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Biomedical subjects

D G McConnell

Publications and source records attributed to D G McConnell.

At least 19 recordsLinked to original sources
PubMed1999

Evidence that MRas1 and MRas3 proteins are associated with distinct cellular functions during growth and morphogenesis in the fungus Mucor racemosus.

The filamentous fungus Mucor racemosus provides a simple and unique model system for defining the function of individual ras genes in a gene family which is closely related to mammalian ras genes. The current study was designed to investigate the role of Mras1 and Mras3 in different stages of fungal morphogenesis, including sporangiospore germination, sporulation, and dimorphic transitions. The overall patterns of Mras1 and Mras3 transcript and protein accumulation were markedly different but, in general, transcripts and proteins were present at low levels during spherical growth and their accumulated level increased severalfold during polar growth (germ tube emergence and elongation). In contrast to Mras1, relatively high levels of Mras3 transcript accumulated during sporulation and MRas3 protein accumulated in sporangiospores. Transformation of M. racemosus with an activated allele of Mras3 reduced growth rate during aerobic sporangiospore germination, while a dominant-negative allele of Mras3 caused a 40% decrease in viable asexual spores. An activated allele of Mras1 increased growth rate during sporangiospore germination but neither activated nor dominant-negative alleles of Mras1 affected total number of asexual spores. Expression of MRas3 and MRas1 proteins appear to be subject to different regulatory mechanisms: exogenous dibutyryl-cAMP and fusidienol caused a strong repression of the level of MRas3 protein (but not MRas1) concurrent with the inhibition of polar growth. Differential posttranslational modification and intracellular localization of MRas1 and MRas3 proteins were also observed. The data strongly suggest that Mras3 and Mras1 play different roles in regulation of cell growth and morphogenesis in Mucor.

Alkyl and Aryl Transferases↗
PubMed1991

Activation of bovine rod outer segment phosphatidylinositol-4,5-bisphosphate phospholipase C by calmodulin antagonists does not depend on calmodulin.

Calmodulin antagonists stimulated phosphatidylinositol-4,5-bisphosphate phospholipase C in soluble and particulate fractions of bovine rod outer segments. Antagonists tested include trifluoperazine, melittin, calmidazolium, compound 48/80, W-13 [N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide], and W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. All were effective, but W-7 was chosen for further characterization of the effect, which was most pronounced in the soluble fraction. Phospholipase C activity in the soluble fraction did not increase linearly with the quality of enzyme assayed, suggesting the presence of an endogenous inhibitor or an inhibitory self-association of the enzyme. W-7 appeared to counteract this inhibition, resulting in a linear activity-quantity relationship. Stimulation by W-7 was therefore largest when large amounts of crude enzyme were assayed and small or nil when small amounts were assayed. The effect of W-7 was also dependent on [Ca2+], with half-maximal stimulation occurring between 0.1 and 1 microM. W-7 and W-13 were much more effective than their nonchlorinated analogues W-5 and W-12 at increasing phospholipase C activity. While this pattern of effectiveness is typical of calmodulin-mediated processes, the absence of any effect by added calmodulin and the retention of W-7 sensitivity by purified CaM-free enzyme argue against regulation by CaM. Octyl glucoside, a nonionic detergent, mimicked some of the effects of CaM antagonists, suggesting that the antagonists act by interfering with protein-protein interactions. It appears likely that CaM antagonists prevent an inhibitory multimerization or aggregation of at least one form of ROS phospholipase C.

Animals↗
PubMed1990

Molecular characterization of human and bovine rod photoreceptor cGMP phosphodiesterase alpha-subunit and chromosomal localization of the human gene.

Defects in proteins that function in photoreceptor signal transduction are prime suspects as causes of some human hereditary retinal degenerations. We have characterized cDNA clones encoding the alpha-subunit of human and bovine rod cell cGMP phosphodiesterase a key phototransduction enzyme. Clones from both species contain an open reading frame capable of coding for an approximately 100-kDa polypeptide of 859 amino acids, 94% of which are identical. Two or more transcripts were detected in both human and bovine retinal poly(A)+ RNA preparations, although the human transcripts ranging from 5.3 to 4.9 kb are significantly larger than the two bovine transcripts of 4.6 and 4.0 kb. The bovine and human genes appear to exist in single copy, with the bovine gene spanning more than 140 kb of genomic DNA. Somatic cell hybrids were used to map the human gene to the long arm of chromosome 5 (5q31.2----q34). Finally, the use of the candidate gene approach in the study of hereditary retinal dystrophies is discussed.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1984

Rapid diglyceride phosphorylation in isolated bovine rod outer segments.

When isolated bovine rod outer segment fragments were incubated with [gamma-32P]ATP, 32P, as revealed by autoradiography, was rapidly incorporated into rhodopsin bands on sodium dodecyl sulfate polyacrylamide gels, and into a low Mr lipid band. Incorporation of 32P into rhodopsin was light-dependent, but labeling of the lipid band was not. A single phosphorylated product, phosphatidic acid, was identified by 2-dimensional thin layer chromatography and by high pressure liquid chromatography of the corresponding glycerophosphate ester. Incorporation of label into phosphatidic acid was detected as early as 15 sec following start of incubation and the product was stable for at least 30 min. No other products were detected, indicating that under the experimental conditions phosphatidic acid was not metabolized to other phospholipids. Up to 1 mol phosphatidic acid was formed per 18 to 40 mol rhodopsin present.

Adenosine Triphosphate↗
PubMed1981

The light-activated GTP-dependent cyclic GMP phosphodiesterase complex of bovine retinal rod outer segments. Dark resolution of the catalytic and regulatory proteins.

A cyclic GMP phosphodiesterase associated with retinal rod outer segment (ROS) membranes is fully activated only in the presence of light and GTP. Activity can be easily depleted from the membranes by hypotonic washing, which in darkness removes two major soluble proteins. One of these has cGMP phosphodiesterase activity which is no longer activated by light or GTP. The other lacks phosphodiesterase activity but copurifies with the catalytic protein unless special measures are taken. In the present report, these 2 proteins removed from the ROS in darkness were resolved in a manner which was qualitatively and quantitatively dependent upon concentrations of 2-mercaptoethanol and EDTA. We designated the catalytic protein as P and the other protein G, because it has been reported to exhibit GTP-related activities. The unresolved P and G proteins behaved as a single complex on native gels, analytical ultracentrifugation, sucrose gradient sedimentation, and gel filtration. The P:G complex had the same Mr as purified P and more stable phosphodiesterase activity. Removal of G from P destabilized the catalytic activity and allowed aggregation of P. With loss of activity by purified P, multiple slow migrating protein bands appeared upon native gel electrophoresis. Destabilization of P could be partially prevented by addition of Mg2+ before physical separation of P from G. If 2-mercaptoethanol was removed simultaneously, dissociation of the P:G complex and destabilization of P were prevented. These findings imply that the G protein is essential to the catalytic stability of P when both are removed in darkness from the ROS membrane.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1977

Circular dichroism, optical rotatory dispersion, and absorption studies on the conformation of bovine rhodopsin iw situ and solubilized with detergent.

Circular dichroism, optical rotatory dispersion and absorption of rhodopsin, the visual pigment of bovine rod outer segment membranes, were studied in situ and in membranes solubilized with various detergents. The alpha-helical content of the membrane protein is approximately 30%. The membrane protein possesses little beta-structure. Solubilization of the membrane by the detergents, Emulphogene BC-720 and cetyltrimethylammonium salts, results in loss of protein helical structure and perturbation of aromatic residues. These effects are not observed on digitonin solubilization. In regard to the structural stability of the membrane during bleaching, the following conclusions were reached: (1) Delocalized conformational changes of rhodopsin in situ involving secondary and/or tertiary structure are very unlikely. (2) Localized conformational changes of rhodopsin in situ involving secondary structure must be limited to the involvement of no more than three amino acid residues and localized conformational changes involving tertiary structure must be limited to very short segments of the protein chain containing, at the most, only a few aromatic residues. (3) Large changes in the interaction of lipid and protein moieties of the membrane are unlikely. (4) The detergents, Emulphogene, cetyltrimethylammonium salts, and digitonin, significantly decrease the conformational stability of rhodopsin as compared to the in situ conditions. The effect is smaller with digitonin. Evidence is presented against a proposed mechanism by which optical activity of the prosthetic group, retinal, is induced by resonance coupling of the transition dipoles of retinal and the lowest energy transitions of the aromatic groups of the apoprotein, opsin. A mechanism in which atropisomers of retinal are preferentially bound by opsin is consistent with the present results. The optical activity of the prosthetic group is markedly changed upon solubilization of the membrane by detergent. This change in optical activity is probably coupled to changes in conformation of the protein moiety induced by solubilization.

Animals↗
PubMed1975

Cyclic nucleotide phosphodiesterases associated with bovine retinal outer-segment fragments.

ATP-dependent cyclic GMP phosphodiesterase activity (EC 3.1.4.16) associated with bovine retinal outer-segment fragment preparations was stimulated an order of magnitude by light, confirming the results of Miki et al. (1973) Proc. Natl. Acad. Sci. U.S. 70, 3820-3824 at Yale for the frog system. In contrast to the results of the Yale group, however, light stimulation was not observed for cyclic AMP as substrate. A direct relationship of bovine rhodopsin bleaching to phosphodiesterase activation differs from a previous report by the Yale group that full activation of the frog enzyme was achieved by bleaching of a maximum of 2% rhodopsin. Phosphodiesterase activity could be qualitatively removed from the fresh outer-segment preparations with isotonic sucrose which apparently did not disrupt the plasmalemma or discs. Activity recovered from the washing was not light sensitive. Two Km values were determined for cyclic AMP, 5 and 0.05 mM; for cyclic GMP a Km of 0.22 mM was found. All Km values were determined in the presence of 1 mM ATP in the dark. Sonication of fresh outer segments or storing at -20 degrees C abolished the light response. However, storage at -76 degrees C fully preserved it.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1975

Relationship of the light-induced proton uptake in bovine retinal outer segment fragments to triton-induced membrane disruption and to volume changes.

Light-induced proton uptake in bovine retinal outer segment (ROS) fragments was shown to be closely related to pH, salt concentration, membrane integrity, and perhaps secondarily to the volume of osmotic compartments. The principal findings were as follows: 1. As pH increased, both the discs and the plasmalemma swelled, and proton uptake markedly diminished. 2. As the discs were disrupted by increasing concentrations of Triton, proton uptake at slightly alkaline pH was supplanted by proton release. 3. Increasing the concentration of chloride salts caused increased H+ uptake roughly proportional to osmotic shrinkage of the ROS. Buffering by acetate prevented the measurement of proton uptake in the presence of acetate salts, although osmotic behavior of the ROS was similar to that observed in chloride salts. Although increasing the concentration of sucrose also resulted in osmotic shrinkage of the ROS, it was not accompanied by a systematic increase in the magnitude of proton uptake. 4. Light-induced H+ uptake was accompanied by small but reproducible changes in volume, probably of the discs. The magnitude and direction of these rapid volume changes were subject to influence by pH, solute, and other variables.

Animals↗