Biomedical subjects

V A Fried

Publications and source records attributed to V A Fried.

At least 37 records · Page 2Linked to original sources

PubMed1991

Structure of the 116-kDa polypeptide of the clathrin-coated vesicle/synaptic vesicle proton pump.

A 116-kDa polypeptide has recently been found to be a common component of vacuolar proton pumps isolated from a variety of sources. The 116-kDa subunit of the proton pump was purified from clathrin-coated vesicles of bovine brain, and internal sequences were obtained from proteolytic peptides. Oligonucleotide probes designed from these peptide sequences were utilized in polymerase chain reactions to isolate partial bovine cDNA clones for the protein. Sequences from these were then utilized to isolate rat brain cDNA clones containing the full-length coding region. RNA blots indicate the presence of an abundant 3.9-kilobase message for the 116-kDa subunit in brain, and primer extension analysis demonstrates that the cloned sequence is full-length. The rat cDNA sequences predict synthesis of a protein of 96,267 Da. Analysis of the deduced amino acid sequence of the 116-kDa subunit suggests that it consists of two fundamental domains: a hydrophilic amino-terminal half that is composed of greater than 30% charged residues, and a hydrophobic carboxyl-terminal half that contains at least six transmembrane regions. The structural properties of the 116-kDa proton pump polypeptide agree well with its proposed function in coupling ATP hydrolysis by the cytoplasmic subunits to proton translocation by the intramembranous components of the pump.

Amino Acid Sequence↗

PubMed1991

Abnormal phosphorylation of tau precedes ubiquitination in neurofibrillary pathology of Alzheimer disease.

On tissue sections of Alzheimer brain, 4 antibodies to tau immunolabel not only neurofibrillary tangles, neuritic plaques and neuropil threads but also the tangle-free cytoplasm of a subset of hippocampal and cortical neurons we believe to be at a stage of alteration preceding the formation of paired helical filaments (PHF). Pretreatment of tissue sections with alkaline phosphatase leads to an increase in staining intensity and in number of immunoreactive lesions with antibodies directed to an amino terminal and to a mid-region of the tau molecule. The diffuse neuronal staining could not be observed with any of 7 monoclonal antibodies recognizing ubiquitin. We conclude (1) that abnormal phosphorylation of tau occurs prior to its incorporation into PHF and leads to its accumulation in the nerve cell body and (2) that ubiquitin is seen associated only when a neurofibrillary tangle is already formed.

Aged↗

PubMed1990

Genetic linkage studies suggest that Alzheimer's disease is not a single homogeneous disorder.

Alzheimer's disease, a fatal neurodegenerative disorder of unknown aetiology, is usually considered to be a single disorder because of the general uniformity of the disease phenotype. Two recent genetic linkage studies revealed co-segregation of familial Alzheimer disease with the D21S1/S11 and D21S16 loci on chromosome 21. But two other studies, one of predominantly multiplex kindreds with a late age-of-onset, the other of a cadre of kindreds with a unique Volga German ethnic origin, found absence of linkage at least to D21S1/S11. So far it has not been possible to discern whether these conflicting reports reflect aetiological heterogeneity, differences in methods of pedigree selection, effects of confounding variables in the analysis (for example, diagnostic errors, assortative matings), or true non-replication. To resolve this issue, we have now examined the inheritance of five polymorphic DNA markers from the proximal long arm of chromosome 21 in a large unselected series of pedigrees with familial Alzheimer's disease. Our data suggest that Alzheimer's disease is not a single entity, but rather results from genetic defects on chromosome 21 and from other genetic or nongenetic factors.

Alzheimer Disease↗

PubMed1990

Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C.

Neurotransmitters are released at synapses by the Ca2(+)-regulated exocytosis of synaptic vesicles, which are specialized secretory organelles that store high concentrations of neurotransmitters. The rapid Ca2(+)-triggered fusion of synaptic vesicles is presumably mediated by specific proteins that must interact with Ca2+ and the phospholipid bilayer. We now report that the cytoplasmic domain of p65, a synaptic vesicle-specific protein that binds calmodulin contains an internally repeated sequence that is homologous to the regulatory C2-region of protein kinase C (PKC). The cytoplasmic domain of recombinant p65 binds acidic phospholipids with a specificity indicating an interaction of p65 with the hydrophobic core as well as the headgroups of the phospholipids. The binding specificity resembles PKC, except that p65 also binds calmodulin, placing the C2-regions in a context of potential Ca2(+)-regulation that is different from PKC. This is a novel homology between a cellular protein and the regulatory domain of protein kinase C. The structure and properties of p65 suggest that it may have a role in mediating membrane interactions during synaptic vesicle exocytosis.

Amino Acid Sequence↗

PubMed1990

Taurine and hypotaurine content of human leukocytes.

Taurine (T) was reported to be at high concentrations in human leukocytes. It was proposed that T is a scavenger for chlorinated oxidants produced by the myeloperoxidase system of monocytes and neutrophils. Hypotaurine (HT) would be a more effective scavenger, and HT could also detoxify products of bromide or iodide oxidation produced by the eosinophil peroxidase system. Methods previously used to measure T in leukocytes might oxidize HT to T or fail to separate T and HT. Therefore, we examined T and HT content, uptake, and biosynthesis in isolated blood cells and cultured tumor cells derived from hematopoietic/lymphoid cells. Platelets and all leukocytes including monocytes, lymphocytes, neutrophils, and eosinophils had high T levels (10-20 mM), and all except eosinophils had substantial HT levels (0.3-1 mM). Intracellular levels were 500-times higher than in plasma. Erythrocytes were the only blood cells with low levels of both T and HT. Tumor cells from lymphoid (CCRF-CEM) and myeloid (HL-60, K-562, RWLeu4, HEL) lineages took up and concentrated T and HT from the bovine calf serum in the culture medium, and intracellular levels were similar to those in leukocytes. When cells were cultured in HT-supplemented media, HT almost completely replaced T, and HT was not converted to T. Levels of T were not raised by culturing cells with possible precursors, but HT levels were raised when cysteine sulfinic acid was present. Washed tumor cells took up T and HT by way of a beta-amino acid transport system, but uptake by leukocytes was very low. Therefore, leukocytes may acquire T and HT by active uptake rather than biosynthesis, and uptake may be completed during differentiation in the bone marrow. Though HT is low relative to T, HT levels may be sufficient to protect leukocytes from toxic oxidants.

Alanine↗

PubMed1989

Purification and complementary DNA cloning of a receptor for basic fibroblast growth factor.

Basic fibroblast growth factor (bFGF) participates in many processes including early developmental events, angiogenesis, wound healing, and maintenance of neuronal cell viability. A 130-kilodalton protein was isolated on the basis of its ability to specifically bind to bFGF. A complementary DNA clone was isolated with an oligonucleotide probe corresponding to determined amino acid sequences of tryptic peptide fragments of the purified protein. The putative bFGF receptor encoded by this complementary DNA is a transmembrane protein that contains three extracellular immunoglobulin-like domains, an unusual acidic region, and an intracellular tyrosine kinase domain. These domains are arranged in a pattern that is different from that of any growth factor receptor described.

Amino Acid Sequence↗

PubMed1989

Phosphoamino acid analysis of protein immobilized on polyvinylidene difluoride membrane.

The direct analysis of phosphorylated proteins bound to polyvinylidene difluoride membrane (PVDFm) has been examined. Use of 14C-methylated marker proteins demonstrated that proteins electroblotted on PVDFm were quantitatively retained through a series of test conditions, which included 1 M hydroxylamine (25 degrees C, 30 min), 0.1 M NaOH (37 degrees C, 30 min), 0.1 M HCl (55 degrees C, 2 h), and 6 U/ml alkaline phosphatase (pH 9.5, 37 degrees C, 24 h). Approximately half the protein remained bound following 2-h treatment in 1 M KOH (55 degrees C). The same series of test conditions were employed to assess the stability of phosphorylated residues in 32P-labeled protein immobilized on PVDFm, in order to assign them as carboxyl-,N-, or O-linked groups. The properties of phosphorylated proteins as determined by this method were comparable to the properties that have been reported for soluble proteins. Use of the PVDFm immobilization step affords simplification of the experimental procedures and permits rapid, quantitative sample recovery using submicrogram quantities of protein. Further, the PVDFm-bound phosphoproteins could be subjected to partial acid hydrolysis directly on the membrane and required no further purification for subsequent identification of the labeled phosphohydroxyamino acids. Definitive identification of labeled phosphoserine residues in histone, phosphoserine and phosphothreonine residues in myelin basic protein and insulin receptor, and phosphotyrosine residues in autophosphorylated insulin receptor was accomplished with as little as 0.2 nCi in about 50 ng of phosphorylated protein.

Amino Acids↗

PubMed1989

Human endomembrane H+ pump strongly resembles the ATP-synthetase of Archaebacteria.

Preparations of mammalian H+ pumps that acidify intracellular vesicles contain eight or nine polypeptides, ranging in size from 116 to 17 kDa. Biochemical analysis indicates that the 70- and 58-kDa polypeptides are subunits critical for ATP hydrolysis. The amino acid sequences of the major catalytic subunits (58 and 70 kDa) of the endomembrane H+ pump are unknown from animal cells. We report here the complete sequence of the 58-kDa subunit derived from a human kidney cDNA clone and partial sequences of the 70- and 58-kDa subunits purified from clathrin-coated vesicles of bovine brain. The amino acid sequences of both proteins strongly resemble the sequences of the corresponding subunits of the vacuolar H+ pumps of Archaebacteria, plants, and fungi. The archaebacterial enzyme is believed to use a H+ gradient to synthesize ATP. Thus, a common ancestral protein has given rise to a H+ pump that synthesizes ATP in one organism and hydrolyzes it in another and is highly conserved from prokaryotes to humans. The same pump appears to mediate the acidification of intracellular organelles, including coated vesicles, lysosomes, and secretory granules, as well as extracellular fluids such as urine.

Amino Acid Sequence↗

PubMed1989

Immunochemical properties of ubiquitin conjugates in the paired helical filaments of Alzheimer disease.

Immunocytochemical and peptide sequencing studies indicate that the regulatory protein ubiquitin (Ub) is incorporated into the paired helical filaments (PHF) of Alzheimer disease. In this study, we showed that some antibodies raised to PHF recognize epitopes of Ub. Analysis of the Ub sequences recognized by the antibodies raised to PHF, along with the known specificity of several monoclonal antibodies raised to artificial Ub conjugates, indicates the immunochemical representation of Ub residues 34-76 in PHF. The Ub epitopes recognized by antibodies raised to PHF are distinct from those recognized by antibodies raised to artificial Ub conjugates in two respects. First, antibodies that are raised to PHF and that recognize Ub react with PHF equally, whether denatured or not, whereas those raised to artificial Ub conjugates show greater reaction after denaturation. Second, mapping of the epitopes recognized by two monoclonal antibodies to PHF onto Ub indicates a distinction in the Ub residues recognized, compared with monoclonal antibodies raised to artificial Ub conjugates. The proximity of their epitopes to the site of conjugation, as well as their affinity for PHF polypeptides, suggests that the PHF antibodies that recognize Ub may be directed specifically to Ub epitopes defined by the protein conjugated to Ub.

Alzheimer Disease↗

PubMed1989

Ubiquitin: a multifunctional regulatory protein associated with the cytoskeleton.

The discovery that ubiquitin is associated with the neurofibrillary tangles of Alzheimer's disease and inclusion bodies of other neurodegenerative disorders raises the possibility that ubiquitin is playing a role in the pathology. Ubiquitin is a small, highly conserved protein that is found both free and covalently attached to other proteins in all eukaryotic cells. Ubiquitin is conjugated with some proteins that are rapidly degraded and with a select set of regulatory proteins that are relatively stable, implying that ubiquitin is multifunctional. We have produced monoclonal antibodies to ubiquitin and identified the epitopes. Different monoclonal antibodies recognize different ubiquitin-protein conjugates; this could be due to different conformational states of ubiquitin or to steric interference by the conjugated protein. These observations suggest that ubiquitin is structurally and therefore functionally different in different contexts. Ubiquitin is localized in several compartments in cells and this may also affect function. We discovered that it is a normal component of the microtubule network and have identified ubiquitinated proteins in bovine brain that appear associated with microtubules. We discovered that ubiquitin has intrinsic proteolytic activity and proposed that ubiquitin-protein conjugates could function as ad hoc proteases. This proteolytic activity offers a heuristic basis from which to explore ubiquitin's cellular functions. The association of ubiquitin with the cytoskeletal networks suggests that proteolytic processing is a function of these structures. Disruption of these structures in neurodegenerative disorders, improper localization of ubiquitinated components or appearance of abnormal conjugates could lead to altered proteolytic processing pathways and contribute to the pathology of these diseases.

Alzheimer Disease↗

PubMed1988

Human insulin-degrading enzyme shares structural and functional homologies with E. coli protease III.

A proteinase with high affinity for insulin has been proposed to play a role in the cellular processing of this hormone. A complementary DNA (cDNA) coding for this enzyme has been isolated and sequenced. The deduced amino acid sequence of the enzyme contained the sequences of 13 peptides derived from the isolated protein. The cDNA could be transcribed in vitro to yield a synthetic RNA that in cell-free translations produced a protein that coelectrophoresed with the native proteinase and could be immunoprecipitated with monoclonal antibodies to this enzyme. The deduced sequence of this proteinase did not contain the consensus sequences for any of the known classes of proteinases (that is, metallo, cysteine, aspartic, or serine), but it did show homology to an Escherichia coli proteinase (called protease III), which also cleaves insulin and is present in the periplasmic space. Thus, these two proteins may be members of a family of proteases that are involved in intercellular peptide signaling.

Amino Acid Sequence↗

PubMed1988

The structure of cytochrome b561, a secretory vesicle-specific electron transport protein.

Cytochrome b561 is a transmembrane electron transport protein that is specific to a subset of secretory vesicles containing catecholamines and amidated peptides. This protein is thought to supply reducing equivalents to the intravesicular enzymes dopamine-beta-hydroxylase and alpha-peptide amidase. We have purified cytochrome b561 from bovine adrenal chromaffin granules by reverse phase chromatography and have determined internal amino acid sequences from peptides. Complementary oligonucleotides were used to isolate two cDNA clones from a bovine brain library. The structure predicted by the sequences of these cDNAs suggests a highly hydrophobic protein of 273 amino acids which spans the membrane six times with little extramembranous sequence. Cytochrome b561 is not homologous to any other cytochrome and thus represents a new class of electron carriers. RNA blotting experiments indicate that cytochrome b561 is expressed in the adrenal medulla and all brain regions of the cow, but not in visceral organs. This result agrees well with the putative function of this unique cytochrome and with the notion that this protein is localized to large dense-core synaptic vesicles.

Amino Acid Sequence↗

PubMed1988

Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases.

Several degenerative diseases of the nervous system are characterized by the presence of neuronal inclusions. Most of these inclusions are made of abnormal filaments and share epitopes with cytoskeletal proteins. One of these inclusions, the neurofibrillary tangle of Alzheimer disease, has recently been shown to contain ubiquitin, a regulatory protein thought to play a role in the degradation of abnormal proteins. We carried out light and electron microscopic immunocytochemistry with several polyclonal and monoclonal antibodies to investigate the presence of ubiquitin in neuronal inclusions of neurodegenerative diseases. Ubiquitin was present not only in paired helical filaments that form the neurofibrillary tangle of Alzheimer disease, but also in the filamentous components of the inclusion characteristic of Parkinson disease, Pick disease, and progressive supranuclear palsy. In contrast, ubiquitin was not detected in other neuronal inclusions often found in aging and in Alzheimer disease, such as Hirano bodies and granulovacuolar degeneration. Reactivity with monoclonal antibodies suggests differences in the ubiquitin-acceptor proteins present in the inclusions studied. It is concluded that ubiquitin is selectively present in neuronal inclusions of degenerative diseases.

Alzheimer Disease↗

PubMed1988

Ubiquitin is a component of the microtubule network.

Immunofluorescence microscopy was used to study the intracellular localization of ubiquitin. Baby hamster kidney cells (BHK cells) and several other cell lines were probed with a well characterized monoclonal antibody to ubiquitin. The antibody stained a complex cellular structure that we identified as the microtubule network. The anti-ubiquitin antibody bound to the microtubule network at all stages of the cell cycle, and we showed that the apparent association of ubiquitin with the microtubule network is not an artifact of crosslinking of free ubiquitin to the cell structure. Immunoblot procedures demonstrated that tubulin itself was not ubiquitinated. We propose that ubiquitin and/or ubiquitin-protein conjugates are associated with those networks as a new class of microtubule-associated protein. The targeting of ubiquitin to specific sites within the cell by its association with the microtubule network may regulate some of the functions of ubiquitin.

Animals↗

PubMed1987

Interactions of the receptor for insulin-like growth factor II with mannose-6-phosphate and antibodies to the mannose-6-phosphate receptor.

Recently, the sequence of the human receptor for insulin-like growth factor II (IGF-II) was found to be 80% identical [Morgan et al., (1987) Nature 329, 301-307] to the sequence of a partial clone of the bovine cation-independent mannose-6-phosphate receptor [Lobel et al., (1987) Proc. Natl. Acad. Sci. USA 84, 2233-2237]. In the present study, the purified receptor for insulin-like growth factor II (IGF-II) was found to react with two different polyclonal antibodies to the purified mannose-6-phosphate receptor. Moreover, mannose-6-phosphate was found to stimulate the binding of labeled IGF-II to the IGF-II receptor by two-fold. This effect had the same specificity and affinity as the reported binding of mannose-6-phosphate to its receptor; mannose-1-phosphate and mannose had no effect on the binding of labeled IGF-II to its receptor, and the half-maximally effective concentration of mannose-6-phosphate was 0.3 mM. Also, mannose-6-phosphate did not affect labeled IGF-II binding to the insulin receptor. These results support the hypothesis that a single protein of Mr-250,000 binds both IGF-II and mannose-6-phosphate. Furthermore, they indicate that mannose-6-phosphate can modulate the interaction of IGF-II to its receptor.

Carrier Proteins↗

PubMed1987

Differential reactivities of lysines in calmodulin complexed to phosphatase.

Calmodulin and calmodulin complexed with calcineurin phosphatase were trace labeled with [3H]acetic anhydride and the incorporation of [3H]acetate into each epsilon-amino lysine of calmodulin was measured. The relative reactivities of calmodulin lysines were higher in the presence of Ca2+ than in the presence of EGTA, and the order was: Lys-75 greater than Lys-94 greater than Lys-148 greater than or equal to Lys-77 greater than Lys-13 greater than or equal to Lys-21 greater than Lys-30. The changes in relative reactivity implied a change in conformation. When calmodulin was complexed with the phosphatase, Lys-21, Lys-77, and Lys-148 were most protected, implying that these residues are at or near the interaction sites or are conformationally perturbed by the interaction. Lys-30 and Lys-75 were slightly protected, lysine 13 showed no change, while lysine 94 significantly increased in reactivity. Comparison with results obtained from myosin light chain kinase using a similar technique (Jackson, A. E., Carraway, K. L., III, Puett, D., and Brew, K. (1986) J. Biol. Chem. 261, 12226-12232) reveals that calmodulin may interact with each of the two enzymes similarly at or near Lys-21, Lys-75, and Lys-148; one difference with phosphatase is that complex formation also involved Lys-77. These findings suggest that calmodulin interacts differently with its target enzymes.

Acetates↗

PubMed1987

Early steps initiating a degradation pathway in Escherichia coli. Characterization of the first intermediate.

Our previous studies demonstrated that a site-specific cleavage event initiates the degradation of large premature termination polypeptides of beta-galactosidase in Escherichia coli. We have isolated the first cleavage intermediate, the "B" polypeptide, by elution from sodium dodecyl sulfate-polyacrylamide gels. The NH2 terminus of this protein, determined by automated Edman degradation, was that of the wild-type molecule and thus established that the first cleavage event was at the COOH-terminal end. The sequence of the COOH-terminal end of the B polypeptide was determined by using the enzyme carboxypeptidase Y. Direct assignment of COOH-terminal residues was made by using o-phthaldialdehyde derivatization and the stoichiometry confirmed by a double-label analysis. The COOH-terminal end of the B polypeptide is at position 837 in the beta-galactosidase sequence. If a single endoproteolytic cleavage event was responsible, the cleavage would have occurred between 2 threonine residues (at positions 837 and 838) that are located within a hydrophobic domain. We have observed other covalent modifications that precede the appearance of the B polypeptide, but these do not appear to participate in signaling the first cleavage event. The structure of the COOH-terminal end of B suggests a high degree of specificity by the initial cleavage enzyme. We propose that this unique site serves as a specific signal and that exposure of this site to the specific cleavage enzyme controls the event initiating the degradation pathway.

Amino Acid Sequence↗

PubMed1987

In vivo activation of quiescent B cells by anti-immunoglobulin. I. Induction of latent VH allotype production in adult rabbits by treatment with heterologous antibodies or antibody fragments.

We demonstrated previously that injection of adult rabbits with homologous anti-VHa1 allotype antibody induces the appearance in serum of genetically unexpected (latent) a1 immunoglobulin (Ig) and a1-like internal images. We have now investigated the mechanism for this induction effect and have found that treatment of animals with polyclonal goat or monoclonal mouse anti-a1 antibody similarly results in production of unexpected a1 determinants. The N-terminal amino acid sequences of deblocked heavy chains showed that latent a1 Ig induced by monoclonal antibody treatment was identical to nominal a1 Ig at 18 of 19 residues, with the one amino acid difference at position 13 probably due to a single nucleotide change. Like nominal a1, these molecules expressed multiple VHa1 framework epitopes, as demonstrated by direct immunoelectron microscopic visualization of immune complexes. Whereas F(ab)'2 fragments of rabbit anti-a1 antibody retained inducing activity, F(ab) fragments were effective only when administered in an aggregated form; this result suggests that cross-linking of surface Ig receptors plays a critical role in the induction process. We conclude that all rabbits contain dormant B cells expressing germ-line-encoded latent allotypes, and that in vivo administration of anti-Ig antibody causes activation of these cells directly rather than through perturbation of an allotype regulatory network.

Amino Acid Sequence↗