Biomedical subjects

D A Amorese

Publications and source records attributed to D A Amorese.

4 recordsLinked to original sources

PubMed1990

Cloning and characterization of molecular isoforms of the catalytic subunit of calcineurin using nonisotopic methods.

The cloning and characterization of cDNAs for the catalytic subunit of calcineurin (CN) from murine and human brain libraries were carried out using nonisotopic methods. A murine cDNA clone encoding a protein of 521 amino acids (Mr approximately 58,650) was isolated; overlapping clones established a 3'-untranslated region of 554 base pairs preceding the poly(A) tail. Homologous cDNAs from human brain showed greater than 92% nucleotide sequence identity in both coding and non-coding regions with greater than 99% conservation of amino acid sequence. A second class of cDNAs lacking a specific 30-base pair region following the calmodulin-binding domain was found in four murine and human libraries. Oligonucleotide probes for both cDNA isoforms hybridized to mRNA from several brain regions indicating the existence of transcripts in vivo. The nucleotide sequences of the two forms were identical except for the inserted sequence, and Southern blot analysis of mouse and rat DNA was consistent with their having originated from the same gene; these data suggest that alternative splicing may give rise to molecular isoforms of the catalytic subunit in brain. Northern blots showed a predominant mRNA for CN in most tissues of approximately 4.0 kilobases (kb) with lower amounts of a 3.6-kb species. Brain showed 10 times more of these mRNAs than skeletal muscle while other tissues had less than or equal to 5% that in brain. In testis, multiple mRNAs were observed, with the major forms being approximately 2.8 and 1.6 kb; the total amount of CN message was about 15% that in brain. The presence of mRNA isoforms of the catalytic subunit may provide for isoenzymes of this phosphatase having distinct phosphoprotein substrate specificities or regulatory properties. The structural relatedness of CN to other mammalian serine/threonine protein phosphatases was highest over a region of approximately 240 amino acids near the amino terminus of this subunit, with greater similarity to protein phosphatase 2A than protein phosphatase 1. The conservation of many regions found in lambda phage phosphatase (Cohen, P.T.W., and Cohen, P. (1989) Biochem. J. 260, 931-934) indicates a common origin for the catalytic domain of this enzyme.

Amino Acid Sequence↗

PubMed1988

Properties of purified actin depolymerizing factor from chick brain.

Actin depolymerizing factor (ADF) from 19-day embryonic chick brains has been purified to greater than 98% homogeneity with a yield of 7.2 mg/100 g of brain. Quantitative immunoblotting with a monospecific antibody to ADF indicated that ADF comprises 0.3% of the total brain protein, resulting in an actual purification yield of about 20%. Brain ADF migrates as a single polypeptide of 19,000 kDa on SDS-containing polyacrylamide gels. The molecular weight of the native protein determined from sedimentation equilibrium in buffers containing from 50 to 200 mM KCl is 20,000. The secondary structure of ADF calculated from the circular dichroic spectrum consists of about 22% alpha-helix, 24% beta-sheet, and 18% beta-turn. ADF contains a blocked N-terminus, a single tryptophan residue located about one-third of the way from one end of the protein, and six cysteine residues (all in reduced form in the native protein). All six cysteine residues could be chemically modified with eosinylmaleimide under nondenaturing conditions; however, ADF activity was lost when more than one cysteine residue was modified. ADF microheterogeneity has been observed upon nonequilibrium pH gradient electrophoresis in polyacrylamide gels containing 9 M urea, the major isoform having a pI of congruent to 7.9-8.0. ADF can interact with either monomeric or filamentous actin to give a complex which can be isolated by gel filtration chromatography. Both major and minor isoforms of the ADF are found in the complex. Assembly-competent actin and active ADF can both be recovered from the complex by chromatography on ATP-saturated DEAE-cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Depolymerizing Factors↗

PubMed1982

In vitro labeling of proteins by reductive methylation: application to proteins involved in supramolecular structures.

Actin and tropomyosin, purified from both muscle and brain, and alpha-actinin, purified from muscle, have been labeled in vitro by reductive methylation to specific activities of greater than 10(5) dpm/micrograms protein. Actin so modified bound DNase I and polymerized identically to unmodified actin. Furthermore, the spectral properties of actin did not change after labeling. The interactions of labeled tropomyosin and alpha-actinin with F-actin were nearly identical to those of the unmodified proteins. These modified proteins comigrated with their unmodified counterparts in both SDS-containing polyacrylamide gels and isoelectric focusing gels. The labeled actin was quantitatively extracted from SDS-containing polyacrylamide gels (yield greater than 98% of radioactivity applied demonstrating that all of the radioactivity was protein bound. The reductive methylation procedure worked well at pH 8.0-8.5 in either pyrophosphate buffer or Bicine buffer using formaldehyde with [3H]-sodium borohydride as the reducing agent. The procedure could also be performed at pH 7.0 in phosphate buffer using (14C]-formaldehyde with sodium cyanoborohydride as the reducing agent. Proteins so labeled are ideal for use in quantitative experiments involving protein-protein interactions.

Actinin↗

PubMed1982

Extracts from the brains of hibernating and alert ground squirrels: effects on cells in culture.

Aqueous extracts were prepared from pulverized, acetone-dehydrated brains of hibernating and alert ground squirrels. Addition of these extracts to Chinese hamster ovary cells in suspension culture resulted in a decrease in the amount of [3H]thymidine incorporated into acid-precipitable material without affecting the transport or phosphorylation of the nucleoside. The inhibition was time- and dose-dependent and full recovery occurred about 2 hr after exposure of the cells to the active extract. The active factor is readily oxidized during storage at -70 degrees C but full activity can be restored by treatment with 2-mercaptoethanol. The peptide nature of the active material is indicated by its susceptibility to proteases and by loss of activity after alkylation. Fasted or cold-acclimated rats also develop increased levels of active substance in their brains; however, brains of hibernating squirrels contain 10- to 50-fold more of the active substance than brains from either alert squirrels or rats. A time-dependent increase in activity of extracts from hibernating brain incubated with a homogenate from alert brain suggests that the peptide is activated or generated in the mixture.

Animals↗