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R Amthauer

Publications and source records attributed to R Amthauer.

18 recordsLinked to original sources
PubMed1992

Phosphatidylinositol-glycan (PI-G)-anchored membrane proteins: requirement of ATP and GTP for translation-independent COOH-terminal processing.

Placental alkaline phosphatase (PLAP) belongs to a class of proteins that are anchored to the plasma membrane by a COOH-terminal phosphatidylinositol-glycan (PI-G) moiety. Nascent forms of such proteins undergo NH2- and COOH-terminal processing to yield the mature PI-G-tailed proteins. We previously introduced a shortened engineered form of preproPLAP (preprominiPLAP) that permits monitoring in cell-free preparations its sequential processing to the pro form and then to the mature PI-G-tailed form. Previous studies were carried out by synthesizing the preproprotein cotranslationally in the presence of rough microsomal membranes (RM). Because of the complexity of the cotranslational system it was not possible to determine whether cofactors were required for processing. We have now prepared RM that are preloaded with prominiPLAP but contain little mature PI-G-tailed miniPLAP. Maximal processing requires supplementation with both ATP and GTP. Inhibitors of PI-G biosynthesis do not affect processing. Since cleavage and PI-G addition are presumably catalyzed by a transamidase, the nucleoside triphosphate requirements suggest that there are additional steps in prominiPLAP processing prior to transamidation with PI-G. These may involve translocation of the pro protein in a proper conformational state to the transamidase site.

Adenosine Triphosphate↗
PubMed1992

Biosynthesis of phosphatidylinositol-glycan (PI-G)-anchored membrane proteins in cell-free systems: PI-G is an obligatory cosubstrate for COOH-terminal processing of nascent proteins.

It is generally recognized that nascent proteins destined to be processed to a phosphatidylinositol-glycan (PI-G)-anchored membrane form contain a hydrophobic signal peptide at both their NH2 and COOH termini. In previous studies we showed that rough microsomal membranes (RM) prepared from CHO cells can carry out COOH-terminal processing. We have now investigated RM prepared from many additional cell types, including frog oocytes, B cells, and T cells, and found that all are competent with respect to COOH-terminal processing. Exceptions were certain mutant T cells that had been shown to be defective at various steps of PI-G anchor biosynthesis [Sugiyama, E., De Gasperi, R., Urakaze, M., Chang, H.-M., Thomas, L. J., Hyman, R., Warren, C. D. & Yeh, E. T. H. (1991) J. Biol. Chem. 266, 12119-12122]. In one such defective mutant, COOH-terminal processing activity of RM could be restored either by transfecting the intact cells with the gene for the deficient step in PI-G synthesis or by adding PI-G extracts to the RM in vitro. Cleavage of the COOH-terminal signal peptide in the RM is therefore dependent on the presence of intact PI-G incorporated into the mature protein.

Acyltransferases↗
PubMed1992

Biosynthesis of phosphatidylinositol-glycan (PI-G)-anchored membrane proteins in cell-free systems: cleavage of the nascent protein and addition of the PI-G moiety depend on the size of the COOH-terminal signal peptide.

Nascent translation products of PI-G-anchored membrane proteins contain both NH2- and COOH-terminal signal sequences of approximately 15-30 residues that are removed during processing. Removal of the latter occurs concomitant with the addition of the PI-G moiety to the newly formed COOH terminus. In human placental alkaline phosphatase (PLAP) the COOH-terminal signal peptide contains 29 residues. An engineered form of PLAP, miniPLAP 208, containing the same NH2- and COOH-terminal signal peptides as PLAP, was used as a substrate for cell-free processing. A comparison was made with mutants (delta 202, delta 197, delta 184, and delta 179) truncated at the COOH terminus. Intact preprominiPLAP 208 and truncated delta 202 were processed to yield the same mature product which, by size and distribution between Triton X-114 and water before and after treatment with inositol-specific phospholipases, indicates that it contained the PI-G moiety. Mutants that were further truncated at the COOH terminus, miniPLAPs delta 197, delta 184, and delta 179, were processed only at their NH2 termini. Those portions of the COOH-terminal sequence in miniPLAPs delta 197 and delta 1984 that extended beyond residue 179 were not removed during processing.

Alkaline Phosphatase↗
PubMed1992

Placental alkaline phosphatase: a model for studying COOH-terminal processing of phosphatidylinositol-glycan-anchored membrane proteins.

Placental alkaline phosphatase (PLAP) has been used as a model for studying the biosynthesis of the phosphatidylinositol-glycan (PI-G)-protein linkage in intact cells and in cell-free systems. However, for the study of processing in cell-free systems, a small protein devoid of glycosylation sites is preferable. A PLAP-derived cDNA was engineered that codes for a nascent protein (mini-PLAP) of 28 kDa in which the NH2- and COOH-termini are retained but most of the interior of PLAP is deleted. In vitro translation of mini-PLAP mRNA in the presence of rough microsomal membranes yields mature PI-G-tailed mini-PLAP. Processing of nascent mutant proteins occurs only when a small amino acid is located at the site of cleavage and PI-G attachment (omega site). Mutations adjacent and COOH-terminal to the omega site have revealed that the omega + 1 site is promiscuous in its requirements but that only glycine and alanine are effective at the omega + 2 site. Rough microsomal membranes from T cells deficient in PI-G biosynthesis do not support processing of mini-PLAP; addition of exogenous PI-G restores activity. Translocation of the proprotein, most likely requiring ATP and GTP, precedes COOH-terminal processing.

Alkaline Phosphatase↗
PubMed1992

Effect of seasonal acclimatization on estrogen-induced vitellogenesis and on the hepatic estrogen receptors in the male carp.

The levels of circulating vitellogenin in the plasma of male carp after induction by estradiol-17 beta was examined in summer- and winter-acclimatized fish. During the warm season male fish exhibited a clear vitellogenic response, whereas the fish adapted to the cold season did not. The evaluation of the hepatic estrogen receptors revealed that although steroid-binding affinity is not affected by seasonal acclimatization, the concentration of estrogen receptors certainly is. This was evidenced in estrogen treated fish. Summer-acclimatized carp featured a concentration of hepatic estrogen receptors 2.5 fold higher than the cold season adapted fish. The results suggest that the failure of estrogen to induce vitellogenesis in cold adapted carp may be due; at least in part, to a deficient up-regulation of hepatic estrogen receptors in the corresponding acclimatized state.

Acclimatization↗
PubMed1992

Cell-free processing of nascent proteins destined to be linked to the plasma membrane by a phosphatidylinositol-glycan anchor.

Certain proteins are anchored to the outer plasma membrane by a phosphatidylinositol-glycan (PI-G) linker. Nascent forms of PI-G anchored proteins contain both NH2- and COOH-terminal signal peptides. The function and structural requirements of the COOH-terminal signal peptide as discussed and some studies on the cell-free processing of a nascent protein to its mature PI-G tailed form are presented.

Amino Acid Sequence↗
PubMed1988

Interaction of cibacron blue and anilinonaphthalenesulphonate with lipoproteins provides a new means for simple isolation of these plasma proteins.

The selective interaction of serum proteins with immobilized Cibacron Blue and the binding properties of the dye anilinonaphthalenesulphonate has been used to separate albumin and lipoproteins by affinity chromatography. The novel binding of anilinonaphthalenesulphonate to lipoproteins from the sera of lamprey, fish and mammals provides a simple procedure for the isolation of these plasma proteins, and permit preparation of specific antisera, tools particularly relevant for evolutionary and clinical studies.

Anilino Naphthalenesulfonates↗
PubMed1984

Fish liver protein synthesis during cold acclimatization: seasonal changes of the ultrastructure of the carp hepatocyte.

A cell-free system, active in protein synthesis, was constructed using ribosomes and supernatant factors isolated from the livers of winter-acclimatized carps. Upon seasonal adaptation, the hepatocytes of these fishes exhibited distinctive morphological features that were not sex dependent. The cytoarchitecture of the liver cell from summer carps is consistent with the cellular arrangements found when the rate of gene expression is high. During winter, several morphological features reveal that macromolecular synthetic activity may be clearly diminished.

Acclimatization↗
PubMed1984

Effects of insulin on the fine structure of hepatocytes from winter-acclimatized carps: studies on protein synthesis.

Male and female winter-acclimatized carps were injected with insulin. This treatment resulted in a sharp decrease in the liver glycogen content. Although an increase in the ribosomal RNA level was also observed, a cell-free system obtained from the hormone-treated fish exhibited less amino acid incorporation activity as compared to the control fish. However, polysomes from insulin-treated fish exhibited a higher amino acid incorporating activity when a soluble fraction of untreated winter carps was used. Insulin induced a profound change in the cytoarchitecture of the winter carp hepatocyte. The cytoplasm and nuclei showed all the features of the summer carp liver cell. The nucleolar components were totally intermingled suggesting a high rate of gene expression as in the case of the summer-acclimatized fish.

Acclimatization↗
PubMed1984

Cloning, physical mapping and genome organization of mitochondrial DNA from Cyprinus carpio oocytes.

The mitochondrial genome from Cyprinus carpio oocytes is a 10.5 megadalton, circular DNA molecule. The carp mitochondrial DNA was cloned in pBR325. Three recombinant plasmids accounted for the entire genome. Mapping of this DNA using 11 different restriction endonucleases is reported here. Both the large and small rRNA genes were then localized using Southern blot analysis. The subunit I of the cytochrome oxidase, the cytochrome b, the tRNAGlu and the URF 4 genes were localized by nucleotide sequence analysis and homology studies with human mtDNA. Our results suggest that a similar gene order has been maintained in the mitochondrial genomes of Chordata and support the hypothesis of a common ancestor for all vertebrate organelle genomes. This study constitutes the first report on the genome organization of a fish mtDNA and provides information for further investigation in connection with sequence determination, replication, and gene expression in carp mitochondria.

Animals↗
PubMed1984

Analys. DNA: a computer program for nucleic acid sequence data processing.

A computer program written in BASIC language is described. The program allows processing and analysis of DNA data and has been designed to be used by persons with little or no computer experience. The operator using different options can search for direct homologies with varying degrees of matching, generate complementary strands, find restriction sites, invert the polarity of the sequence and edit a print-out.

Animals↗
PubMed1982

Behavior of RNA and protein synthesis during the acclimatization of the carp. Studies with isolated hepatocytes.

1. Carp hepatocytes were isolated by dissociating the liver tissue with collagenase. The procedure yields viable cells with highly preserved ultrastructural and metabolic features. The isolated cells were able to self-aggregated and form tissue. 2. RNA and protein synthesis activity was significantly higher in the carp hepatocytes from summer acclimatized fish compared to the activity present in the cold adapted animals. 3. RNA synthesis assayed in carp hepatocytes suspensions obtained from summer and winter acclimatized fish exhibited a behaviour consistent with an inverse compensation to the cold acclimatization state, being apparently repressed, whereas protein synthesis did not show a compensatory activity.

Acclimatization↗