward orienting ability. The shell's weight was compensated for by a neutral float and increased by weights. The crabs perceive an inclination of less than 5°, and do so with increasing certainty as the slope increases. Perception of the slope seems to be both by statocyst and proprioceptive input, but the latter is not essential for upward orientation.     

An investigation of Chromatium vinosum high-potential iron-sulfur protein by EPR and Mossbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions.

The high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum contains a cubane prosthetic group that shuttles between the [4Fe-4S]3+,2+ states. We find that the EPR spectra from this protein can be explained as a sum of two components, a major one with g = 2.02; 2.04; 2.12, and a minor one with g = 2.04; 2.07; approximately 2.13. In the presence of 0.1-2.0 M NaCl, freezing induces polymerization of the protein (presumably dimers), which is detected as intercluster spin-spin interaction in the EPR. The observed spin-spin interactions are interpreted as being due to two very similar dimeric structures in an approx. 1:2 ratio. Computer simulation of the X- and Q-band EPR spectra shows that the z-components of the g-tensors in each dimer pair must be co-linear, with center-to-center distances between the clusters of approximately 13 A and approximately 16 A. Inspection of possible dimeric structures of C. vinosum HiPIP by standard molecular graphics procedures revealed that the Fe/S cluster is exposed toward a flattened surface and is accessible to solvent. Moreover, the Fe/S clusters in two HiPIP molecules can easily achieve a center-to-center distance of approximately 14 A when approaching along a common 3-fold axis that extends through the S4 sulfur atom of the cubane; the z-component of the EPR g-tensor is co-linear with this symmetry axis.     

Transfer, expression, and inheritance of salmonid growth hormone genes in channel catfish, Ictalurus punctatus, and effects on performance traits.

We examined expression and inheritance of salmonid growth hormone genes RSVLTR-rtGH1 cDNA and RSVLTR-csGH cDNA, transferred to channel catfish (Ictalurus punctatus) by microinjection. One to 9 copies of the foreign DNA were inserted in either head-to-tail tandem array at single insertion sites or single copies at multiple insertion sites. All P1 transgenic catfish evaluated produced salmonid growth hormone regardless of the construct. Five P1 x P1 matings were accomplished. The spawning rate and fertility of these P1 transgenics in artificial spawning conditions were comparable to those of normal channel catfish. In two of three years, 100% spawning and 100% hatch were obtained. Percent transgenic progeny observed in the five matings were 20, 52, 7, 47, and 0%, which was lower (P < 0.001, chi 2) than the 75% inheritance expected assuming the P1 brood stock had at least one copy of the foreign gene integrated and were not mosaics in the germ line. At least 7 of 10 P1 were mosaics, and a minimum of 2 of 10 P1 did not possess the salmonid growth hormone genes in their germ line. P1 transgenics grew at the same rate as their nontransgenic full siblings, which is not surprising because the P1 were mosaics. F1 transgenic progeny in two families possessing RSVLTR-csGH cDNA grew 26% faster, to 40 to 50 gm, than their nontransgenic full siblings when evaluated communally. One F1 progeny group produced by RSVLTR-rtGH1 cDNA x RSVLTR-csGH cDNA mating and one F1 progeny group (parents either RSVLTR-rtGH1 cDNA or RSVLTR-csGH cDNA) grew at the same rate as normal full siblings when grown communally to 25 gm and 60 mg, respectively. In families where F1 progeny grew faster than controls, the range in body weight and coefficient of variation for the transgenic full siblings were less than those for controls. In families where F1 progeny grew at the same rate as controls, range in body weight and coefficient of variation were similar for transgenic and normal individuals. The percent deformities observed in P1 transgenics (13.6%) was higher (P < 0.05) than in microinjected P1 nontransgenics (5.1%). Percent deformities in transgenics and control F1 channel catfish was not different (p > 0.05; 0.5 and 2.8%, respectively).     

The initial characterization of the iron environment in lipoxygenase by M?ssbauer spectroscopy

The incorporation of 57Fe into two lipoxygenase isoenzymes from soybeans has been achieved making possible the first observations of the iron environment in these proteins using M?ssbauer spectroscopy. Immature soybean seeds were grown in tissue culture medium supplied with 57Fe. The iron in the active lipoxygenases that were isolated from the cultured seeds was readily detected in M?ssbauer measurements. It was unequivocally demonstrated that the native enzyme contains high-spin Fe(II). Based on the sign of the electric field gradient, the most likely ligand sphere for the iron in native lipoxygenase consists of oxygen and nitrogen ligands in a roughly octahedral field of symmetry. It was possible to detect M?ssbauer signals in highly concentrated samples of native lipoxygenases containing 57Fe at natural abundance. The spectra obtained for enriched and natural abundance native enzyme had the same high-spin Fe(II) M?ssbauer parameters. This confirmed that the environment of the iron in enzymes isolated from cultured seeds and dry soybeans were the same. The M?ssbauer spectra (4.2-250 K) for samples of both isoenzymes after oxidation of the iron in native enzyme by the product of lipoxygenase catalysis were extremely broad (20 mm/s) with no obvious narrow resonance lines. This was the result of the existence of paramagnetically broadened spectra for such samples even at relatively high temperature as evidenced by the appropriate EPR signal. A small molecule containing an iron site sharing many of these M?ssbauer and electron paramagnetic resonance properties with lipoxygenase was identified: Fe(II)/(III).diethylenetriaminepentaacetic acid.     

Properties of detergent-dispersed myocardial adenylate cyclase

Adenylate cyclase from rabbit ventricle was solubilized in 30 to 50% yield by the nonionic detergent Lubrol PX. The detergent, when present in the assay at concentrations above 0.05%, rapidly inactivated the enzyme in assays conducted above 26 °C; assays were valid only when conducted below this temperature. The solubilized enzyme was eluted from diethylaminoethyl (DEAE)-Bio-Gel A (DEAE-agarose) with 100 mm NaCl in a yield of 25% and was free of detergent. Several properties of the solubilized detergent-free enzyme were similar to properties of the native membrane-bound species. The K_m for substrate was 0.1 mm, the K_a for Mg²⁺ was 2.5 mm, and ATP in excess of Mg²⁺ was inhibitory. The enzyme was activated by F^? and guanyl-5′-yl imidodiphosphate [Gpp(NH)p] in a time- and temperature-dependent manner, and activation by the latter was persistent. Activation by F^? and Gpp(NH)p reduced the K_a for Mg²⁺. Activation by Gpp(NH)p was increased by Mg²⁺; the apparent K_a for activation was 0.1 μm. Multiple binding sites for Gpp(NH)p were present: one class with a K_d value of 0.11 μm was probably associated with activation of the enzyme. The soluble enzyme was insensitive to catecholamines, in both the presence and the absence of Gpp(NH)p. Sensitivity to catecholamines was not restored by the addition of phospholipids, particularly phosphatidyl inositol, in either the presence or the absence of Gpp(NH)p, and this phospholipid did not increase the sensitivity of the membrane-bound enzyme to epinephrine. Catecholamine binding sites were present, and their association with adenylate cyclase was seemingly not affected by phospholipids.     

A physical explanation of the EPR spectrum observed during catalysis by enzymes utilizing coenzyme B12.

We have proposed that the "doublet" EPR spectra observed during catalysis by a number of coenzyme B12-requiring enzymes arises from a weak electrostatic exchange interaction between an organic free radical and low spin Co(II), B12r. By varying the magnitude of the exchange of coupling we have quite accurately simulated the published EPR spectra from the enzyme systems: diol dehydrase, glycerol dehydrase, ribonucleotide reductase, and ethanolamine ammon-ia lyase. A dipolar model was shown to be incompatible with the observed properties of these systems.     

Active potassium transport in reticulocytes of high-K and low-K sheep

The kinetics of active K⁺ transport were studied in immature red blood cells cells from high-K⁺ and low-K⁺ sheep, particularly with respect to the effects of varying intracellular K⁺ concentration, [K]_i. Comparison was made with active transport, or pump, activity in mature high-K⁺ and low-K⁺ red cells. Reticulocytes from both types of sheep had much higher maximal active K⁺ influxes than did mature cells. In both types of reticulocytes, and in mature high-K⁺ cells as well, the pump was relatively insensitive to increasing [K]_i. In contrast, intracellular K⁺ markedly inhibited the pump in mature low-K⁺ cells. Active K⁺ transport in low-K⁺ reticulocytes, however, as in mature low-K⁺ cells, is stimulated by specific isoimmune anti-L serum. Therefore the K⁺ pumps of high-K⁺ and low-K⁺ reticulocytes have similar kinetic properties. Maturation of the red cells, involving inactivation of most of the pump activity in both cell types, results in mature high-K⁺ and low-K⁺ cells with K⁺ pumps of very different kinetic characteristics.