Biomedical subjects

D Guerini

Publications and source records attributed to D Guerini.

44 records · Page 3Linked to original sources

PubMed1989

Isolation and sequence of a cDNA clone for human calcineurin B, the Ca2+-binding subunit of the Ca2+/calmodulin-stimulated protein phosphatase.

We have identified and cloned human cDNA for the Ca2+-binding subunit of calcineurin, the brain isozyme of the Ca2+/calmodulin-stimulated protein phosphatase. The 2.5-kb cDNA has an open reading frame of 510 bp, a leader sequence of at least 500 bp, and a 1,277-bp 3'-noncoding sequence. The deduced sequence of the human protein differs from bovine brain calcineurin B by an additional valine at the carboxyl terminus and substitution of Met-11 and Ser-153 by cysteine. A partial clone of the mouse protein corresponding to amino acids 75-150 was also isolated. This portion of the human and mouse protein sequence is identical, with the DNA sequences showing 94% identity. The respective mRNAs in human and mouse are also of similar size. As was observed with protein levels, mRNA abundance in brain is 20-60 times that found in other tissues with the exception of HeLa cells which, like brain, contain abundant calcineurin B mRNA.

Amino Acid Sequence↗

PubMed1987

Stimulation of the erythrocyte Ca2+-ATPase and of bovine brain cyclic nucleotide phosphodiesterase by chemically modified calmodulin.

Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+-ATPase whereas the stimulation of the bovine brain cyclic-nucleotide phosphodiesterase was reduced up to 50%. Different species of carbamoylated calmodulin have been isolated but no differences were detected in their interaction with the cyclic-nucleotide phosphodiesterase. Modification of arginine residues by 1,2-cyclohexanedione had no effect of the stimulation of the phosphodiesterase but reduced by 40% the stimulation of the erythrocyte Ca2+ ATPase. Mild oxidation of methionines by N-chlorosuccinimide produced a number of differently modified calmodulins. The different species have been purified and the modified residues have been identified. They affected the two different test enzymes to different extents indicating that methionines in the central helix of calmodulin are of greater importance for the interaction with the phosphodiesterase, whereas methionines located in the C-terminal half of calmodulin are more important for the interaction with the Ca2+-ATPase.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1987

Effect of tryptic calmodulin fragments on guanylate cyclase activity from Paramecium tetraurelia.

Tryptic bovine brain calmodulin fragments 1-77 or 1-106 reactivated La-inactivated ciliary guanylate cyclase from Paramecium dose-dependently up to 60%. They were 20-fold less potent compared to bovine brain calmodulin. Fragment 78-148 was even less active. Concomitant addition of fragments 1-77 and 78-148 had no additive effect. Genetically engineered calmodulin lacking a blocked amino terminus and trimethyllysine at position 115 reactivated La-treated guanylate cyclase as good as bovine brain calmodulin. After detergent solubilization of La-inactivated guanylate cyclase intact bovine brain calmodulin and calmodulin fragments 1-77 and 78-148 were equipotent. 80% Reactivation was obtained with 40 microM of either fragment.

Animals↗

PubMed1987

Characterization of heart cytosolic proteins capable of modulating calcium uptake by the sarcoplasmic reticulum. 1. Isolation of a protein with protective activity and its identification as muscle albumin.

A new type of regulation of the Ca-pumping activity of isolated sarcoplasmic reticulum membranes has been investigated. An inhibitory and an antagonistic fraction were obtained after (NH4)2SO4 fractionation of cardiac muscle cytosol according to a published procedure [Narayanan et al. (1983) Biochem. Biophys. Acta 735, 53-66]. The former fraction inhibited Ca uptake by sarcoplasmic reticulum vesicles in a concentration-dependent way. The inhibition could be prevented and even reversed by addition of the antagonistic fraction. The protein components of this latter fraction were resolved and separated using an anion-exchange chromatographic procedure (mono Q column in an FPLC system). A pure protein component with antagonistic properties was isolated. Biochemical (molecular mass, tryptic digestion pattern and antagonistic activity) and immunological (cross-reactivity with specific antibodies) analysis resulted in the identification of the purified antagonist protein as muscle albumin, a serum-albumin-like protein which is localized near the A/I junctions in the striated muscle cells. The protein may be involved in the regulation of Ca fluxes across the cisternal compartments of the sarcoplasmic reticulum.

Animals↗

PubMed1985

Separation of various calmodulins, calmodulin tryptic fragments, and different homologous Ca2+-binding proteins by reversed-phase, hydrophobic interaction, and ion-exchange high-performance liquid chromatography techniques.

Reversed-phase, hydrophobic interaction, and ion-exchange high-performance liquid chromatography techniques have been used to separate different Ca2+-binding proteins and their proteolytic fragments. An alkali-stable ion-exchange column permitted the baseline separation of calmodulin fragments which differed only by one to three charged amino acids. The new hydrophobic interaction chromatography system displayed a high-resolution power separating calmodulins from different sources and calmodulin fragments obtained by trypsin proteolysis. The properties and advantages of the different systems are discussed in detail.

Animals↗

PubMed1984

Stimulation of the purified erythrocyte Ca2+-ATPase by tryptic fragments of calmodulin.

Highly purified tryptic peptides of calmodulin have been obtained by high-performance liquid chromatography. Tryptic cleavage of calmodulin in the presence of Ca2+ results in two main fragments which have been identified by analysis of the amino acid composition as 1-77 and 78-148. In the absence of Ca2+, trypsin cleavage yields fragments 1-106, 1-90, and 107-148. Only fragments 78-148 and 1-106 are still able to stimulate the purified Ca2+-ATPase of erythrocytes, albeit much less efficiently on a molar basis, than intact calmodulin. On the other hand, the same fragments were unable to stimulate the calmodulin-dependent cyclic nucleotide phosphodiesterase, even at 1000-fold molar excess (shown also by Newton, D.L., Oldewurtel, M.D., Krinks, M.H., Shiloach, J., and Klee, C.B. (1984) J. Biol. Chem. 259, 4419-4426). This points to the importance of the carboxyl-terminal half of calmodulin and especially of Ca2+-binding region III in the interaction of calmodulin with the Ca2+-ATPase and provides clear evidence that calmodulin interacts differently with different targets. Oxidation of methionine(s) of fragment 78-148 with N-chlorosuccinimide removes the ability of this fragment to stimulate the ATPase.

Amino Acids↗

PubMed1984

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a hydrophobic, photoreactive probe, labels calmodulin and calmodulin fragments in a Ca2+-dependent way.

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID), a highly hydrophobic, carbene-generating photoreactive probe, labels calmodulin and some of its proteolytic fragments in the Ca2+-bound conformation only. It is assumed that [125I]TID labels hydrophobic sites exposed by the binding of Ca2+. The finding offers a new and powerful means to characterize calmodulin sites that play a role in the interaction with targets.

Amino Acids↗

PubMed1983

Influence of temperature and denaturing agents on the structural stability of calmodulin. A 1H-nuclear magnetic resonance study.

The structural stability of calmodulin was studied by 1H-nuclear magnetic resonance spectroscopy under different denaturing conditions. The presence of Ca2+ stabilizes the structural properties of the native protein. In the absence of calcium the structural integrity of calmodulin can easily be affected by elevated temperatures or by high concentrations of denaturing agents. The unfolding process under various denaturing conditions is reversible underlining the high degree of structural flexibility of this protein.

Animals↗