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M M Pratt

Publications and source records attributed to M M Pratt.

15 recordsLinked to original sources
PubMed1993

Cyclic AMP-dependent phosphorylation of a 26 kD axonemal protein in ovine cilia isolated from small tissue pieces.

To study cyclic adenosine monophosphate (cAMP)-dependent phosphorylation events in ovine cilia in vitro, we adapted published axonemal isolation methods to obtain pure mammalian axonemal proteins from small ovine tracheal mucosa pieces with a surface area of only 1 cm2. The isolated axonemes could be reactivated in vitro upon ATP addition, thereby attesting to their functional integrity. The axonemal protein yield from these small mucosa pieces was high enough to allow protein concentration measurements of each sample and axonemal polypeptide analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). cAMP is known to increase ciliary beat frequency, possibly through a phosphorylation event in the axoneme. To study cAMP-dependent phosphorylation events in ovine tracheal cilia, these axonemal preparations were exposed to [gamma-32P]ATP under conditions that stimulated or inhibited kinase activity. Analysis of axonemal polypeptides by SDS-PAGE and subsequent autoradiography showed that an axonemal protein with a M(r) of 26 kD is the only polypeptide consistently phosphorylated in a cAMP-dependent manner. The phosphorylation of this protein could be diminished by a highly specific inhibitor of cAMP-dependent protein kinase, KT-5720. The addition of calcium did not affect label incorporation into this protein during cAMP treatment. In the presence of cAMP and calcium, inhibitors of protein kinase C and calcium/calmodulin-dependent kinase did not change the level of phosphorylation of the 26 kD protein. We conclude that cAMP treatment of isolated mammalian cilia results in the phosphorylation of a single protein with a M(r) of 26 kD (p26).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Protein kinase C-dependent phosphorylation of a ciliary membrane protein and inhibition of ciliary beating.

The present study examined whether protein kinase C phosphorylated a ciliary protein and whether this phosphorylation event was temporally correlated with a decrease in ciliary beat frequency. Activation of protein kinase C decreased ciliary beat frequency of sheep tracheal epithelium, an effect fully blockable by pretreatment of the tissue pieces with H-7, a protein kinase inhibitor. Using cilia removed from these epithelial surfaces and incubated in solutions containing stimulators of protein kinase C along with [gamma-32P]ATP or [gamma-35S]ATP, a single protein target of ciliary protein kinase C activity was identified. The protein is a polypeptide of molecular mass 37 kDa (p37) as estimated by SDS-polyacrylamide gel electrophoresis. Protein kinase C dependency of p37 phosphorylation was proven by showing that Calphostin C, a specific protein kinase C inhibitor, blocked label incorporation into p37 completely, and by demonstrating that purified protein kinase C phosphorylated p37. Inhibitors of cAMP-dependent kinase and calcium/calmodulin-dependent kinase did not change the phosphorylation of p37 in the presence of protein kinase C activators. p37 was recovered in a Triton X-100-extractable fraction of this ciliary preparation, suggesting that p37 is membrane associated. This hypothesis was further supported by the fact that p37 was present in a pellet representing reconstituted membranes. Thin-layer electrophoresis revealed that p37 was phosphorylated on serine and tyrosine residues, suggesting that the activation of protein kinase C also stimulated tyrosine kinase activity. p37 did not precipitate with annexin I or II antibodies. These results show that sheep tracheal cilia contain protein kinase C activity and that activated protein kinase C phosphorylates a membrane-associated ovine ciliary target, an effect temporally related to a protein kinase C-mediated decrease in ciliary beat frequency.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Activation of ATPase activity of 14S dynein from Tetrahymena cilia by microtubules.

The ATPase activity of 14S dynein was activated by the presence of microtubule-associated-protein-free microtubules. The activation was 2.5-3.5 fold at 10 mg microtubule/ml, and the activity increased further with increasing microtubule concentration. The microtubule-14S-dynein complex, microtubule bundles with 14S dynein, was treated with a zero-length chemical cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). The ATPase activity of the complex responded to EDC in a biphasic, concentration-dependent manner and, at most, it was enhanced 5-10 fold. The complex treated with EDC was no longer unbundled by addition of ATP, as revealed by electron-microscopic observation. Several ATP analogues, which support in vitro microtubule translocation mediated by 14S dynein, were turned over faster by this mechanochemical enzyme in the presence of microtubules than in their absence. However, some ATP analogues which do not support the translocation were also turned over faster in the presence of microtubules. Thus, microtubule-dynein motility and substrate-turnover activation are not tightly coupled, which indicates that all three major motor systems, actin- heavy-meromyosin, microtubule-kinesin [Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F. & Vale, R. D. (1991) J. Cell Biol. 112, 1189-1197] and microtubule-dynein, have this characteristic property in common.

Adenosine Triphosphatases↗
PubMed1992

Mechanism of hydrogen peroxide-induced inhibition of sheep airway cilia.

To study the effect of the inflammatory mediator hydrogen peroxide (H2O2) on airway ciliary activity, we measured ciliary beat frequency (CBF) in cultured tracheal explants from sheep. Addition of H2O2 (10(-8) to 10(-4) M) produced a concentration-dependent mean (+/- SEM) decrease in CBF between 11.1 +/- 0.4% (P less than 0.01) and 100 +/- 0% (P less than 0.001); at each concentration, the maximal effect was reached by 20 to 25 min. Between 10(-8) and 10(-6) M H2O2, the decrease in CBF was reversible, lactate dehydrogenase (LDH) release was not significantly increased, and major morphologic lesions were not seen. At higher concentrations of H2O2, incomplete recovery of CBF (10(-5) M) or irreversible ciliostasis (10(-4) M) developed, and a significant increase in LDH and morphologic lesions were present. Catalase (2,000 U/ml) and H-7 (10(-5) M), a protein kinase inhibitor, abolished cilioinhibition produced by H2O2 at 10(-6) M and lower concentrations but not at 10(-5) M and higher concentrations. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, caused a dose-dependent (10(-11) to 10(-5) M), reversible decrease in CBF; this effect was abolished by H-7. We suggest that at nonlethal concentrations, H2O2 inhibits the beat frequency of airway epithelial cilia reversibly, through the activation of second messengers, including protein kinase C. This mechanism might contribute to the previously demonstrated impairment of mucociliary clearance in airway inflammation.

Animals↗
PubMed1992

A novel vesicle-associated protein (VAP-1) in sea urchin eggs containing multiple RNA-binding consensus sequences.

We have identified a novel high molecular weight, vesicle-associated protein (VAP-1) in the eggs of the sea urchin Strongylocentrotus purpuratus. Biochemical fractionation and immunofluorescence analysis of unfertilized eggs indicate that VAP-1 is a peripheral membrane protein associated with microsomal membrane fractions. Sequence analysis of partial VAP-1 cDNA clones reveals that the protein contains at least four RNA-binding consensus sequences. The RNA-binding sequences are separated by several glycine rich domains and this organization, RNA-binding domains separated by glycine rich sequences, is common to several RNA-binding proteins including the heterogeneous ribonuclear protein A1 and nucleolin. The characteristics of VAP-1 suggest that the protein may function as a multidomain RNA-binding protein. The possibility that VAP-1 may play a role in nuclear RNA processing is also discussed.

Amino Acid Sequence↗
PubMed1986

Homology of egg and flagellar dynein. Comparison of ATP-binding sites and primary structure.

Unfertilized sea urchin eggs contain a Mg2+-ATPase which shares physical and enzymatic characteristics with dynein, the enzyme which powers ciliary and flagellar movement. To further investigate the homology of the egg ATPase and axonemal dynein, ATP-binding subunits in preparations of each of the enzymes were identified using a photoaffinity probe of ATP, 8-azido-ATP (8-N3ATP), and three high molecular weight (HMW) polypeptide components of the two enzymes were compared by one-dimensional peptide mapping. Two heavy chains (A and B) of both the flagellar and egg ATPases bound [alpha-32P]8-N3ATP. The labeling of the HMW bands was specifically inhibited by ATP or ADP. Both the cytoplasmic ATPase and flagellar dynein utilized 8-N3ATP as a substrate indicating that the reagent binds to the active site. The two HMW ATP-binding polypeptides and one other HMW component of the egg ATPase were compared to flagellar dynein heavy chains by peptide mapping. Digestion of the egg versus flagellar HMW polypeptides with Staphylococcus V8 protease or alpha-chymotrypsin produced a highly similar group of peptides, and each pair of heavy chains was qualitatively estimated to be over 85% homologous. These data support the identification of the egg ATPase heavy chains as components of a cytoplasmic dynein and suggest that the HMW polypeptides form active enzymatic sites in flagellar and egg dynein which are substantially homologous.

Actins↗
PubMed1986

Stable complexes of axoplasmic vesicles and microtubules: protein composition and ATPase activity.

Fast transport of axonal vesicles and organelles is a microtubule-associated movement (Griffin, J. W., K. E. Fahnestock, L. Price, and P. N. Hoffman, 1983, J. Neuroscience, 3:557-566; Schnapp, B. J., R. D. Vale, M. P. Sheetz, and T. S. Reese, 1984, Cell, 40:455-462; Allen, R. D., D. G. Weiss, J. H. Hayden, D. T. Brown, H. Fujiwake, and M. Simpson, 1985, J. Cell Biol., 100:1736-1752). Proteins that mediate the interactions of axoplasmic vesicles and microtubules were studied using stable complexes of microtubules and vesicles (MtVC). These complexes formed spontaneously in vitro when taxol-stabilized microtubules were mixed with sonically disrupted axoplasm from the giant axon of the squid Loligo pealei. The isolated MtVCs contain a distinct subset of axoplasmic proteins, and are composed primarily of microtubules and attached membranous vesicles. The MtVC also contains nonmitochondrial ATPase activity. The binding of one high molecular mass polypeptide to the complex is significantly enhanced by ATP or adenyl imidodiphosphate. All of the axoplasmic proteins and ATPase activity that bind to microtubules are found in macromolecular complexes and appear to be vesicle-associated. These data allow the identification of several vesicle-associated proteins of the squid giant axon and suggest that one or more of these polypeptides mediates vesicle binding to microtubules.

Adenosine Triphosphatases↗
PubMed1984

Calmodulin interaction with cytoplasmic and flagellar dynein: calcium-dependent binding and stimulation of adenosinetriphosphatase activity.

Hisanaga and Sakai [Hisanaga, S., & Sakai, H. (1983) J. Biochem. (Tokyo) 93, 87-98] demonstrated that cytoplasmic dynein could be purified, in part, by chromatography on a calmodulin-Sepharose 4B affinity column and that the adenosinetriphosphatase (ATPase) activity of the enzyme was stimulated by Ca2+-calmodulin. In the present study, we have investigated, in detail, the interaction of cytoplasmic and flagellar dynein from the sea urchin Hemicentrotus pulcherrimus with calmodulin (CaM) isolated from porcine brain or sea urchin egg. The dynein Mg2+-ATPase activity is stimulated 3-8-fold by calmodulin from either source. The stimulation is dependent on calcium ions and is inhibited by trifluoroperazine. CaM stimulation is sensitive to physiologically regulatory calcium ion concentrations around 1 microM. Activation is also sensitive to pH and occurs maximally at physiological pH near 7.0. Calmodulin binds directly to cytoplasmic dynein as judged by cosedimentation in a sucrose density gradient. The binding and enzymatic stimulation occur at calmodulin:dynein ratios of 150:1 to 300:1, which are consistent with estimates of in vivo ratios. Cytoplasmic and flagellar dynein ATPase activities are also stimulated by Triton X-100, a nonionic detergent, and by limited protolysis with trypsin. Both of these treatments abolish further activation by calmodulin. The possibility of a trypsin-labile, CaM binding subunit of the enzyme is discussed. In addition, since both CaM and dynein are localized in the mitotic apparatus, we suggest that CaM may regulate possible mitotic dynein activity.

Adenosine Triphosphatases↗
PubMed1984

An improved purification method for cytoplasmic dynein.

An improved method has been devised for the purification of cytoplasmic dynein from sea urchin eggs (Strongylocentrotus droebachiensis and S purpuratus). This protocol introduces three changes over a previously published procedure (Hisanaga and Sakai: J Biochem 93:87, 1983)--the substitution of diethylaminoethyl (DEAE)-cellulose for hydroxylapatite chromatography, the elimination of sucrose density gradient centrifugation, and the use of phosphocellulose chromatography. These changes reduce the time and increase the efficiency of the purification procedure. The purified egg cytoplasmic dynein has enzymatic properties in common with axonemal dynein, including ionic specificity (Ca++ATPase/Mg++ ATPase = 0.8) and inhibition by sodium vanadate and erythro-9-2,3-hydroxynonyl adenine (EHNA). As assayed by silver staining of polyacrylamide gels, the cytoplasmic dynein is composed of two high molecular weight polypeptides (greater than 300 kilodaltons) that comigrate with flagellar dynein heavy chains, and lesser amounts of three lower molecular weight bands. None of these polypeptides appears to contain bound carbohydrate. The purification procedure can be modified slightly to allow the preparation of cytoplasmic dynein in only 2 days from as little as 3-5 ml of packed eggs, a 20-fold reduction over the previous method. This more rapid and efficient method will facilitate the investigation of cytoplasmic dynein in other systems where starting material is limited, including tissue culture cells and nerve axoplasm.

Adenosine Triphosphatases↗
PubMed1980

Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase.

Photochemical cross-linking of both Tetrahymena and Aequipecten ciliary membrane proteins with the lipophilic reagent 4,4'-dithiobisphenylazide links together a high molecular weight dynein-like ATPase, membrane tubulin, and at least two other proteins. Electron microscopy of detergent-extracted cilia reveals that the cross-linked complex remains attached to the outer-doublet microtubules by a microtubule-membrane bridge. Cleavage of the reagent's disulfide bond releases the bridge-membrane complex and the dynein-like membrane-associated ATPase. Electron microscopy was used to ensure that the dynein-like protein did not result from the solubilization of the dynein arms attached to the outer-doublet microtubules. The dynein-like protein has been isolated using sucrose gradients and is similar to axonemal dynein with respect to its sedimentation characteristics nucleotide specificity, and divalent cation requirements. Photochemical cross-linking of ciliary membrane porteins in vivo results initially in the modification of ciliary beat and, eventually, in the cessation of ciliary movement. These results suggest that a dynein-like ATPase comprises the bridge which links the ciliary membrane to the outer-doublet microtubules and that this bridge is involved in the modulation of normal ciliary movement.

Adenosine Triphosphatases↗
PubMed1980

Dynein-like Mg2+-ATPase in mitotic spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis).

Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg2+-ATPase resembling axonemal dynein, and a Ca2+-ATPase postulated to be bound in membranes. To examine the nature of the Mg2+-ATPase, we isolated membrane-free mitotic spindles from Stronglylocentrotus droebachiensis embryos by rapidly lysing these in a calcium-chelating, low-ionic-strength buffer (5 mM EGTA, 0.5 mM MgCl2, 10 mM PIPES, pH 6.8) that contained 1% Nonidet P-40. The fibrous isolated mitotic spindles closely resembled spindles in living cells, both in general morphology and in birefringence. In electron micrographs, the spindles were composed primarily of microtubules, free from membranes and highly extracted of intermicrotubular cytoplasmic ground substance. As analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the pelleted spindles contain 18% tubulin, variable amounts of actin (2-8%), and an unidentified protein of 55 kdaltons in a constant weight ratio to tubulin (1:2.5). The isolated spindles also contained two polypeptides, larger than 300 kdaltons, that comigrated with egg dynein polypeptides, and ATPase activity (0.02 mumol Pi/mg . min) that closely resembled both flagellar and egg dynein. The spindle Mg2+-ATPase showed a ratio of Ca2+-/Mg2+-ATPase = 0.85, had minimal activity in KCl and EDTA, and cleaved GTP at 35% of the rate of ATP. The Mg2+-ATPase was insensitive to ouabain or oligomycin. The spindle Mg2+-ATPase was inhibited by sodium vanadate but, like egg dynein, was less sensitive to vanadate than flagellar dynein. The spindle Mg2+-ATPase does not resemble the mitotic Ca2+-ATPase described by others. We propose that the spindle Mg2+-ATPase is egg dynein. Bound carbohydrate on the two high-molecular-weight polypeptides of both egg dynein and the spindle enzyme suggest that these proteins may normally associate with membranes in the living cell.

Adenosine Triphosphatases↗
PubMedDate not supplied

Severity index scores correlate with survival of AIDS patients.

A major concern of hospices treating AIDS patients is survival prognosis. Patients are eligible for government hospice benefits only if they are in the last six months of life, but for AIDS patients who present at different disease stages, it is often difficult to predict survival. We have tested an index of AIDS severity developed by Alemi et al. (1991, Interfaces, 21(3), 105) for its ability to predict survival in hospice-AIDS patients. Using retrospective analysis of medical records, a severity index (SI) score was determined for 26 AIDS patients who were admitted at different disease stages to a South Florida hospice. The length of stay for each patient was also recorded. The patients fell clearly into two groups, those with stays of six months or less and those with stays of more than six months. The mean SI scores of the two groups were .9188 and .7845, respectively. These scores were significantly different at the p = .005 level. In this preliminary study, the severity score correlated well with survival prognosis. Based on these results, it appears that the severity index may have great utility in predicting survival for AIDS patients seeking hospice admission.

Acquired Immunodeficiency Syndrome↗