Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Phoborhodopsin (also called sensory rhodopsin II) is a photoreceptor protein which mediates photophobic responses of Halobacterium halobium to blue-green light. Under conditions where the synthesis of the chromophore retinal is inhibited, the photophobic system is reconstituted in vivo by incorporation of all-trans retinal or retinal analogs into the apoprotein of phoborhodopsin. Retinal analogs which retard the cyclic photoreaction kinetics of phoborhodopsin increase significantly the sensitivity of the photophobic response. This supports the previously reported hypothesis that signal amplification occurs during the lifetime of intermediate states of the photocycle. The sensitivity increase caused by the chromophore substitution is observed in cells at several different growth stages, i.e. the naturally occurring chromophore (all-trans retinal) does not produce maximal sensitivity at any stage of the culture growth. These results are difficult to interpret in terms of the proposal by Marwan et al. (J. Mol. Biol. 199, 663-664, 1988) that only a single photon is sufficient to cause the photobehavioral response in cells containing native phoborhodopsin. A new interpretation for the fluence-response curves is described based in part on their Poisson statistical analysis. Further, a kinetic model which relates the receptor photochemical reaction cycle to the behavioral response is developed, which accounts for both the sensitivity increase and the shape of the fluence-response curves.
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PMID:Sensitivity increase in the photophobic response of Halobacterium halobium reconstituted with retinal analogs: a novel interpretation for the fluence-response relationship and a kinetic modeling. 149 28

Two and three-dimensional solution nuclear magnetic resonance studies of the 11K apoprotein from natural antitumor agent neocarzinostatin (NCS) were extended to elucidation of the high-resolution structure by the use of restrained molecular dynamics computations. The refined structures attained convergency upon three steps of iterative calculations, in which more distance restraints were extracted from experimental data, and the existing distance bounds were optimized on the basis of computed structures. The solution structures of apo-NCS contain seven antiparallel beta-strands, which form two closely located beta-sheets and a short beta-segment. This protein lacks any alpha-helical component. The alignment of the seven beta-strands gives rise to a beta-barrel with an elongated diameter in one direction. The global structure of apo-NCS resembles that of the Ig-fold domain found in immunoglobulins and other structurally related beta-proteins. Residues responsible for side-chain packing and the possible salt-bridge formation important for protein folding were identified. Neocarzinostatin and the analogous proteins are known to exert their biological activity through the interaction of DNA with a chromophoric molecule, which is non-covalently bound to the apo-proteins. This molecular chromophore-binding site in apo-NCS is made of a cavity consisting of residues from the four-beta-stranded sheet and the short beta-segment. Although the solution structures of apo-NCS are similar to that of the analogous apoauromomycin in the crystalline state, difference in the shape of the binding cavities between the two was found. This study provides a structural basis for characterization of the specific recognition and molecular mechanism of the antitumor NCS chromophore binding to its host protein.
J Mol Biol 1992 May 05
PMID:Three-dimensional solution structure of apo-neocarzinostatin. 153 78

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.
Plant Mol Biol 1992 Feb
PMID:Isolation and characterization of a cDNA-clone coding for potato type A phytochrome. 153 28

Pulmonary surfactant is critical for gas exchange and is composed of both phospholipids and specific surfactant-associated proteins. The most abundant surfactant protein is termed surfactant apoprotein A (SP-A). This protein is thought to be important in the formation of tubular myelin, in absorption of surfactant to the air-liquid interface, in recycling of surfactant in alveolar type II cells, and in the regulation of secretion. We have examined the expression and localization of SP-A mRNA in streptozotocin-induced diabetic rats by in situ hybridization using a specific rat cDNA probe. Diabetes was induced by intraperitoneal injection of 60 mg/kg streptozotocin. After 10 wk, lungs were excised and examined by in situ hybridization and by light and electron microscopy. The ultrastructural examination demonstrated the marked changes of endoplasmic reticulum of alveolar type II cells, as reported previously. Immunohistostaining of SP-A in diabetic lungs was weak in alveolar type II cells. However, by autoradiographs of in situ hybridization, compared with the control lungs, a larger number of silver grains for the SP-A mRNA were shown in alveolar type II cells and also in some bronchiolar epithelial (Clara) cells from the diabetic lungs. Alveolar type II cells having high contents of silver grains were also increased in number. These results were confirmed by measurement of the SP-A content and by Northern blot analysis. The present study demonstrates an overexpression of SP-A mRNA despite the ultrastructural changes in the endoplasmic reticulum of alveolar type II cells in the diabetic lungs, which will provide new information on the regulatory mechanism of SP-A gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1992 Mar
PMID:Overexpression of pulmonary surfactant apoprotein A mRNA in alveolar type II cells and nonciliated bronchiolar (Clara) epithelial cells in streptozotocin-induced diabetic rats demonstrated by in situ hybridization. 154 Mar 94

Pulmonary surfactant isolated from bronchoalveolar lavage fluid of rat lung contained a high content of surfactant protein A (SP-A) in starved for 2 days compared to fed controls, but this phenomena returned to baseline following more than 4 days starvation. As determined by immunoperoxidase staining of lung sections using SP-A antibody, SP-A could be consistently observed in nonciliated bronchiolar (Clara) cells, alveolar type II cells and some alveolar macrophages (AM). Fc receptor-mediated phagocytosis of AM was enhanced by SP-A, which was dependent on the dosis and reached a maximum at 10 micrograms of SP-A/ml. Antibody to SP-A completely inhibited the enhanced response of phagocytosis. When exposed AM subpopulations, separated into four fractions (I, II, III and IV) by discontinuous Percoll gradient, to SP-A or pulmonary surfactant prepared from rats fed and starved for 2 days enhanced their phagocytic activity in high dense cells (III and IV), particularly to SP-A and pulmonary surfactant from rats starved for 2 days. Whereas little change in lower dense fractions (I and II) were seen in all exposures except for SP-A that enhanced the cells of fraction II. These results supported the concept that pulmonary surfactant and its apoprotein, SP-A, are a factor to regulate lung defense system including activation of AM that undergo different processes following starvation.
Cell Mol Biol 1992 Apr
PMID:Effects of pulmonary surfactant and surfactant protein A on phagocytosis of fractionated alveolar macrophages: relationship to starvation. 157 41

Amastigotes of Leishmania major were isolated from infected mice and radiolabeled for 2 h with [3H]galactose. An acidic [3H]glycoconjugate was extracted from a dilipidated residue fraction with the solvent water/ethanol/diethylether/pyridine/NH4OH (15:15:5:1:0.017). The radioactivity labeled glycoconjugate was found to possess the following characteristics that were similar to the lipophosphoglycan extractable from promastigotes: (i) migrated as a broad band upon electrophoresis on SDS polyacrylamide gels; (ii) deaminated with nitrous acid; and (iii) hydrolyzed with phosphatidylinositol-specific phospholipase C. Furthermore, analysis of the aqueous soluble material released by the latter enzyme revealed a negatively-charged [3H]polysaccharide intermediate in size compared to the analogous portions of LPG isolated from non-infective and metacyclic promastigotes. Most importantly, the [3H]polysaccharide was found to contain phosphate and was susceptible to mild acid hydrolysis, establishing that the intact molecule is a lipophosphoglycan. A structural difference, however, was found in the major, mild acid-generated fragment of the amastigote phosphoglycan, which was larger in size and not as anionic as the analogous fragment from the promastigote phosphoglycans. These results indicate that the amastigotes do express a lipophosphoglycan, but that it is structurally distinct from its promastigote counterparts.
Mol Biochem Parasitol 1991 Mar
PMID:Expression of a stage-specific lipophosphoglycan in Leishmania major amastigotes. 164 60

Specific receptor and internalization process for low density lipoprotein (LDL) and modified LDL (acetyl-LDL) have been well characterized in placental microvilli and in trophoblastic cells in culture. The aim of this study was to investigate high density lipoprotein (HDL3) binding and its eventual subsequent internalization in both these purified placental preparations. Isolated term placental microvilli were used for binding of [125I]HDL3 (devoid of apoprotein E). HDL3 were conjugated to colloidal gold for ultrastructural visualization of binding and internalization in syncytiotrophoblast in culture. Saturable binding of HDL3 was identified. Scatchard analysis revealed a Kd value of 24.2 +/- 8.0 micrograms HDL3 protein/ml and a maximum binding capacity at 4 degrees C of 128.2 +/- 54.5 micrograms HDL3 protein/mg of membrane protein. These sites have broad specificity: both LDL and acetyl-LDL were able to partially inhibit the HDL3 binding. Ultrastructural study confirms that gold-HDL3 bind specifically to syncytiotrophoblast membrane. However, after incubation at 37 degrees C, an internalization process similar to those described for gold-LDL and gold-acetyl-LDL was not observed for gold-HDL3. These results demonstrate specific HDL3 binding without internalization. The physiological significance of an HDL3 membranous interaction and the placental steroidogenesis remains to be established.
Mol Cell Endocrinol 1991 May
PMID:High density lipoprotein interaction with human placenta: biochemical and ultrastructural characterization of binding to microvillous receptor and lack of internalization. 166 66

The human choriocarcinoma cell line, BeWo, synthesizes the glycoprotein hormone, human chorionic gonadotropin (hCG). We have undertaken this study to compare the synthesized and secreted forms of hCG and their alpha- and beta-subunits in cell cultures of BeWo cells to those forms of normal placental cells by immunobinding techniques. BeWo cells appeared to synthesize and secrete one species of the respective hCG subunit. The immature alpha- and beta-subunits, synthesized in BeWo cells as well as those of placental cells, were digested by endoglycosidase H indicating N-linked sugar chain(s) to be the high-mannose type. The mature alpha- and beta-subunits, secreted by BeWo cells as well as subunits of urinary hCG which are usually used as a standard hCG secreted by normal placental cells, were sensitive to neuraminidase treatment indicating that these subunits have terminal sialic acid(s). Contrary to placental cells, mature subunits of BeWo hCG could not be found in any subcellular fraction indicating a rapid secretion rate or supporting the hypothesis that BeWo cells secrete hCG subunits without the formation of secretory granules. The alpha-subunit synthesized in BeWo cells had a slightly lower molecular weight than that of placental cells; however, the alpha-subunit secreted by BeWo cells had a slightly higher molecular weight than the alpha-subunit of urinary hCG. The beta-subunits synthesized and secreted by BeWo cells had slightly higher molecular weights than beta-subunits of both placental cells and urinary hCG. Even after digestion by N-glycanase as well as endoglycosidase H, molecular weights were still different between the respective subunits of BeWo and placental cells indicating that the apoprotein structures of BeWo hCG subunits may differ from those of placental cells. Moreover, urinary beta-subunit was sensitive to endo-alpha-N-acetylgalactosaminidase treatment but the beta-subunit secreted by BeWo cells was not, indicating that the structure of O-linked sugar chain(s), if present, may be unusual. Analysis of assembled and free forms of subunits of BeWo cell cultures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by immunobinding methods revealed that subunits are associated intracellularly and then secreted to the media as hCG. Moreover, only free beta-subunits, but not alpha-subunits, of BeWo hCG were found intra- and extracellularly.
Mol Cell Endocrinol 1990 Mar 05
PMID:Synthesis and secretion of human chorionic gonadotropin and its subunits in choriocarcinoma cells: a comparative study with normal placental cells. 169 20

A cDNA library constructed using mRNAs isolated from pituitary glands of estradiol-treated eels was screened with a cDNA fragment for the rat glycoprotein hormone alpha-subunit. Three out of 10,000 cDNA clones were revealed and subcloned in pUC13 for characterization and sequencing. All three had the same nucleotide sequence except for a single, silent change in the coding sequence for one of them, and for the location of the poly(A) tail. Analysis of the deduced amino acid sequence strongly suggests that these cDNA clones encode the precursor for the eel common glycoprotein hormone alpha-subunit. This precursor would therefore consist of a 93 amino acid apoprotein preceded by a 24 amino acid long signal peptide. Alignment with glycoprotein hormone alpha-subunits from fish and mammals reveals high homology, ranging from 60 to 90%. Particularly, the ten cysteines and the two putative N-linked glycosylation sites were at the same position. Comparison between fish and mammals shows also that two regions are highly conserved, comprising about half of the protein length. This high conservation rate through evolution argues for the importance of these regions in the conservation of biological properties of the alpha-subunits. In contrast, other regions are highly variable and could be responsible for the immunological specificity. Northern blot analysis of pituitary RNA from control and estradiol-treated eels showed that estradiol treatment strongly increases the pituitary content of mRNA encoding the glycoprotein hormone alpha-subunit.
Mol Cell Endocrinol 1990 Jul 09
PMID:Cloning and sequence analysis of the cDNA for the pituitary glycoprotein hormone alpha-subunit of the European eel. 169 69

Apolipoprotein (apo) E, a major protein component of plasma lipoproteins, is a physiological ligand for the low density lipoprotein (LDL) receptor as well as for a specific apoE receptor; it is therefore an important modulator of lipoprotein metabolism. In this study we cloned and sequenced bovine apoE complementary DNA. Comparison of nucleotide substitution rates shows that apoE is less conservative than apoA-I and evolves about 30% faster than an average mammalian protein. Although apoE is not a conservative protein, several regions have been well conserved among all eight mammalian sequences now available. These include a 33-amino-acid block immediately upsteam from the third intron/exon junction and the LDL receptor binding region. We have also compared published apoC-I and apoC-II sequences. Both proteins are less conservative than apoE. In particular, apoC-I shows no well-conserved region except for a small region in the common 33-amino-acid block, suggesting that the function of apoC-I does not have stringent structural requirements. On the other hand, in apoC-II the region encoded by exon 4, which consists of the last 29 amino acids of the polypeptide, has been rather well conserved, probably because this region is important for the activation of lipoprotein lipase and chylomicron and very low density lipoprotein metabolism.
J Mol Evol 1991 Jun
PMID:Cloning and sequencing of bovine apolipoprotein E complementary DNA and molecular evolution of apolipoproteins E, C-I, and C-II. 190 18


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