A E Levin | PubMed Health

PubMed1994

"Checkerboard" DNA-DNA hybridization.

A method is introduced for hybridizing large numbers of DNA samples against large numbers of DNA probes on a single support membrane. Denatured DNA from up to 43 samples was fixed in separate lanes on a single membrane mounted in a Miniblotter 45. The membrane was then rotated 90 degrees in the same device, which enabled simultaneous hybridization with 43 different DNA probes. Hybridizations were also performed on lysates of bacterial cells blotted to membranes. A MiniSlot device allowed lysates loaded in parallel channels to be aspirated through the membrane, depositing horizontal lanes on the membrane surface. Hybridizations were performed in vertical lanes with either digoxigenin-labeled whole genomic probes or 16S rRNA-based oligonucleotide probes directly conjugated to alkaline phosphatase. The method permits the simultaneous determination of the presence of multiple bacterial species in single or multiple dental plaque samples, thus suggesting its usefulness for a range of clinical or environmental samples.

Base Sequence↗

PubMed1989

Purification and characterization of a calcium-dependent ATPase from Paramecium tetraurelia.

We report the purification of a CaATPase of high specific activity from Paramecium tetraurelia. The enzyme is preferentially released into solution upon deciliation of cells by a Ca2+ shock procedure. Purification by ion exchange and gel filtration chromatography yields major peptides of 68 and 53 kDa and a minor peptide of 58 kDa, as determined by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. These three peptides yield similar proteolytic peptide maps. Rabbit antisera to the purified enzyme inhibit enzyme activity and specifically label 68- and 53-kDa bands on nitrocellulose blots of the deciliation supernatant from which the enzyme is isolated. Concanavalin A-Sepharose precipitates about 60% of ATPase activity; only the 53-kDa band binds concanavalin A on nitrocellulose blots. The purified enzyme has a specific activity of 620 +/- 70 mumol/min/mg with ATP as substrate in the presence of Ca2+, which is required for enzyme activity. As substrates, ATP and GTP are strongly preferred to UTP and CTP. The Km for ATP in the presence of 3 mM Ca2+ is approximately 20 microM. Enzyme activity is strongly inhibited by the calmodulin antagonists trifluoperazine, fluphenazine, W7, and calmidazolium. However, calmodulin is not associated with the purified enzyme, based on the enzyme's inability to bind anti-calmodulin antibodies or to stimulate brain phosphodiesterase. The intracellular origin of this ATPase, its possible function, and its relationship to several other ATPases of Paramecium are discussed.

Adenosine Triphosphate↗

PubMed1987

Assembled clathrin in erythrocytes.

Clathrin cages were isolated from rat erythrocytes. These structures exist in the intact cell as demonstrated by immunofluorescence and were not formed during the isolation procedure. The cages were largely devoid of membrane but contained the assembly protein complex and both the 50-kDa kinase (pp50) and casein kinase II activities found previously in clathrin-coated vesicles.

Animals↗