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M F Schmidt

Publications and source records attributed to M F Schmidt.

At least 55 records · Page 3Linked to original sources
PubMed1994

Self-recognition: a constraint on the formation of electrical coupling in neurons.

Electrical coupling between specific neurons is important for proper function of many neuronal circuits. Identified cultured neurons from the snail Helisoma show a strong correlation between electrical coupling and presence of gap junction plaques in freeze-fracture replicas. Gap junction plaques, however, were never seen between overlapping neurites from a single neuron, even though those same neurites formed gap junctions with neurites from another essentially identical identified neuron. This observation suggests that a form of self-recognition inhibits reflexive gap junction formation between sibling neurites. When one or both of those growth cones had been physically isolated from the neuronal cell body, both electrical coupling and gap junction plaques, between growth cones from the same neuron, were observed to form rapidly (within 30 min). Thus, inhibition of electrical coupling between sibling neurites apparently depends on cytoplasmic continuity between neurites, and not the molecular composition of neurite membrane. The formation of gap junctions is not likely due to the isolation process; rather, the physical isolation appears to release an inhibition of reflexive gap junction formation. These data demonstrate the existence of a previously unknown constraint on the formation of electrical synapses.

Animals↗
PubMed1993

Timing of palmitoylation of influenza virus hemagglutinin.

The timing of the attachment of fatty acids to the hemagglutinin (HA) of influenza A virus was studied. Treatment of virus infected cells with brefeldin A (BFA), a drug which blocks intracellular transport along the exocytic pathway at a pre-Golgi site, does not prevent palmitoylation of HA. The relationship of HA-palmitoylation to the oligomerisation and to the proteolytical cleavage of the protein revealed that the uncleaved trimer of HA is the substrate for the acylating enzyme in virus infected cells. The results are discussed with regard to the intracellular site of palmitoylation.

Animals↗
PubMed1993

Role of intracellular calcium in NI-35-evoked collapse of neuronal growth cones.

A myelin-associated protein from the central nervous system, the neurite growth inhibitor NI-35, inhibits regeneration of lesioned neuronal fiber tracts in vivo and growth of neurites in vitro. Growth cones of cultured rat dorsal root ganglion neurons arrested their growth and collapsed when exposed to liposomes containing NI-35. Before morphological changes, the concentration of free intracellular calcium ([Ca2+]i) showed a rapid and large increase in growth cones exposed to liposomes containing NI-35. Neither an increase in [Ca2+]i nor collapse of growth cones was detected in the presence of antibodies to NI-35. Dantrolene, an inhibitor of calcium release from caffeine-sensitive intracellular calcium stores, protected growth cones from collapse evoked by NI-35. Depletion of these caffeine-sensitive intracellular calcium stores prevented the increase in [Ca2+]i evoked by NI-35. The NI-35-evoked cascade of intracellular messengers that mediates collapse of growth cones includes the crucial step of calcium release from intracellular stores.

Animals↗
PubMed1993

Fibroblast growth factors, depolarization, and substratum interact in a combinatorial way to promote neuronal survival.

Neuronal survival in vivo may be determined by the combined effects of multiple agents rather than simply by the effect of an individual agent. This idea is supported by experimental evidence showing that neuronal survival can independently be influenced by target-derived factors as well as afferent inputs. To test this idea directly, cultured chick ciliary ganglion neurons were used to study the potentially interactive and combinatorial effects of trophic factors (acidic and basic fibroblast growth factor; FGF), depolarization (as would be expected from afferent activity), and substrate (laminin and collagen IV). Our results were consistent with the idea that combinatorial interactions between multiple agents may be critical in the regulation of cell survival. Exposure to either basic FGF (bFGF) or depolarization on a laminin substrate promoted neuron survival. However, bFGF did not promote survival and depolarization-mediated survival was significantly reduced when neurons were plated on collagen. The simultaneous addition of FGF and depolarization affected survival synergistically when plated on both laminin and collagen. Surprisingly, while survival by FGF or depolarization alone was greatly dependent on substrate, the simultaneous addition of FGF and depolarization appeared to greatly reduce this dependency on substrate. Taken together, these data demonstrate the potential importance of synergistic interactions between trophic factors and depolarizing stimuli.

Animals↗
PubMed1993

Structure, function, and intracellular localization of glycoprotein B of herpesvirus simian agent 8 expressed in insect and mammalian cells.

The cloned gene of glycoprotein B (gB) of herpesvirus simian agent 8 (SA 8) was expressed with a baculovirus system in insect cells. Expression of gB was easily detectable over the cellular background by Coomassie staining of electrophoretically separated proteins. Endoglycosidase digestion of immunoprecipitated gB revealed that the gene product is N-glycosylated, but only with unprocessed, endoglycosidase-H sensitive carbohydrates. The lack of terminal glycosylation of gB is consistent with the observation that gB expressed in insect cells has a molecular weight slightly lower than gB synthesized during an SA 8 infection in mammalian cells. The truncated carbohydrates of gB from insect cells have no measurable effect on the tertiary structure of gB. Immunofluorescence studies on mammalian cells expressing gB from a simian virus 40 based vector revealed that the glycoprotein is localized to cytoplasmic membranes, to the plasma membrane and to the nuclear envelope. Cells expressing gB were fused to polykaryons, which shows that gB has cell fusing activity in the absence of any other SA 8 gene product.

Animals↗
PubMed1991

Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin.

The hemagglutinin (HA) of influenza virus is a type I transmembrane glycoprotein which is acylated with long-chain fatty acids. In this study we have used oligonucleotide-directed mutagenesis of cloned cDNA and a simian virus 40 expression system to determine the fatty acid binding site in HA and to examine possible functions of covalently linked fatty acids. The results show that the HA is acylated through thioester linkages at three highly conserved cysteine residues located in the cytoplasmic domain and at the carboxy-terminal end of the transmembrane region, whereas a cysteine located in the middle of the membrane-spanning domain is not acylated. Mutants lacking fatty acids at individual or all three attachment sites acquire endoglycosidase H-resistant oligosaccharide side chains, are cleaved into HA1 and HA2 subunits, and are transported to the plasma membrane at rates similar to that of wild-type HA. All mutants are membrane bound and not secreted into the medium. These results exclude transport signal and membrane-anchoring functions of covalently linked fatty acids for this integral membrane glycoprotein. Furthermore, lack of acylation has no obvious influence on the biological activities of HA: cells expressing fatty acid-free HA bind to and, after brief exposure to mildly acidic pH, fuse with erythrocytes; the HA-induced polykaryon formation is not impaired, either. Other possible functions of covalently linked fatty acids in integral membrane glycoproteins which cannot be examined in conventional cDNA expression systems are discussed.

Acylation↗
PubMed1991

Sustained increase in intracellular calcium promotes neuronal survival.

Ciliary ganglion neurons, half of which normally suffer developmental death in the embryo, will survive in culture in medium supplemented with depolarizing concentrations of potassium. It is not known how elevated potassium acts inside the cell to promote survival. We report here that depolarizing concentrations of extracellular potassium promote neuronal survival by causing a sustained increase in intracellular calcium. Raising extracellular potassium from 5 to 40 mM, an optimal concentration for survival, caused a sustained increase in intracellular calcium from 250 nM to greater than 600 nM. By 26 hr, at which time greater than 90% of neurons in 5 mM potassium had died, the calcium concentration of neurons in 40 mM potassium was still above 400 nM. Reduction of extracellular potassium from 40 to 5 nM, which prevents the increase in survival, also reduced intracellular calcium back to rest levels. PN200-110, a dihydropyridine calcium channel blocker that inhibits the survival-promoting effect of elevated potassium, also prevented and reversed the potassium,-mediated increase in intracellular calcium. In addition, there was a strong, quantitative correlation between the percentage of neuronal survival and the intracellular calcium concentration over a wide range of extracellular potassium concentrations. These results suggest that elevated potassium opens dihydropyridine-sensitive calcium channels, causing a sustained increase in intracellular calcium that quantitatively determines the number of surviving neurons.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1991

On the enzymes which make "fatty proteins".

Twelve years have passed since the initial report on the modification of viral proteins with covalently linked fatty acids appeared (Schmidt et al., 1979). With gratitude the first author and his students look back on a number of fruitful and happy years in Professor Rott's department, during which we contributed to furthering insight into the fatty acylation of proteins. Following the initial discovery this area has expanded tremendously, and as reflected by a great number of review articles (e.g. Schmidt 1982a; Low 1987, Schulz et al., 1988; Towler et al., 1988; Grand 1989; Schmidt 1989; Schmidt and Schlesinger, 1991) research on "hydrophobic modifications of proteins" has occupied virologists, cell-biologists and biochemists alike. Rather than duplicating the circulating reviews at this stage we take this occasion to report new data on the enzymatic nature of fatty acylation. We believe that the knowledge of the acylating enzymes will provide the cornerstone for full understanding of this hydrophobic modification of proteins.

Acylation↗
PubMed1990

The hemagglutinating glycoproteins of influenza B and C viruses are acylated with different fatty acids.

We present evidence that the hemagglutinin (HA) of influenza B virus and the glycoprotein of influenza C virus (HEF) are acylated. The fatty acid linkage is sensitive to treatment with hydroxylamine and mercaptoethanol, which points to a labile thioester-type linkage. The HA of influenza B virus contains mainly palmitic acid, whereas the HEF glycoprotein of influenza C virus is acylated with stearic acid which has not been observed before as the prevailing fatty acid in viral or cellular acyl proteins.

Acylation↗
PubMed1989

Different palmitoylation of paramyxovirus glycoproteins.

Different paramyxoviruses were analyzed for the covalent attachment of fatty acids into their structural proteins. The fusion protein (F) of Newcastle diseases virus and the hemagglutinin-neuraminidase (HN) of Simian virus 5 are fatty acylated, whereas the glycoproteins of Sendai virus are fatty acid free. The fatty acid linkage is labile to treatment with hydroxylamine. SDS-PAGE in the presence of mercaptoethanol releases some of the covalently bound acyl chains.

Amino Acid Sequence↗
PubMed1989

Myristoylation of budgerigar fledgling disease virus capsid protein VP2.

We present evidence that the structural protein VP2 of budgerigar fledgling disease virus, an avian polyomavirus, is specifically modified by covalent attachment of myristic acid. The fatty acid linkage is insensitive to hydroxylamine treatment and thus represents the amide type of fatty acylation of proteins.

Animals↗
PubMed1988

Chemical identification of cysteine as palmitoylation site in a transmembrane protein (Semliki Forest virus E1).

The palmitoylation site of the membrane glycoprotein E1 of Semliki Forest virus (SFV) has been identified by chemical analysis of an acylpeptide. 3H-Palmitoylated E1 isolated from SFV grown in baby hamster kidney cells was digested with chymotrypsin and the resulting peptides subjected to high performance liquid chromatography on a wide-pore column. The 3H-acylated peptide fraction peaked at above 60% 2-propanol in the eluent, indicating its hydrophobic character. Polyacrylamide gel electrophoresis analysis revealed a molecular weight of about Mr = 6000 for the radiolabeled peptide. Manual sequencing of this material by the 4-N,N'-dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate procedure on solid phase revealed the amino-terminal sequence Ala-Ala-Ser-His-Ser-Asn-Val-Val-Phe-Pro. The same peptide also labels with [35S]cysteine. Comparison with the deduced amino acid sequence of E1 revealed that the palmitoylated peptide contains at least 43 amino acid residues, and thus includes the membrane spanning region down to the only cysteine residue five positions up from the carboxyl terminus of E1. Since [3H]palmitic acid was cleaved from E1 with thiol reagents, and since the peptide labels with [14C]iodoacetamide only after the release of fatty acids by hydroxylamine treatment, cysteine in position 433 represents the palmitoylation site in SFV E1.

Amino Acid Sequence↗
PubMed1987

Cell-free fatty acylation of microsomal integrated and detergent-solubilized glycoprotein of vesicular stomatitis virus.

An enzymatic activity associated with intracellular membrane fractions of Merwin plasma cell tumor II, baby hamster kidney, and chicken embryo fibroblast cells and bovine kidney has been characterized which covalently links fatty acids onto the G protein of vesicular stomatitis virus. Exogenous G protein extracted from native vesicular stomatitis virus particles can be acylated in vitro only after it has been previously deacylated. The fatty acids transferred in vitro are sensitive to treatment with hydroxylamine, indicating an ester linkage. Cell-free acyl transfer was also observed with endogenous G protein present in membrane fractions prepared from vesicular stomatitis virus-infected cells. In this case, the fatty acids become linked to a G protein species (G1) which is not terminally glycosylated and therefore has not entered the trans-Golgi compartment. The same G protein species also becomes acylated in infected cells during short pulses with radioactive palmitic acid. Acylation of the G protein in vitro with free palmitic or myristic acid is energy-dependent, and the addition of ATP is specifically required. Other nucleoside triphosphates cannot substitute for ATP in the activation of free acyl chains. Alternatively, activated fatty acids linked in a high energy thioester bond to coenzyme A, e.g. [14C] palmitoyl-CoA, are suitable lipid donors in the in vitro acylation reactions. Palmitic acid transfer onto G protein shows the typical characteristics of an enzyme-catalyzed reaction.

Acyltransferases↗
PubMed1986

Characterization of a protein fatty acylesterase present in microsomal membranes of diverse origin.

A microsomal activity of baby hamster kidney cells which cleaves ester-type bound fatty acids from acyl proteins in vitro has been characterized. This activity is also present in microsomal membranes from pig liver, calf kidney, and human mucous cells. Cell free deacylation is described for the Semliki Forest virus acyl proteins E1 and E2 and the precursor of E2 designated p62. Acyl chain cleavage operates with both exogenous and endogenous viral acyl protein substrates. The in vitro cleavage requires microsomes solubilized by detergents of which various kinds are equally effective (Nonidet P-40, Tween 20, sodium deoxycholate, Triton X-100, or octyl-beta-D-glucoside). If microsomes are boiled for 15 min prior to the incubation, deacylation is abolished completely and no radioactivity is released from the palmitoylated acyl proteins during incubation with either detergents or microsomes alone. No changes in the molecular structure of the deacylated Semliki Forest virus proteins were detected, and the cleavage product was identified as free fatty acid. Deacylation is time- and temperature-dependent and can be enhanced by increasing the concentration of microsomal protein in the incubation mixture. It is completely inhibited under acidic conditions (pH 5) and at low temperature (4 degrees C). Deacylation also occurs in the presence of EDTA and bivalent cations such as Mg2+, Mn2+, and Ca2+ which influence the reaction marginally. On the other hand, fatty acid release is drastically reduced with a mixture of Co2+, Zn2+, and Hg2+ ions. The activity is not identical with protein fatty acyltransferase operating in the reverse direction, since a partially purified preparation of this acyltransferase failed to cleave fatty acids from fatty acylated substrate proteins. Taken together, these data lead us to postulate an enzymatic activity which cleaves fatty acids from ester-type fatty acylated proteins, and we propose to designate this enzyme a protein fatty acylesterase.

Animals↗
PubMed1986

Membrane fusion induced by influenza virus hemagglutinin requires protein bound fatty acids.

The low pH-dependent fusion of lipid membranes induced by two types of the fatty acylated influenza viral hemagglutinin has been studied by use of an energy transfer assay. When protein bound fatty acids were released from the hemagglutinin by hydroxylamine treatment viral fusion activity was inhibited. The extent of fusion inhibition correlates with the amount of fatty acids cleaved from the hemagglutinin. Virosomes prepared from fowl plague virus containing fatty acid free hemagglutinin showed a much lower fusion activity than control virosomes containing fatty acylated hemagglutinin. The hydroxylamine treatment applied has no detectable effects on the virus other than fatty acid release from its spike glycoproteins. These results support our previous hypothesis that protein bound fatty acids are involved in the induction of membrane fusion by the influenza hemagglutinin.

Animals↗