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PubMed2005

Electrochemical structure of the crowded cytoplasm.

The current view of the cytoplasm as a 'bustling and well-organized metropolitan city' raises the issue of how physicochemical forces control the macromolecular interactions and transport of metabolites and energy in the cell. Motivated by studies on bacterial osmosensors, we argue that charged cytoplasmic macromolecules are stabilized electrostatically by their ionic atmospheres. The high cytoplasmic crowding (25-50% of cell volume) shapes the remaining cell volume (50-75%) into transient networks of electrolyte pathways and pools. The predicted 'semi-conductivity' of the electrolyte pathways guides the flow of biochemical ions throughout the cytoplasm. This metabolic and signaling current is powered by variable electrochemical gradients between the pools. The electrochemical gradients are brought about by cellular biochemical reactions and by extracellular stimuli. The cellular metabolism is thus vectorial not only across the membrane but also throughout the cytoplasm.

Animals↗
PubMed2005

Cytoplasmic CD24 expression in advanced ovarian serous borderline tumors.

OBJECTIVES: CD24, originally described as a B-cell marker, has been revealed as one of the candidate molecular markers of epithelial ovarian cancer. We aimed to determine the pattern and extent of CD24 expression in ovarian serous tumors and to clarify its relationship with pathological parameters, especially those associated with the early events of tumor progression in serous tumors of borderline malignancy. METHODS: A total of 114 ovarian serous tumors, including 9 adenomas, 34 borderline, and 71 carcinomas, were analyzed immunohistochemically using a CD24 monoclonal antibody on paraffin blocks. RESULTS: In normal epithelium and serous cystadenomas, the CD24 expression was localized to the apical membranous portion. In some of borderline tumors (26.4%), additional cytoplasmic expression was observed. The cytoplasmic expression of CD24 in borderline tumors was associated with microinvasion (P = 0.001) and omental implants (P = 0.033) with statistical significance. Serous adenocarcinomas showed strong diffuse cytoplasmic expression of CD24, which was significantly associated with shortened survival rate both in univariate (P = 0.011) and multivariate (P = 0.009) analysis. CONCLUSION: The loss of apical localization with the acquisition of the cytoplasmic staining of CD24 protein is a surrogate marker of stromal invasion in ovarian serous tumors of borderline malignancy. Furthermore, the increase in the cytoplasmic expression of CD24 protein is a strong independent molecular marker for shortened survival rate of patients with ovarian serous adenocarcinomas.

Adult↗
PubMed1999

Parkinson's disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra neurons.

Oxidative damage, including modification of nucleic acids, may contribute to dopaminergic neurodegeneration in the substantia nigra (SN) of patients with Parkinson's disease (PD). To investigate the extent and distribution of nucleic acid oxidative damage in these vulnerable dopaminergic neurons, we immunohistochemically characterized a common product of nucleic acid oxidation, 8-hydroxyguanosine (8OHG). In PD patients, cytoplasmic 8OHG immunoreactivity was intense in neurons of the SN, and present to a lesser extent in neurons of the nucleus raphe dorsalis and oculomotor nucleus, and occasionally in glia. The proportion of 8OHG immunoreactive SN neurons was significantly greater in PD patients compared to age-matched controls. Midbrain sections from patients with multiple system atrophy-Parkinsonian type (MSA-P) and dementia with Lewy bodies (DLB) also were examined. These showed increased cytoplasmic 8OHG immunoreactivity in SN neurons in both MSA-P and DLB compared to controls; however, the proportion of positive neurons was significantly less than in PD patients. The regional distribution of 8OHG immunoreactive neurons within the SN corresponded to the distribution of neurodegeneration for these three diseases. Nuclear 8OHG immunoreactivity was not observed in any individual. The type of cytoplasmic nucleic acid responsible for 8OHG immunoreactivity was analyzed by preincubating midbrain sections from PD patients with RNase, DNase, or both enzymes. 8OHG immunoreactivity was substantially diminished by either RNase or DNase, and completely ablated by both enzymes. These results suggest that oxidative damage to cytoplasmic nucleic acid is selectively increased in midbrain, especially the SN, of PD patients and much less so in MSA-P and DLB patients. Moreover, oxidative damage to nucleic acid is largely restricted to cytoplasm with both RNA and mitochondrial DNA as targets.

Aged↗
PubMed2002

Role of the C-terminus and the long cytoplasmic loop in reduced folate carrier expression and function.

The reduced folate carrier (RFC1), a member of the major facilitative superfamily, generates uphill transport of folates into cells through an exchange mechanism with intracellular organic anions. RFC1 has twelve transmembrane domains with N- and C-termini, and the long loop connecting the 6th and 7th transmembrane domains, directed to the cytoplasm. To elucidate the role of the C-terminus and the long cytoplasmic loop in carrier function, mutants with deletion of the entire C-terminus or with progressive deletions of the loop region were constructed and stably transfected into the murine MTX(r)A cell line, which lacks functional RFC1. While expression of the C-terminus-deleted RFC1 protein could not be detected in the cell lysate, the RFC1 mutant lacking 57 of 66 amino acid residues of the long cytoplasmic loop appeared to be inserted into the cytoplasmic membrane but was not functional. In cell lines in which 17 or 31 amino acids were deleted from the carboxyl half of the loop, there was partial preservation of methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate transport. The loss of 5-formyltetrahydrofolate transport activity in the delta31 and delta17 mutants was due primarily to a decrease in substrate binding to the carrier. Mutants with partially truncated internal loops demonstrated an anion responsiveness similar to that of wild-type RFC1, indicating that this region of the carrier does not contain a site(s) that plays a role in anion exchange. This is the first study to describe the important role of the long cytoplasmic loop in substrate binding and the crucial role of the C-terminus in maintaining stability of RFC1.

Amino Acid Sequence↗
PubMed2002

Alterations in membrane-bound and cytoplasmic K-ras protein levels in mouse lung induced by treatment with lovastatin, cholestyramine, or niacin: effects are highly mouse strain dependent.

Agents that either increase (cholestyramine, CS) or decrease (lovastatin, Lov) de novo peripheral cholesterol synthesis may increase (CS) or decrease (Lov) ras protein membrane localization by altering protein prenylation, and potentially have pro- or anti-carcinogenic effects. Male A/J, Swiss, and C57/BL6 mice were treated with 2 or 4% CS, 1% dietary niacin, or 25mg/kg of Lov three times per week (Lov-3X) or five times per week (Lov-5X). After 3 weeks, serum cholesterol and triglycerides were determined enzymatically. Membrane and cytoplasmic K-ras proteins in lung were determined by immunoprecipitation followed by western blotting with a K-ras specific antibody. Results confirmed the hypothesis only in isolated instances. A/J mice had a significant 30% increase in cytoplasmic K-ras and a 40% decrease in membrane K-ras from Lov treatment, as predicted. C57/BL6 mice had a significant 77% increase in membrane K-ras, as expected from CS feeding. At variance with the hypothesis, Swiss mice had increased levels (3-28%) of membrane K-ras with all treatments (including Lov), and C57/BL6 mice treated with Lov had a 58-78% increase in cytoplasmic K-ras without any reduction in the levels of membrane K-ras. Niacin, predicted to have no effect on ras membrane localization, decreased cytoplasmic K-ras in A/J mice, increased both membrane and cytoplasmic K-ras in Swiss mice, and had no effect in C57/BL6 mice. Results may have differed from those predicted because of strain-dependent differences in response to the cholesterol-lowering agents. A difference in response among the mouse strains suggests that individual genetic differences may alter the effect of hypocholesterolemic agents on K-ras membrane localization, and potentially the risk of ras-dependent cancer.

Animals↗
PubMed1986

The mechanics of motility in dissociated cytoplasm.

We stimulate the dynamical behavior of dissociated cytoplasm using the Reactive Flow Model (Dembo, M., and F. Harlow, 1986, Biophys. J., 50:109-121). We find that for the most part the predicted dynamical behavior of the cytoplasm is governed by three nondimensional numbers. Several other nondimensional parameters, the initial conditions, and boundary conditions are found to have lesser effects. Of the three major nondimensional parameters, one (D#) controls the percentage of ectoplasm, the second (C#) controls the sharpness of the endoplasm-ectoplasm boundary, and the third (R#) controls the topological complexity of the endoplasm-ectoplasm distribution. If R# is very small, then the cytoplasm contracts into a single uniform mass, and there is no bulk streaming. If R# is very large, then the cytoplasmic mass breaks up into a number of clumps scattered throughout the available volume. Between these clumps the solution undergoes turbulent or chaotic patterns of streaming. Intermediate values of R# can be found such that the mass of cytoplasm remains connected and yet undergoes coherent modes of motility similar to flares (Taylor, D.L., J.S. Condeelis, P.L. Moore, and R.D. Allen, 1973, J. Cell Biol., 59:378-394) and rosettes (Kuroda, K., 1979, Cell Motility: Molecules and Organization, 347-362).

Algorithms↗
PubMed2002

Vasopressin-induced cytoplasmic and nuclear calcium signaling in cultured cortical astrocytes.

We sought to determine whether vasopressin V(1a) receptor (V(1a)R) mRNA detected in cortical astrocytes [Brain Res. Mol. Brain Res. 45 (1997) 138] was translated into functional receptors by investigating the effector calcium signaling cascade associated with the vasopressin V(1a) receptor subtype. Analysis of intracellular calcium dynamics using the calcium-sensitive dye fura-2 AM indicated that exposure of cortical astrocytes to V(1) vasopressin receptor agonist, [Phe(2),Orn(8)]-oxytocin, induced a marked dose-dependent increase in intracellular calcium which was abolished by depletion of extracellular calcium. V(1) agonist treatment induced a rapid increase in calcium signal in both the cytoplasm and nucleus, which was followed by an accumulation of the calcium signal in the nucleus, suggesting translocation of cytoplasmic calcium into the nucleus. The nuclear calcium signal was sustained for several seconds followed by translocation back to the cytoplasm. Following the nuclear-to-cytoplasmic calcium translocation, total free intracellular calcium concentration decreased. The dynamic calcium cytoplasmic and nuclear localization was confirmed by laser scanning confocal microscopy coupled with the calcium-sensitive dye fluo-3 AM. To determine the source of calcium, V(1) agonist-induced (45)Ca(2+) uptake and [(3)H]IP(1) accumulation were investigated. V(1) agonist induced significant and rapid uptake of (45)Ca(2+) and a significant dose-dependent increase in [(3)H]IP(1) accumulation in cortical astrocytes. To our knowledge, this is the first documentation of a vasopressin receptor-induced calcium signaling cascade in cortical astrocytes and the first documentation of vasopressin receptor induction of nuclear calcium signaling.

Animals↗
PubMed2003

Basolateral sorting of transforming growth factor-alpha precursor in polarized epithelial cells: characterization of cytoplasmic domain determinants.

In polarized Madin-Darby canine kidney (MDCK) cells, newly synthesized transforming growth factor-alpha precursor (proTGFalpha) is directly sorted to the basolateral cell surface where it is sequentially cleaved and released into the basolateral conditioned medium (Dempsey, P.J., Coffey, R.J., J. Biol. Chem. 269 (1994) 16878-16889). In the present study, the role of the proTGFalpha cytoplasmic domain in basolateral sorting has been investigated using deletional and site-directed mutagenesis, as well as chimeric analyses of different TGFalpha constructs stably expressed in MDCK cells. The loss of polarized secretion of a proTGFalpha secretory mutant (TGFsec88) indicated that the proTGFalpha transmembrane and/or cytoplasmic domains contain essential basolateral sorting information. Using reporter chimeras with two apically sorted membrane proteins, p75 neurotrophin growth factor receptor and placental alkaline phosphatase, we show that the proTGFalpha cytoplasmic domain contains dominant basolateral sorting information. Analysis of proTGFalpha cytoplasmic domain truncation and internal deletion mutants, together with site-directed mutagenesis studies within the full-length proTGFalpha cytoplasmic domain, revealed redundant basolateral sorting motifs. Importantly, the C-terminal type I PDZ-binding motif was not required for basolateral sorting as determined by the integrity of basolateral sorting in deletion mutants lacking this motif. ProTGFalpha basolateral sorting may have important consequences for ligand presentation and spatial compartmentalization of epidermal growth factor receptor signaling networks in polarized epithelial cells.

Amino Acid Motifs↗
PubMed1997

The p80 TNF receptor-associated kinase (p80TRAK) associates with residues 354-397 of the p80 cytoplasmic domain: similarity to casein kinase.

The cytoplasmic domain of the p80 TNF receptor associates with a protein kinase, termed p80TRAK, that phosphorylates both the p60 and p80 TNF receptors. To determine the region of the cytoplasmic domain that is necessary for binding of p80TRAK and the region that it phosphorylates, a series of deletions of the p80 cytoplasmic domain were constructed and expressed as glutathione-S-transferase fusion proteins. These fusions were then used to examine the binding of p80TRAK derived from cellular extracts. We found that out of 174 residues (266-439) in the cytoplasmic domain of p80 receptor, 44 residues (354-397) were sufficient for binding of p80TRAK. Interestingly, this was also the region that contained the phosphorylation site for p80TRAK. Phosphoamino acid analysis of this region revealed phosphorylation primarily on serine residues. Furthermore, we found that, like p80TRAK, purified casein kinase 1 (CK1) also binds to residues 354-397 of the p80 TNF receptor and causes its phosphorylation. Additionally, the activity of p80TRAK was inhibited by CK1-7, the CK1-specific inhibitor. Thus, our results indicate that p80TRAK associates with a short stretch of approximately 44 residues located in the cytoplasmic domain of the p80 TNF receptor and that this kinase is similar to CK1.

Antigens, CD↗
PubMed1998

Changes in cytoplasmic ATP concentration parallels changes in ATP-regulated K+-channel activity in insulin-secreting cells.

Changes in cytoplasmic ATP concentration were monitored in intact insulin-producing cells and correlated to changes in the activity of ATP-sensitive K+-channels (KATP channels). Luciferase was introduced into HIT M2.2 cells and whole pancreatic islets by transient expression of firefly (Photinus pyralis) luciferase cDNA. In transfected cells, extracellular addition of luciferin increased the luminescence signal to a maximum within 50-120 s. Addition of 1 microM of the mitochondrial uncoupler FCCP decreased the luminescence, an effect partly reversed upon withdrawal of the compound. High concentrations of glucose increased cytoplasmic free ATP concentration. Changes in the luminescence signal were accompanied by changes in activity of the ATP-sensitive K+-channel. Transfection per se did not deteriorate cell function, as verified by experiments showing similar changes in cytoplasmic free Ca2+-concentration, [Ca2+li, in both transfected and non-transfected cells. By measuring the cytoplasmic ATP concentration and KATP channel activity under similar experimental conditions, it was possible to establish, for the first time, a direct relationship between these two parameters. This indeed suggests that the cytoplasmic ATP concentration has a crucial role in the regulation of KATP channel activity under physiological conditions.

Adenosine Triphosphate↗
PubMed1999

Calpain cleavage of integrin beta cytoplasmic domains.

We showed previously that the calcium-dependent protease, calpain, cleaves the cytoplasmic domain of the integrin beta3 subunit. To investigate whether susceptibility to calpain is a common feature of all integrin beta subunits, and to map calpain cleavage sites in different integrin beta tails, we treated recombinant cytoplasmic domains of integrin beta1A, beta1D, beta2, beta3 and beta7 subunits with purified calpain in vitro. We found that the cytoplasmic domains of all these integrin chains were cleaved by calpain. HPLC followed by mass spectrometry was used to identify calpain cleavage sites. These sites were clustered in the C-terminal half of the integrin beta cytoplasmic domains in regions flanking the two NXXY motifs, suggesting the possibility that the structural framework provided by these motifs is recognized by calpain. We used the knowledge of these cleavage sites to develop cleavage site-specific antibodies and to demonstrate cleavage of the beta1A cytoplasmic domain in intact platelets stimulated with calcium ionophore or thrombin. Thus susceptibility to calpain cleavage is common to integrin beta subunits, can be induced in intact cells, and appears to favor regions surrounding two conserved NXXY motifs.

Amino Acid Sequence↗
PubMed2000

Varicocele is associated with abnormal retention of cytoplasmic droplets by human spermatozoa.

OBJECTIVE: To determine whether varicocele is associated with retention of sperm cytoplasmic droplets in infertile men. DESIGN: Retrospective study. SETTING: University infertility clinic. PATIENT(S): Nonazoospermic men with idiopathic (n = 69) and varicocele-associated infertility (n = 73), and 20 fertile controls presenting for vasectomy. INTERVENTION(S): None. MAIN OUTCOME MEASURES(S): Standard semen parameters and percentage of spermatozoa with cytoplasmic droplets on Papanicolaou smears. RESULT(S): No statistically significant differences were found between the fertile and infertile groups with respect to semen volume. Fertile controls had significantly greater mean percent sperm motility and normal morphology than infertile men. The mean percentage of sperm with residual cytoplasm was statistically significantly different in all three groups. Infertile men with varicocele had the highest percentage of sperm with cytoplasmic droplets, the next highest level being in men with idiopathic infertility and the lowest level in fertile controls (11.7 +/- 1.0, 8.1 +/- 0.9 and 3.2 +/- 0.4%, respectively, P<.0001). CONCLUSION(S): Our data show that idiopathic and even moreso, varicocele-related male infertility are conditions associated with impaired disposal of residual sperm cytoplasm by the testis and/or epididymis. These data provide a possible mechanism for the observed semen abnormalities and reduced fertility potential associated with varicocele and idiopathic male infertility.

Cytoplasm↗
PubMed2001

Cytoplasmic accumulation of flavonoids in flower petals and its relevance to yellow flower colouration.

It is widely accepted that the mix of flavonoids in the cell vacuole is the source of flavonoid based petal colour, and that analysis of the petal extract reveals the nature and relative levels of vacuolar flavonoid pigments. However, it has recently been established with lisianthus flowers that some petal flavonoids can be excluded from the vacuolar mix through deposition in the cell wall or through complexation with proteins inside the vacuole, and that these flavonoids are not readily extractable. The present work demonstrates that flavonoids can also be compartmented within the cell cytoplasm. Using adaxial epidermal peels from the petals of lisianthus (Eustoma grandiflorum), Lathyrus chrysanthus and Dianthus caryophyllus, light and laser scanning confocal microscopy studies revealed a significant concentration of petal flavonoids in the cell cytoplasm of some tissues. With lisianthus, flavonoid analyses of isolated protoplasts and vacuoles were used to establish that ca 14% of petal flavonoids are located in the cytoplasm (cf. 30% in the cell wall and 56% in the vacuole). The cytoplasmic flavonoids are predominantly acylated glycosides (cf. non-acylated in the cell wall). Flavonoid aggregation on a cytoplasmic protein substrate provides a rational mechanism to account for how colourless flavonoid glycosides can produce yellow colouration in petals, and perhaps also in other plant parts. High vacuolar concentrations of such flavonoids are shown to be insufficient.

Chromatography, High Pressure Liquid↗
PubMed1998

Characterization of cytoplasmic secretory granules (PSG), in prostatic epithelium and their transformation-induced loss in dysplasia and adenocarcinoma.

Cytoplasmic clarity is a histological feature of normal prostatic secretory cells, but in this study, tissue fixation in strong (>2.5%) glutaraldehyde dramatically altered cytological staining. Secretory cytoplasm appeared red and granular on routine stains because of myriad intensely staining eosinophilic granules (PSG). Immunostaining for prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) showed their exclusive localization to the PSG. Electron microscopy confirmed these findings and also showed that after fixation in many agents, including formaldehyde, PSG appeared empty, accounting for the artefactual "clear cell" appearance on light microscopy. PSG were most densely concentrated apically in a bud-shaped luminal compartment in which cytokeratin was selectively absent. Normal exocrine secretion was visualized as detachment of apocrine buds or their in situ disintegration. Distinctively in dysplasia and almost all carcinomas, PSG were rare to absent, and proteases were free in the cytoplasm, often concentrated beneath the apical membrane. The apocrine compartment was absent, with no observed secretory mechanism. Tumor cells had dark amphiphilic cytoplasm after all fixatives. This provided a reliable method of distinguishing malignant from benign glands in tissues fixed in strong glutaraldehyde. Clear cell carcinomas, whose cytoplasm mimicked routinely fixed normal secretory cells, surprisingly had almost no PSG. Instead, their "granules" were lipid-filled vacuoles reflecting a secretory pathway not seen in normal cells, dysplasia, or the common "dark cell" carcinomas. These observations may define two distinctive biological pathways of prostate cancer evolution and may facilitate diagnostic decisions on needle biopsy samples.

Acid Phosphatase↗
PubMed2007

Impact of assisted reproductive technologies: a mitochondrial perspective of cytoplasmic transplantation.

Many of the assisted reproductive techniques associated with maternal aging, disease states, or implantation failure aim to correct poor developmental capacity. These techniques are highly invasive and require the exchange of nuclear or cytoplasmic material from a donor oocyte to compensate for deficiencies inherent in the affected individual. These techniques are based on the assumption that the cytoplasm of the donor oocyte can effectively substitute the necessary component(s) to enable development to proceed. Several studies have attempted to inject cytoplasm from "normal" (young) donors, into aged eggs, again assuming that beneficial components of the cytoplasm are transferred to restore developmental capacity. These invasive assisted reproduction technology (ART) procedures aim to eliminate chromosomal abnormalities, improve the quality of oocytes deficient in some important cytoplasmic factors necessary for maturation and/or subsequent development, and eliminate maternally inherited diseases (particularly mitochondrial myopathies). However, in order to develop such ART, understanding the processes involving mitochondrial DNA replication and transcription is imperative, as asynchrony between mitochondrial and nuclear genomes may cause problems in mitochondrial function, localization, and biogenesis.

Animals↗
PubMed2000

Can cytoplasm exist without undergoing phase separation?

Studies on such systems as the lens of the eye and theoretical considerations suggest that phase separation may well occur in cytoplasm. In this chapter, several issues relevant to this question are raised. It is suggested that while the interaction between water and the macromolecules in a mixture is proving crucial to their phase separation behavior, the abnormal water that is widely observed in cytoplasm and concentrated protein solutions is unlikely to constitute a thermodynamic phase in the sense of phase separation studies. The role of fixed structures in the cytoplasm, the likelihood that the volume of separated phases would be small and subject to spreading over the fixed structures and the expectation that much of the phase volume could be occupied and dominated by properties of the interface are also discussed. Finally, some experimental approaches to studying the existence of liquid-liquid phases in cytoplasm are proposed. While there is no proof that phase separation exists in cytoplasm, application of some of the techniques outlined might well provide more positive evidence for its presence.

Animals↗
PubMed1997

Nucleoplasmic and cytoplasmic differences in the fluorescence properties of the calcium indicator Fluo-3.

The fluorescent indicator Fluo-3 is widely used to monitor the calcium concentration ([Ca2+]) in the cytoplasm and nucleus of various cells. Estimates of nuclear [Ca2+] are based on the assumption of identical behavior of Fluo-3 in different cellular compartments. The assumption is not valid if the fluorescence properties of the dye are altered by the nuclear environment, independent of the [Ca2+]. To determine the effects of the nucleoplasm on the behavior of Fluo-3, we applied laser scanning confocal microscopy and spectrophotometry to measure fluorescence intensity as well as emission and absorbance spectra of the Ca2+ indicator, Fluo-3. Spectra were measured in intact Xenopus oocytes, neuroblastoma cells, and cytoplasmic and nucleoplasmic homogenates. The fluorescence signal in intact cells loaded with Fluo-3 was approximately 2-times higher in the nucleus when compared to the cytoplasm. The fluorescence intensity of Fluo-3 in nucleoplasmic homogenates was higher than in cytoplasmic homogenates or internal buffers even when [Ca2+] was clamped. Despite identical [Ca2+], pH, and temperature, the emission and absorbance spectra of Fluo-3 from nuclear homogenates displayed a higher fluorescence at each wavelength measured when compared to spectra from cytoplasmic homogenates or internal buffer solutions, and saturated above 100 nM. These findings demonstrate that the composition of the nucleoplasm changes the fluorescence properties of the calcium indicator Fluo-3. Consequently, analysis of nuclear calcium dynamics must take into account the distinct behavior of Fluo-3 in different cellular compartments.

Aniline Compounds↗
PubMed2000

Two approaches to isolate cytoplasmic dynein ATPase from Neurospora crassa.

Cytoplasmic dynein is a force-producing enzyme that, in association with dynactin, conducts minus-end directed transport of various organelles along microtubules. Biochemical analyses of cytoplasmic dynein and dynactin have been conducted primarily in vertebrate systems, whereas genetic analyses have been explored mainly in yeast and the filamentous fungi. To provide a complementary biochemical approach for the study of fungal dynein, we isolated/partially purified cytoplasmic dynein ATPase from the filamentous fungus Neurospora crassa. N. crassa dynein was partially purified by slightly modifying the existing procedures, described for mammalian cytoplasmic dynein that uses dynein-microtubule binding, followed by release with ATP and sucrose gradient fractionation. A novel approach was also used to isolate dynein-specific ATPase by gel filtration (Sepharose CL-4B). The K(m), ATP obtained by isolating dynein ATPase using gel filtration was similar to that obtained by using conventional method, suggests that contaminant proteins do not interfere with the dynein ATPase activity. Like vertebrate dynein, N. crassa dynein is a general NTPase with highest activity toward ATP, and only the ATPase activity is stimulated by microtubules. The K(m), ATP for N. crassa cytoplasmic dynein is 10- to 15-fold higher than that of the vertebrate enzyme.

Adenosine Triphosphate↗