The structural basis of ankyrin function. II. Identification of two functional domains

Human erythrocyte ankyrin was cleaved by restricted proteolysis at 0 degrees C into two distinct chemical domains. The site on ankyrin that binds spectrin was found to be within a 55,000-dalton domain by spectrin affinity chromatography and co-sedimentation with spectrin in a sucrose gradient. A 32,000-dalton fragment of this domain was prepared (tryptic digest, 0 degrees C, 24 h), separated by gel filtration, and shown to inhibit spectrin binding to the membrane. By comparison with previous two-dimensional peptide maps, the spectrin-binding site was located within this 32,000-dalton fragment near the end of the molecule. The band 3-binding site was identified within an 82,000-dalton domain by binding to a band 3 affinity column. Gel electrophoresis in the absence of detergents confirmed these results and demonstrated that a peptide from the cytoplasmic portion of band 3 retained the capacity to bind the 82,000-dalton domain. The binding properties of the structural domains of ankyrin were correlated with a determination of the affinity constant of the intact molecule. Ankyrin bound with a high affinity to the cytoplasmic portion of band 3 (KD = 8 X 10(-8) M) and to spectrin tetramer (KD = 1 X 10(-7) M) but less so to spectrin dimer (KD = 1 X 10(-6) M). These findings are summarized in a preliminary structural and functional model of ankyrin's role in linking spectrin to the membrane.     

The Control of Cadmium Uptake in the Lichen Genus Peltigera

Intra- and extracellular Cd uptake in the lichen genus Peltigerawere investigated, and intracellular uptake found to displayMichaelis-Menten kinetics. Compared with values reported forZn uptake in free-living algae and fungi, Peltigera had lowaffinities for Cd and low maximum uptake rates. Intra-, andto a lesser extent extracellular uptake rates were temperaturedependent. When lichens were incubated concurrently with Cdand equimolar concentrations of a range of other cations, mostwere found to reduce both extra- and intracellular Cd uptake,implying that the Cd uptake systems had low specificities. Mg,though not a strong competitor for extracellular Cd uptake,inhibited intracellular Cd uptake to a similar extent to borderlineelements, and it is postulated that intracellular Cd uptakeoccurs by a system which normally transports Mg. Although concurrently-suppliedcations reduced Cd-induced inhibitions of photosynthesis, thereductions were not proportional to the effect of the cationson intracellular Cd uptake. This indicated that other cationsaffected the toxicity of Cd to photosynthesis by some meansin addition to reducing Cd uptake. Intracellular Cd uptake waslight-stimulated, suggesting that a close relationship existedbetween metal uptake and metabolism. The rate of intracellularCd uptake in the dark was probably not directly linked to thesupply of respirable reserves, as it was unaffected by prolongedstorage in the dark, and was not increased by adding glucose.It is hypothesized that light-stimulated Cd uptake representsactive entry into algal cells, but with uptake in the dark itis not clear which symbiont is involved, and whether energyis required. Key words: Lichen, Cadmium uptake, Temperature, Light, Cation competition     

Whole Plant Studies using Radioactive 13-Nitrogen: II. A COMPARTMENTAL MODEL FOR THE UPTAKE AND TRANSPORT OF NITRATE IONS BY Zea mays

Nitrate ion uptake by the roots of hydroponically grown maizeseedlings was measured using the short-lived isotope ¹³N. Itis shown to be described by a four compartment model, recognizablynitrogen in the root bathing solution, nitrogen which is readilyexchangeable from the root, nitrogen bound in the root, andnitrogen transported from the root. Some of the absorbed activity leaks back into the root bathingsolution with the efflux from the root, as a fraction of theinflux, increasing with concentration to be greater than 0–8at external nitrate ion concentrations above about 1.0 mol m^–3.The capacity of the exchangeable root pool increases with externalnitrate ion concentration, approaching the expected cytoplasmicnitrate ion content at the highest external nitrate ion concentrationsstudied (70 mol m^–3). The investigation has highlighted the problems of interpretinguptake profiles in experiments for which the 10 min half-lifeof ¹³N dictates experimental times that are comparable withthe times for saturation of root pools. Key words: Zea mays, ¹³N, Compartmental model, Nitrate uptake     

Initiation of DNA synthesis by human thrombin: relationships between receptor binding, enzymic activity, and stimulation of 86Rb+ influx

Stimulation of amiloride-sensitive sodium (Na+) influx and the subsequent activation of NA+, K+-ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human alpha-thrombin was converted to gamma-thrombin, nitro-alpha-thrombin, and diisopropylphospho (DIP)-alpha-thrombin. These derivatives retain either the capacity to bind cell surface alpha-thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of alpha-thrombin or the various thrombin derivatives, only alpha-thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP-alpha-thrombin that saturate the alpha-thrombin receptors (up to 2 micrograms/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP-alpha-thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either gamma-thrombin or nitro-alpha-thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.     

Regulation of pyruvate kinase expression and growth in P-815 mastocytoma cells. II. Potential role of prostaglandins

P-815 mastocytoma cells increase the level of pyruvate kinase (PK) expression in response to chloroform-methanol extracts of conditioned media, butyrate, and dibutyryl cyclic AMP (but2cAMP) plus theophylline. The butyrate effect is indomethacin sensitive, suggesting a prostaglandin (PG) is the active signaling factor. Moreover, the chloroform-methanol extracts contain PGE2 and PGF2 alpha and additions of the latter enhance PK activity. PGE2 alone has little or no effect but acts synergistically with PGF2 alpha. These data show that PGF2 alpha can regulate PK levels. On the other hand, other factors may also be active, since the endogeneous and the but2cAMP plus theophylline effects are indomethacin insensitive. Most of the factors that increase PK activity also inhibit cellular growth; however, regulation of PK expression can be uncoupled from growth inhibition.     

Retinoic acid alters subcellular compartmentalization of ATP pools in 3T3 cells but not in HeLa cells

From the Chinese hamster ovary (CHO) cell, genetic variants (MonR-31 and MonR-32) relatively resistant to monensin, an ionophoric antibiotic, have been isolated. Growth of both MonR-31 and MonR-32 clones required higher doses of serum than CHO. Addition of insulin to media containing a low dose of serum restored full colony formation, but growth of MonR-31 or MonR-32 cells required more insulin than CHO cells. Specific binding of [125I]insulin was observed in these cell lines. The two MonR clones bound about one-half or less the [125I]insulin bound by CHO cells. Scatchard analysis for [125I]insulin binding at 4 degrees C and 37 degrees C showed altered number of binding sites, but not insulin affinity: The number of binding sites in the MonR cell was about a half or less that of the parental CHO cell. Down-regulation of insulin receptor was assayed when both CHO and MonR cells were incubated with 1 microgram/ml insulin. A 50-60% decrease in levels of insulin surface binding capacities was observed in CHO after exposure to insulin, whereas there was no decrease in MonR cell. The cellular uptake of 2-[3H]deoxyglucose into CHO cells was significantly enhanced in the presence of insulin, but only slight, if any, increase was observed in MonR cells.     

Cultured fetal rat myoblasts release peptide growth factors which are immunologically and biologically similar to somatomedin

The production of immunologically and biologically active somatomedin activity from isolated myoblasts and fibroblasts from fetal rats of 21 days gestational age was investigated. Myoblast-rich cell populations were derived from primary cultures of dispersed muscle cells by the tendency of myoblasts to become detached from the culture dish in the presence of cytochalasin B. Fibroblasts were obtained from fetal muscle. Culture medium conditioned by exposure to myoblasts for 48 hours produced an increased incorporation of both [35S]sulphate and [3H]thymidine by explants of fetal rat costal cartilage in vitro compared to fresh medium. Myoblast-conditioned medium also contained somatomedin-C-like immunoreactivity which diluted in parallel with partially purified human somatomedin-C (3,271 +/- 446 mU/mg cell protein; mean +/- SEM, seven experiments). Medium conditioned by exposure to fetal rat fibroblasts did not promote isotope uptake by fetal rat cartilage above control values, and contained only low levels of somatomedin-C-like immunoreactivity (343 +/- 89 mU/mg cell protein, three experiments). The release of both somatomedin bioactivity and immunoreactivity into conditioned medium was significantly reduced by the incubation of myoblasts in the presence of rat growth hormone (100 ng/ml and 500 ng/ml). We conclude that fetal rat myoblasts released growth factor activity during culture which exhibited biological and immunologic characteristics of somatomedin. Since the bioactivity was demonstrated on skeletal tissues from rat fetuses of the same gestational age as those that yielded myoblasts such growth factor release may be physiological.     

Characterization and partial purification of keratinocyte growth factor from the hypothalamus

An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum-free culture system were performed. The keratinocyte growth-promoting activity of the hypothalamic extract was unaffected by heating (100 degrees C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G-25 column, and an approximate pl of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150-300 micrograms/ml) increased keratinocyte growth approximately 50-fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat-treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum-free culture system. Keratinocyte growth-promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.     

The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase

A 110-kDa protein present in chicken intestinal brush-border microvilli is believed to laterally link the actin filament bundle that forms the structural core of the microvilli with the microvillar plasma membrane. We have purified a 110-kDa protein to greater than 95% homogeneity by extraction of brush borders with solution containing 0.6 M KCl and 5 mM ATP, followed by gel filtration chromatography, sedimentation as a complex with exogenous actin, and hydroxylapatite chromatography. The 110-kDa protein-calmodulin complex bound F-actin in the absence but not the presence of ATP and had K+,EDTA-ATPase (0.2 mumol/min/mg) and Ca2+-ATPase (0.2 mumol/min/mg) activities and Mg2+-ATPase activity (0.03 mumol/min/mg) that was not activated by F-actin. The actin-binding and ATPase activities of the complex were similar to those of purified brush-border myosin. However, immunoblot analysis showed no reactivity between the 110-kDa protein and polyclonal antibody against purified chicken brush-border myosin. Also, peptide maps of 110-kDa protein and myosin obtained by limited proteolysis with chymotrypsin and Staphylococcus aureus V8 protease had few, if any, peptides in common. Immunoblot analysis also showed that myosin heavy chain was stable under the conditions of the preparation.     

Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface

After 24 h of continuous labeling with radioactive precursors, a high molecular weight heparan sulfate proteoglycan (HS-PG) was isolated from both the medium and cell layer of human colon carcinoma cells (WiDr) in culture. The medium HS-PG eluted from a diethylaminoethyl anion exchange column with 0.45-0.50 M NaCl, had an average density of 1.46-1.49 g/ml on dissociative CsCl density-gradient ultracentrifugation, and eluted from Sepharose CL-2B with a Kav = 0.57. This proteoglycan had an estimated Mr of congruent to 8.5 X 10(5), with glycosaminoglycan chains of Mr = 3 X 10(4) which were all susceptible to HNO2 deaminative cleavage. Deglycosylation of the HS-PG with polyhydrogen fluoride resulted in a 3H-core protein with Mr congruent to 2.4 X 10(5). The cell layer contained a population of HS-PG with characteristics almost identical to that released into the medium but with a larger Mr = 9.5 X 10(5). Furthermore, an intracellular pool contained smaller heparan sulfate chains (Mr congruent to 1 X 10(4)) which were mostly devoid of protein core. In pulse chase experiments, only the large cell-associated HS-PG was released (approximately 58%) into the medium as intact proteoglycan and/or internalized and degraded (approximately 42%), with a t1/2 = 6 h. However, the small intracellular component was never released into the medium and was degraded at a much slower rate. When the cells were subjected to mild proteolytic treatment, only the large cell-associated HS-PG, but none of the small component, was displaced. Addition of exogenous heparin did not displace any HS-PG into the medium. Both light and electron microscopic immunocytochemistry revealed that the cell surface reacted with antibody against an HS-PG isolated from a basement membrane-producing tumor. Electron microscopic histochemistry using ruthenium red and/or cuprolinic blue revealed numerous 10-50-nm diam granules and 70-220-nm-long electron-dense filaments, respectively, on the surface of the tumor cells. The results indicate that colon carcinoma cells synthesize HS-PGs with distinct structural and metabolic characteristics: a large secretory pool with high turnover, which appears to be synthesized as an integral membrane component and localized primarily at the cell surface, and a small nonsecretory pool with low turnover localized predominantly within the cell interior. This culture system offers an opportunity to investigate in detail the mechanisms involved in the regulation of proteoglycan metabolism, and in the establishment of the neoplastic phenotype.