scholarly journals Association of calmodulin and an unconventional myosin with the contractile vacuole complex of Dictyostelium discoideum.

1992 ◽  
Vol 118 (2) ◽  
pp. 347-358 ◽  
Author(s):  
Q Zhu ◽  
M Clarke
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Indirect Immunofluorescence ◽  
Mammalian Cells ◽  
Contractile Vacuole ◽  
Unconventional Myosin ◽  
Calmodulin Binding ◽  
Contractile Vacuoles ◽  
Vesicle Movement ◽  
Cytochemical Marker

mAbs specific for calmodulin were used to examine the distribution of calmodulin in vegetative Dictyostelium cells. Indirect immunofluorescence indicated that calmodulin was greatly enriched at the periphery of phase lucent vacuoles. The presence of these vacuoles in newly germinated (non-feeding) as well as growing cells, and the response of the vacuoles to changes in the osmotic environment, identified them as contractile vacuoles, osmoregulatory organelles. No evidence was found for an association of calmodulin with endosomes or lysosomes, nor was calmodulin enriched along cytoskeletal filaments. When membranes from Dictyostelium cells were fractionated on equilibrium sucrose density gradients, calmodulin cofractionated with alkaline phosphatase, a cytochemical marker for contractile vacuole membranes, at a density of 1.156 g/ml. Several high molecular weight calmodulin-binding proteins were enriched in the same region of the gradient. One of the calmodulin-binding polypeptides (molecular mass approximately 150 kD) cross-reacted with an antiserum specific for Acanthamoeba myosin IC. By indirect immunofluorescence, this protein was also enriched on contractile vacuole membranes. These results suggest that a calmodulin-binding unconventional myosin is associated with contractile vacuoles in Dictyostelium; similar proteins in yeast and mammalian cells have been implicated in vesicle movement.

An Immunoprecipitation Assay for High Molecular Weight Alkaline Phosphatase in Human Serum

1989 ◽  
Vol 26 (2) ◽  
pp. 151-157 ◽  
Author(s):  
Gerald A Maguire ◽  
Halima Adnan
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Liver Disease ◽  
High Molecular Weight ◽  
Solid Phase ◽  
Bone Alkaline Phosphatase ◽  
Intestinal Alkaline Phosphatase ◽  
Alkaline Phosphatases ◽  
Molecular Weight Form ◽  
Hepatic Malignancies

The serum of patients with obstructive liver disease may contain a high molecular weight form of alkaline phosphatase (high Mr alkaline phosphatase). The presence of this form of alkaline phosphatase is associated with hepatic malignancies. We have investigated the use of anti-alkaline phosphatase monoclonal antibodies which do not bind high Mr alkaline phosphatase in assays for high Mr alkaline phosphatase. Direct immunoprecipitation of liver and bone alkaline phosphatase with solid phase anti-liver alkaline phosphatase antibody (which also reacts with bone alkaline phosphatase) and measurement of the residual supernatant alkaline phosphatase activity led to a precise assay. Intestinal alkaline phosphatase interfered in this assay which, consequently, was of little use in the differential diagnosis of liver disease. Indirect precipitation of liver, bone, placental and intestinal alkaline phosphatase by soluble anti-liver alkaline phosphatase (which reacts with liver and bone alkaline phosphatases), soluble anti-intestinal alkaline phosphatase (which reacts with placental and intestinal alkaline phosphatases) and solid phase anti-mouse IgG led to an assay which, although less precise, showed more promise of being useful clinically.

Extracellular alkaline phosphatase from marine bacteria: purification and properties of extracellular phosphatase from a marine Pseudomonas sp.

1980 ◽  
Vol 26 (7) ◽  
pp. 833-838 ◽  
Author(s):  
Hiromi Kobori ◽  
Nobuo Taga
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Marine Bacteria ◽  
Divalent Cations ◽  
Maximal Activity ◽  
Pseudomonas Sp ◽  
Purification And Properties ◽  
Extracellular Alkaline Phosphatase ◽  
Increased Pressure

Extracellular alkaline phosphatase produced by a marine Pseudomonas was purified to electrophoretic homogeneity. The molecular weight of the enzyme was estimated to be 100 000. The enzyme had maximal activity at pH 11.5. The enzyme was completely inhibited by 1 mM EDTA. However, divalent cations reversed the enzyme inhibition and their order of effectiveness on the reaction was Zn2+ > Ca2+ > Mn2+ > Mg2+ > Sr2+ > Co2+. The enzyme activity was affected by the species of anion whose order of effectiveness was demonstrated to follow the lyotrophic series, Cl− > Br− > NO3−> ClO4− > SCN−. The activity of phosphatase was accelerated linearly by increased pressure until up to 1000 atm (1 atm = 101.325 kPa), and the enzyme activity at 1000 atm was 3.2 times higher than that at 1 atm.

Alkaline phosphatase is a useful cytochemical marker for the diagnosis of acute myelomonocytic and monocytic leukemia in the dog

2014 ◽  
Vol 44 (1) ◽  
pp. 79-93 ◽  
Author(s):  
Tracy Stokol ◽  
Deanna M. Schaefer ◽  
Martha Shuman ◽  
Nicole Belcher ◽  
Lynn Dong
Keyword(s):  
Alkaline Phosphatase ◽  
Monocytic Leukemia ◽  
Cytochemical Marker

DNA replication and repair of Tilapia cells. II. Effects of temperature on DNA replication and ultraviolet repair in Tilapia ovary cells

1988 ◽  
Vol 89 (2) ◽  
pp. 263-272
Author(s):  
J.D. Chen ◽  
F.H. Yew
Keyword(s):  
Molecular Weight ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Control Level ◽  
High Molecular Weight Dna ◽  
Wide Range ◽  
Effects Of Temperature ◽  
Fish Cells

TO-2 is a fish cell line derived from the Tilapia ovary. It grows over a wide range of temperature (15–34 degrees C). While most fish cells lack DNA excision repair and are hypersensitive to ultraviolet light (u.v.), Tilapia cells are more u.v.-resistant than mammalian cells. In this paper we report the effects of temperature on DNA replication and u.v. repair in TO-2 cells. When the cells were moved from 31 degrees C to the sublethal high temperature of 37 degrees C, the rate of DNA synthesis first decreased to 60%, then speedy recovery soon set in, and after 8 h at 37 degrees C the rate of DNA synthesis overshot the 31 degrees C control level by 180%. When moved to low temperature (18 degrees C) Tilapia cells also showed an initial suppression of DNA synthesis before settling at 30% of the control level. u.v. reduced but could not block DNA synthesis completely. The inhibition was overcome in 3 h at 37, 31 and 25 degrees C, but not at 18 degrees C. Initiation of nascent DNA synthesis was blocked at 4 J m-2 in TO-2 cells compared with less than or equal to 1 J m-2 in mammalian cells. After 9 J m-2 u.v. irradiation, low molecular weight DNA replication intermediates started to accumulate, and they could be chased into high molecular weight DNA with little delay. TO-2 cells showed low levels of u.v.-induced excision repair; but this was prominent compared with other fish cells. The u.v.-induced incision rate has been measured at various temperatures, and the activation energy of incision estimated to be 13 kcal mol-1 (1 cal approximately equal to 4.184 J).

The Physiology of Contractile Vacuoles

1960 ◽  
Vol 37 (1) ◽  
pp. 73-82
Author(s):  
J. A. KITCHING ◽  
J. E. PADFIELD ◽  
M. H. ROGERS
Keyword(s):  
Body Surface ◽  
Normal Activity ◽  
The Body ◽  
Contractile Vacuole ◽  
Diffusion Constants ◽  
Contractile Vacuoles

1. The suctorian Discophrya collini (Root) has been subjected to D2O-H2O mixtures containing up to 99.7% D2O. 2. In 25% D2O or over there is a rapid but temporary shrinkage of the body. This shrinkage is difficult to estimate owing to the wrinkling of the body surface, but amounts to at least 10% in the undiluted (99.7%)D2O. 3. During the period of temporary shrinkage the contractile vacuole ceases activity. Normal activity is resumed when the normal volume is regained. In concentrations of D2O too low to cause shrinkage there is a temporary fall in the rate of vacuolar output. 4. Return to H2O leads to a brief but often very considerable rise in vacuolar output. 5. It is concluded that D2O penetrates less rapidly than H2O. A difference of at least 10% in the diffusion constants in the membrane would be required to explain our results. We cannot exclude this as unreasonable from our data, although an explanation based on differences in the equilibrium properties of D2O and H2O might also be invoked.

GFP Fusions for Fluorescence Detection of Ca2+ and Ca2+-Calmodulin in Living Cells

1998 ◽  
Vol 4 (S2) ◽  
pp. 1008-1009
Author(s):  
Anthony Persechini
Keyword(s):  
Mammalian Cells ◽  
Dissociation Constants ◽  
Hek 293 ◽  
Fluorescent Indicators ◽  
Calmodulin Binding ◽  
Binding Domains ◽  
293 Cells ◽  
20 Nm ◽  
Binding Sequence ◽  
Expression In Mammalian Cells

We have previously described fluorescent indicators for Ca2+ (FIP-CAs) and (Ca2+)4-calmodulin (FIP-CBs) whose responses are based on a ligand-dependent decrease in fluorescence energy transfer (FRET) between GFP variants. The indicators for (Ca2+)4-calmodulin contain calmodulin-binding domains, those for Ca2+ also contain an integral calmodulin (CaM) domain. We have developed new versions of these indicators constructed with enhanced blue- and red-shifted GFPs suitable for stable and transient expression in mammalian cells, and have begun to use them to investigate the relationships between the free intracellular concentrations of Ca2+ ([Ca2+]i) and (Ca2+)4-CaM ([(Ca2+)4-CaM]i). When the blue-shifted fluorophore is excited at 380 nm these constructs exhibit an emission peak at 505 nm due to FRET to the red-shifted fluorophore.We have made FIP-CBs with dissociation constants for (Ca2+)4-CaM of 0.5 nM, 20 nM, 300 nM and > 20 μM by introducing R →Q substitutions in the CaM-binding sequence, and have stablyexpressed them in HEK-293 cells (Fig. 1).

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