Biomedical subjects

S M Strittmatter

Publications and source records attributed to S M Strittmatter.

At least 55 records · Page 3Linked to original sources

PubMed1993

Mediation by G proteins of signals that cause collapse of growth cones.

During development, motion of nerve growth cones ceases on contact with particular targets. The signaling mechanism is unknown. In culture, growth cone collapse can be caused by solubilized embryonic brain membranes, central nervous system myelin, a 35-kilodalton protein isolated from myelin, and mastoparan. Collapse induced by each of these is blocked by pertussis toxin. Thus, collapse of growth cones is mediated by G protein-coupled receptors, which may be activated by proteins associated with the cell surface as well as by soluble ligands.

Animals↗

PubMed1993

Functional expression of sodium channel mutations identified in families with periodic paralysis.

Two mutations in the sodium channel alpha subunit that have been implicated as the cause of periodic paralysis were studied by functional expression in a mammalian cell line. Both mutations disrupted inactivation without affecting the time course of the onset of the sodium current or the single-channel conductance. This is the same functional defect that was observed in myotubes cultured from affected patients and proves that these mutations are not benign polymorphisms. Unlike the currents in the myotubes, however, there was no consistent potassium dependence for the noninactivating component. These mutations also define new regions of the sodium channel alpha subunit that are involved in the process of inactivation.

Amino Acid Sequence↗

PubMed1992

Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43.

The addition of palmitate to cysteine residues enhances the hydrophobicity of proteins, and consequently their membrane association. Here we have investigated whether this type of fatty acylation also regulates protein-protein interactions. GAP-43 is a neuronal protein that increases guanine nucleotide exchange by heterotrimeric G proteins. Two cysteine residues near the N-terminus of GAP-43 are subject to palmitoylation, and are necessary for membrane binding as well as for G(o) activation. N-terminal peptides, which include these cysteines, stimulate G(o). Monopalmitoylation reduces, and dipalmitoylation abolishes the activity of the peptides. The activity of GAP-43 protein purified from brain also is reversibly blocked by palmitoylation. This suggests that palmitoylation controls a cycle of GAP-43 between an acylated, membrane-bound reservoir of inactive GAP-43, and a depalmitoylated, active pool of protein.

Animals↗

PubMed1992

GAP-43 as a plasticity protein in neuronal form and repair.

Neurons exhibit a remarkable plasticity of form, both during neural development and during the subsequent remodelling of synaptic connectivity. Here we review work on GAP-43 and G0, and focus upon the thesis that their interaction may endow neurons with such plasticity. We also present new data on the role of G proteins in neurite growth, and on the interaction of GAP-43 and actin. GAP-43 is a protein induced during periods of axonal extension and highly enriched on the inner surface of the growth cone membrane. Its membrane localization is primarily due to a short amino terminal sequence which is subject to palmitoylation. Binding to actin filaments may also assist in restricting the protein to specific cellular domains. Consistent with its role as a "plasticity protein," there is evidence that GAP-43 can directly alter cell shape and neurite extension, and several theses have been advanced for how it might do so. Two other prominent components of the growth cone membrane are the alpha and beta subunits of G0. GAP-43 regulates their guanine nucleotide exchange, which is an unusual role for an intracellular protein. We speculate that GAP-43 may adjust the "set point" of responsiveness for G0 stimulation by receptors, thereby altering the neuronal propensity to growth, without actually causing growth. To begin to address how G protein activity affects axon growth, we have developed a means to introduce guanine nucleotide analogs into sympathetic neurons. Stimulation of G proteins with GTP-gamma-S retards axon growth, whereas GDP-beta-S enhances it. This is compatible with G protein registration of inhibitory signals.

Actins↗

PubMed1992

GAP-43 as a modulator of G protein transduction in the growth cone.

Much circumstantial evidence that GAP-43 is involved in neuronal growth cone function has accumulated over the last ten years. The expression of the protein is closely correlated both temporally and spatially with periods of axonal outgrowth, and the protein is highly concentrated in the growth cone membrane. There is direct evidence that overexpression of the protein can alter cell shape. This review focuses on the molecular mechanisms whereby GAP-43 could exert these actions. One important requirement for GAP-43 function is its localization to appropriate regions of the cell. The ability of this hydrophilic protein to associate with membrane fractions is determined by a short 10 amino acid stretch of the amino terminus of the protein, which contains two cysteine residues subject to palmitoylation. Whether this region can direct growth cone targeting in neurons is not yet clear. Once appropriately localized, GAP-43 may modulate complex cellular properties such as growth cone motility, synaptic plasticity and neurotransmitter release. One possible molecular mechanism for these cellular changes in GAP-43 regulation of the GTP-binding protein, G(o). The observation that the growth cone membrane contains extremely high concentrations of G(o) led us to investigate the interaction of G(o) and GAP-43. There is evidence that G protein-mediated transduction systems can control the same cellular functions thought to be altered by GAP-43: growth cone motility, neurotransmitter release and synaptic plasticity. Purified GAP-43 does stimulate guanine nucleotide binding to G(o). Its action in stimulating GDP release is quite similar to that of G protein-coupled transmembrane receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗

PubMed1991

An intracellular guanine nucleotide release protein for G0. GAP-43 stimulates isolated alpha subunits by a novel mechanism.

G protein-coupled membrane receptors activate G proteins by enhancing guanine nucleotide exchange. G0 is a major component of the growing regions (growth cones) of neurons. GAP-43 is a neuronal protein associated with the cytosolic face of the growth cone plasma membrane and stimulates binding of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to Go (Strittmatter, S. M., Valenzuela, D., Kennedy, T. E., Neer, E. J., and Fishman, M. C. (1990) Nature 344, 836-841). Here we have examined the mechanism by which GAP-43 affects G0. Like G protein-coupled receptors, GAP-43 enhances GDP release from G0, increases the initial rate of GTP gamma S binding, and increases the GTPase activity of Go, all without altering the intrinsic kappa cat for the GTPase. Unlike the case for receptors, however, the GAP-43 effect is not blocked by pertussis toxin, nor affected by the presence or absence of beta gamma or of phospholipids. There is specificity to the interaction, in that GAP-43 increases GTP gamma S binding to recombinant alpha o and alpha i1, but not to recombinant alpha s. Thus, GAP-43 is a guanine nucleotide release protein with a novel mechanism of action, potentially controlling membrane-associated G proteins from within the cell.

Animals↗

PubMed1991

The neuronal growth cone as a specialized transduction system.

Neuronal growth and remodelling are guided by both intracellular gene programs and extracellular stimuli. The growth cone is one site where the effects of these extrinsic and intrinsic factors converge upon the mechanical determinants of cell shape. We review the growth cone as a transduction device, converting extracellular signals into mechanical forces. A variety of soluble, extracellular matrix and membrane bound molecules control growth cone behavior. In addition, GAP-43 is discussed as a possible component of the intraneuronal gene program which modulates growth cone activity. The GTP-binding protein, Go, is a major growth cone membrane protein that may transduce signals not only from outside the cell, but from within as well. This may provide a molecular site in the growth cone for the coordination of a genetic growth program with environmental signals.

Animals↗

PubMed1991

Growth cone transduction: Go and GAP-43.

The neuronal growth cone plays a crucial role in forming the complex brain architecture achieved during development, and similar nerve terminal mechanisms may operate to modify synaptic structure during adulthood. The growth cone leads the elongating axon towards appropriate synaptic targets by altering motility in response to a variety of extracellular signals. Independently of extrinsic clues, neurons manifest intrinsic control of their growth and form (Banker and Cowan, 1979). Hence, there must be intracellular proteins which control nerve cell shape, so-called 'plasticity' or 'growth' genes. GAP-43 may be such a molecule (Skene and Willard, 1981; Benowitz and Lewis, 1983). For example, GAP-43 is localized to the growth cone membrane (Meiri et al. 1986; Skene et al. 1986) and can enhance filopodial formation even in non-neuronal cells (Zuber et al. 1989a). It includes a small region at the amino terminus for membrane association and perhaps growth cone targeting (Zuber et al. 1989b, Liu et al. 1991). We have found that Go, a member of the G protein family that links receptors and second messengers, is the major non-cytoskeletal protein in the growth cone membrane (Strittmatter et al. 1990). Double staining immunohistochemistry for GAP-43 and Go shows that the distributions of the two proteins are quite similar. Purified GAP-43 regulates the activity of purified Go (Strittmatter et al. 1990), a surprising observation since GAP-43 is an intracellular protein. We have compared the mechanism of GAP-43 activation of Go with that of G protein-linked receptors.2+ interactions between Go and GAP-43 suggest that Go plays a pivotal role in growth cone function, coordinating the effects of both extracellular signals and intracellular growth proteins.

Animals↗

PubMed1990

G0 is a major growth cone protein subject to regulation by GAP-43.

G0, a GTP-binding protein that transduces information from transmembrane receptors, has been found to be a major component of the neuronal growth cone membrane. GAP-43, an intracellular growth cone protein closely associated with neuronal growth, stimulates GTP-gamma-S binding to G0. It does so through an amino-terminal domain homologous to G-linked transmembrane receptors. Thus, G0 in the growth cone may be regulated by intracellular as well as extracellular signals.

Amino Acid Sequence↗

PubMed1989

A membrane-targeting signal in the amino terminus of the neuronal protein GAP-43.

Neurons and other cells, such as those of epithelia, accumulate particular proteins in spatially discrete domains of the plasma membrane. This enrichment is probably important for localization of function, but it is not clear how it is accomplished. One proposal for epithelial cells is that proteins contain targeting signals which guide preferential accumulation in basal or apical membranes. The growth-cone membrane of a neuron serves as a specialized transduction system, which helps to convert cues from its environment into regulated growth. Because it can be physically separated from the cell soma, it has been possible to show that the growth-cone membrane contains a restricted set of total cellular proteins, although, to our knowledge, no proteins are limited to that structure. One of the most prominent proteins in the growth-cone membrane is GAP-43. Basi et al. have suggested that the N-terminus of GAP-43 might be important for the binding of GAP-43 to the growth-cone membrane. Skene and Virag recently found that the cysteines in the N-terminus are fatty-acylated and that this post-translational modification correlates with membrane-binding ability. We investigated the binding of GAP-43 to the growth-cone membrane by mutational analysis and by laser-scanning confocal microscopy of fusion proteins that included regions of GAP-43 and chloramphenicol acetyltransferase (CAT). We found that a short stretch of the GAP-43 N-terminus suffices to direct accumulation in growth-cone membranes, especially in the filopodia. This supports a previous proposal for the importance of this region of GAP-43 in determining the membrane distribution of GAP-43.

Adrenal Gland Neoplasms↗

PubMed1989

Localization of angiotensin converting enzyme in the ciliary epithelium of the rat eye.

Angiotensin converting enzyme (ACE, E.C. 3.14.5.1) was localized in the rat eye by immunocytochemical staining using anti-rat lung ACE monoclonal antibody, and by autoradiography using the potent ACE inhibitor [3H]captopril. Highest levels of ACE immunoreactivity and [3H]captopril binding were found in the ciliary epithelium (Bmax = 1050 fmol [3H]captopril bound/mg protein) while lower levels were present in the sclera (Bmax = 500 fmol/mg protein). Scattered regions of the choroidal epithelium were weakly immunocytochemically stained by the anti-ACE antibody. No other major sites of labeling of ACE were demonstrated. In the ciliary epithelium, ACE was confined to cells bordering on the posterior chamber of the eye, similar to its presence at the ventricular surface of the choroid plexus of the brain. These findings suggest that ACE may metabolize a peptide involved in the regulation of aqueous humor production.

Animals↗

PubMed1988

Characterization of a neutral, divalent cation-sensitive endopeptidase: a possible role in neuropeptide processing.

A trypsin-like endopeptidase which cleaves the synthetic substrate Dansyl-Phe-Leu-Arg-Arg-Ala-Ser-Leu-Gly-COOH (Dansyl-Phe-Kemptide) primarily at the Arg4-Ala5 bond has been partially purified from bovine adrenal chromaffin granules, brain and liver. The enzyme appears to have a relatively homogeneous tissue distribution, although highest levels were found in brain regions such as the hippocampus and corpus striatum. Sucrose density gradient fractionation established that enzyme activity assayed at pH 8.5 is not associated with lysosomes. Purified enzyme displays a dimeric structure with subunit molecular weights of 40 kDa and 42 kDa and a native molecular weight of 85,000 Da. The endopeptidase has a neutral pH optimum, is sensitive to divalent cations and thiol reagents, and can cleave on either the amino or carboxyl side of some but not all internal basic amino acids.

Adrenal Medulla↗

PubMed1988

Enkephalin convertase: characterization and localization using [3H]guanidinoethylmercaptosuccinic acid.

Enkephalin convertase (carboxypeptidase E,H; EC 3.4.17.10) is a carboxypeptidase B-like enzyme which appears to be physiologically associated with the biosynthesis of the enkephalins and certain other peptides. We have localized enkephalin convertase in the brain and other tissues autoradiographically by labeling studies with [3H]guanidinoethylmercaptosuccinic acid ([3H]GEMSA). In the brain, [3H]GEMSA localizations parallel enkephalin distribution but with certain exceptions, suggesting a role in relation to other peptides. In the pancreas, [3H]GEMSA binding sites are localized to the islets suggesting an involvement in insulin, glucagon, or somatostatin formation. The selective concentration of [3H]GEMSA grains in cardiac atria suggests a link to atrial natriuretic factor.

Animals↗

PubMed1987

Angiotensin converting enzyme immunohistochemistry in rat brain and pituitary gland: correlation of isozyme type with cellular localization.

We have localized angiotensin converting enzyme in rat brain and pituitary gland immunohistochemically with an anti-rat lung angiotensin converting enzyme monoclonal antibody. The distribution of immunoreactive angiotensin converting enzyme is identical with that of binding sites for the angiotensin converting enzyme inhibitor, [3H]captopril. Most intense staining is in the choroid plexus and subfornical organ, with intermediate values in the caudate-putamen, globus pallidus, entopeduncular nucleus, pars reticulata of the substantia nigra, posterior pituitary and anterior pituitary. Lower levels are observed in the supraoptic and paraventricular nuclei of the hypothalamus. Within the basal ganglia angiotensin converting enzyme immunoreactivity is distributed throughout the neuropil; no cell bodies are stained, even after colchicine treatment. The punctate pattern of immunoreactivity in the anterior pituitary corresponds to the distribution of endothelial cells. The posterior pituitary is stained diffusely. Angiotensin converting enzyme is increased by 45% in the posterior lobe after pituitary stalk section, demonstrating that this diffuse staining is associated with pituicytes. Antibody specificity was demonstrated by the immunoaffinity purification of angiotensin converting enzyme to homogeneity from crude tissue extracts using anti-angiotensin converting enzyme antibody and protein A-sepharose. The apparent molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis of lung, choroid plexus and anterior pituitary angiotensin converting enzyme is 175,000. In the substantia nigra and caudate putamen, where angiotensin converting enzyme is localized to neuronal as opposed to epithelial cells, the molecular weight is 165,000. The pituicyte angiotensin converting enzyme of the posterior pituitary is 170,000 daltons.

Animals↗

PubMed1987

Enkephalin convertase in the gastrointestinal tract an associated organs characterized and localized with [3H]guanidinoethylmercaptosuccinic acid.

Enkephalin convertase (carboxypeptidase EH; EC 3.4.17.10), a carboxypeptidase B-like enzyme which processes hormone and neuropeptide precursors, has been characterized in the gastrointestinal tract, submandibular gland, and pancreas using a binding assay with [3H]guanidinoethylmercaptosuccinic acid. Binding to homogenates of the membrane and soluble fractions of stomach, small intestine, colon, and submandibular gland is saturable, with Kd values of about 2 nM. Partial purification of the membrane fractions reveals a Co+2-stimulated carboxypeptidase B activity which is not detectable in crude homogenates. In vitro autoradiography with [3H]guanidinoethylmercaptosuccinic acid localizes enkephalin convertase to the epithelial cells of the stomach, colon, and intestine, the islet cells of the pancreas, and the acinar cells of the submandibular gland. This localization contrasts to the distribution of enkephalins and other neuropeptides in the gastrointestinal tract and associated organs, suggesting that enkephalin convertase may serve functions other than neuropeptide and prohormone processing.

Animals↗

PubMed1986

Differential ontogeny of rat brain peptidases: prenatal expression of enkephalin convertase and postnatal development of angiotensin-converting enzyme.

We quantitated the levels of two peptidases in the developing rat brain as a means to determine their function. Enkephalin convertase (EC 3.4.17.10), a carboxypeptidase B-like enzyme detected by [3H]guanidinoethylmercaptosuccinic acid (GEMSA) autoradiography, is present in high concentration throughout the brains of rat fetuses 3 days prior to birth. During the first 3 postnatal weeks, the density of [3H]GEMSA-labeled enkephalin convertase drops to adult levels. The expression of enkephalin convertase prior to that of most neuropeptides supports a role for this enzyme in propeptide processing. The regional distribution of [3H]GEMSA binding is similar in fetal and adult rats except that the thalamus exhibits the highest levels of [3H]GEMSA binding prenatally, and among the lowest levels in adult rats. Thus, peptide(s) formed in high concentration in the prenatal thalamus may be substrates for enkephalin convertase. Angiotensin-converting enzyme (ACE, EC 3.14.15.1) was visualized in the perinatal period by [3H]captopril autoradiography. Striatonigral ACE is undetectable at birth and increases to adult levels by two weeks of age. The expression of ACE after the initial presence of known peptides in the basal ganglia implies that the enzyme is not essential for peptide synthesis, suggesting instead a degradative role. In contrast to the striatonigral system, the choroid plexus contains high concentrations of ACE prior to birth, consistent with previous proposals of different substrates for ACE in the choroid plexus and the basal ganglia.

Age Factors↗

PubMed1986

Angiotensin-converting enzyme localized in the rat pituitary and adrenal glands by [3H]captopril autoradiography.

We have localized angiotensin-converting enzyme (EC 3.14.5.1) in the rat pituitary and adrenal glands by [3H]captopril autoradiography. The maximal numbers of [3H] captopril-binding sites are: posterior pituitary, 4500 fmol/mg protein; anterior pituitary, 1950 fmol/mg; intermediate pituitary, less than 100 fmol/mg; adrenal medulla, 480 fmol/mg; and adrenal cortex, less than 25 fmol/mg. The distribution within the posterior pituitary and adrenal medulla is homogeneous, whereas that in the anterior pituitary is patchy. Subcellular fractionation of the bovine adrenal medulla reveals enrichment of angiotension-converting enzyme in plasma membrane fractions, but not in chromaffin granules. [3H]Captopril autoradiography in the rat pituitary gland is unaltered by dehydration, adrenalectomy, or reserpine treatment and in Brattleboro rats. [3H]Captopril binding in the adrenal medulla is increased by 75% 3 weeks after hypophysectomy and is elevated by 80% after reserpine treatment. The change after hypophysectomy is not reversed by dexamethasone treatment.

Adrenal Glands↗

PubMed1986

Characterization of angiotensin converting enzyme by [3H]captopril binding.

We demonstrate that [3H]captopril selectively labels angiotensin converting enzyme (EC 3.14.15.1) (ACE) and employ this technique to probe enzyme-inhibitor interactions. [3H]Captopril binding sites copurify with ACE activity from rat lung or rat brain. At each stage of the purification the Vmax/Bmax ratio, or kcat is 17,000 min-1 with hippuryl-L-histidyl-L-leucine as substrate. The specificity of [3H]captopril binding is apparent in the similar pharmacologic profile of inhibition in crude and pure enzyme preparations. Furthermore, binding sites and enzyme activity comigrate in gel filtration and sucrose gradient sedimentation experiments. Equilibrium analysis of [3H]captopril binding to purified ACE reveals a Bmax of 6 nmol/mg of protein (KD = 2 nM), demonstrating the presence of one inhibitor binding site per polypeptide chain. The kinetics of [3H]captopril binding are characterized by monophasic association and dissociation rate constants of 0.026 nM-1 min-1 and 0.034 min-1, respectively. The affinity of ACE for both [3H] captopril and enalaprilat is greater at 37 degrees than at 0 degree, demonstrating that these interactions are entropically driven, perhaps by an isomerization of the enzyme molecule. The ionic requirements for [3H]captopril binding and substrate catalysis differ. Chloride and bromide ion, but not fluoride, are about 100-fold more potent stimulators of binding than catalysis. When the active site Zn2+ ion is replaced by Co2+, catalysis was stimulated 2-fold, whereas binding activity was decreased by 70%.

Angiotensin-Converting Enzyme Inhibitors↗