Ruth S. Gurd

Human glucagon-like peptides 1 and 2 activate rat brain adenylate cyclase.

Two human glucagon‐like peptides, GLP‐1 and GLP‐2, which are coencoded with pancreatic glucagon in the preproglucagon gene, do not significantly inhibit [125I]monoiodoglucagon binding to rat liver and brain membranes and do not activate adenylate cyclase in liver plasma membranes. Nevertheless, GLP‐1 and GLP‐2 were each found to be potent stimulators of both rat hypothalamic and pituitary adenylate cyclase. Only 30–50 pM concentrations of each peptide elicited half‐maximal adenylate cyclase stimulation. Our data suggest that GLP‐1 and GLP‐2 may be neurotransmitters and/or neuroendocrine effectors, which would account for their high degree of sequence conservation through vertebrate evolution.

Separation of adrenergic and cholinergic synaptosomes from immature rat brain

The modern quantitative molecular neurobiology had its inception with the isolation and characterization of synaptosomes in the laboratories of Whittaker and of DeRobertis [ 1, 21. These pinched off terminal boutons of axons, frequently still retaining subsynap tic and postsynaptic elements, constitute the most appropriate starting material for the purification of the various molecules and supramolecular aggregates confined by and defining the synapse. Synaptosomes are usually isolated by a combination of rate and equilibrium sedimentation techniques on sucrose or Ficoll gradients. They consist of a mixed population varying, for instance, in the identities of their chemical transmitters. Clearly, inquiries into synaptosomal structure, function, and biogenesis would be facilitated if this population could be subfractionated into defined types according to the transmitters and the cognate enzymes they contain. This communication shows that synaptosomes from cerebral cortices of 1.5 day old

Biological activities of catfish glucagon and glucagon-like peptide

The ability of catfish glucagon and glucagon-like peptide to bind and activate mammalian glucagon receptors was investigated. Neither catfish peptide binds to glucagon receptors of rat liver, hypothalamus or pituitary. Neither stimulates adenylate cyclase activity in liver membranes. Catfish glucagon fails to activate adenylate cyclase in hypothalamic or pituitary membranes in contrast to mammalian glucagon. However, catfish glucagon-like peptide does stimulate hypothalamic and pituitary adenylate cyclase (EC50 approximately 1 pM) possibly through mammalian glucagon-like peptide receptors.

Ultrapure cyanogen bromide-cleaved glucagon: Isolation in high yield by ion-exchange chromatography☆

Abstract Cyanogen-bromide cleaved glucagon has been extensively purified in yields of 80–85% by the use of gel filtration and by cation-exchange chromatography at pH 4.5–5.2. This pH range maintains a charge difference between the holohormone and its cleavage product, the truncated homoserine lactone derivative, yet maintains the integrity of the lactone ring. Purity is determined by the lack of methionine and the presence of homoserine following peptide hydrolysis. The homoserine lactone is opened by treatment with 0.2 n triethylamine at pH 9.5. The lactone can be reformed by treatment with trifluoroacetic acid for 1 h at room temperature although protection against photooxidation of tryptophan-25 must be provided. The homoserine lactone form binds less well to glucagon receptors than does the homoserine form. Adenylate cyclase is activated by the lactone to an extent comparable to that obtained by native hormone but at elevated concentrations. The procedures described may be useful for purification of other cyanogen bromide cleavage products and is useful for semisynthetic methods based upon cyanogen bromide-cleaved derivatives of glucagon.

Quantitative analysis of internalization of glucagon by isolated hepatocytes

Biochemical methods have been used to quantitate total, acid-stable and acid-labile association of (mono[125I]iodoTyr10) glucagon with rat hepatocytes in suspension to evaluate internalization of glucagon and its receptors. Internalization is inhibited by low temperature, phenylarsine oxide, and by blocking receptor binding, consistent with receptor-mediated endocytosis. Approximately 30% of the total cell-associated hormone is internalized at 30 min of incubation. The rate declines until 90 min when the internalization of glucagon ceases, although the cells remain competent to internalize asialofetuin. From 90 min to 4 h, 27% of the maximum label internalized at 30 min remains within cells. The number of cell surface receptors decreases but the affinity of those remaining is unchanged. However, 1.7-2.7 surface receptors are lost to binding for each molecule of radiolabeled glucagon internalized. Uptake occurs according to a rate constant of 0.183 min-1 (t1/2 = 3.8 min). We conclude that (i) hepatocytes internalize a finite quantity of glucagon, implying the existence of undefined regulatory mechanisms; (ii) hormone is retained for greater than 2 h within cells and may play a physiological role within cells; and (iii) both occupied and unoccupied receptors become inaccessible to extracellular hormone as internalization proceeds; rapid recycling of receptors does not occur.

Guanine nucleotide regulation of the interconversion of the two-state hepatic glucagon receptor system of rat

To investigate whether guanine nucleotides regulate interconversion of the two-state hepatic glucagon receptor we have utilized kinetic assays of glucagon binding to partially purified rat liver plasma membranes. Dissociation of glucagon at 30 degrees C exhibited biexponential character in either the absence or presence of GTP, indicating that the system previously seen in intact hepatocytes is independent of intracellular modulators. In each case the receptors underwent a time-dependent conversion from a low affinity to a high affinity state. However, GTP decreased the fraction of receptors in the high affinity state. The rank order for stabilizing the low affinity state was Gpp(NH)p greater than GTP greater than GDP much greater than GMP = no nucleotides. Data from competition binding assays with increasing concentrations of GTP allow calculation of equilibrium constants which are 3.32 nM for glucagon and receptor in the absence of GTP, 18.6 nM for glucagon and receptor in the presence of GTP, 1.55 microM for the association of receptor and GTP presumably linked to an N protein, and 8.86 microM for the association of the glucagon-receptor complex and GTP again presumably linked to an N protein, Glucagon binding to receptor is noncooperative in both the absence and presence of GTP, distinguishing this system from the beta-adrenergic system. With GTP, binding to the low affinity state is favored because of the relative affinities reported. Therefore, GTP regulates the activation by slowing the conversion of the receptor from a low affinity to high affinity form.

Differences in protein patterns on polyacrylamide-gel electrophoresis of neuronal membranes from mice of different strains

OVER 400 mutant strains of mice have been catalogued, including many neurological mutants associated with pathological development of definite areas of the brain (SIDMAN, GREEN and APPEL, 1965; GREEN, 1966). Within the more nearly normal range, certain strains show definite variations in behavioural characteristics (WIMER and FULLER, 1966; GREEN, 1968). In our experiments, strains C57B1/6J, C3HeB/FeJ and DBA/U were used. The first two are at opposite extremes in response to chlorpromazine (HUFF, 1962); C57 and DBA have been reported to display genetically determined differences in learning ability (BOVET, BOVET-NITTI and OLTVERIO, 1969), but the factors responsible for such behavioural differences may require re-interpretation (WERNER, SYMINGTON, FARMER and SCHWARTZKROIN, 1968). We used male animals, 7-10 weeks old. In any pair of experiments the age difference was 5 2 days and animals were maintained under uniform conditions. Electrophoresis was carried out in 7.5 per cent polyacrylamide gels crosslinked with 0.15 per cent ethylene diacrylate (TAKAYAMA, MACLENNAN, TZAGALOFF and STONER, 1966). Samples of membrane fractions obtained from brain tissue (approx. 100 pg protein per sample) were resuspended in 0.25 M-mercaptoethanol and after 1 h at 20°C dissolved in a medium containing by volume two parts phenol, one part glacial acetic acid and one part 5.0 M-urea (COTMAN and MAHLER, 1967). Electrophoresis proceeded at 100 V for 3.0 h in glass tubes 65 mm long by 5 mm i.d. The gels were stained with amido black and destained by diffusion in 7 per cent acetic acid. In the preliminary experiments, sets of three to six animals were decapitated under ether anaesthesia and the brains separated into cerebral cortex, cerebellum and a residual region called ‘stem’. Samples of 0.4 g tissue were homogenized and fractionated by centrifugation on discontinuous sucrose gradients by a standard procedure (WHIITAKER and GRAY, 1962). Samples consisting mainly of mitochondria, of synaptosomes (pinched-off nerve endings) and of myelin were. examined by gel electrophoresis. We could not distinguish any differences between material from C57 and C3 mice when comparisons were made of corresponding subcellular fractions from the same brain region. The gel patterns derived from DBA animals, however, differed from C57 or C3 in the synaptosomal fractions, the differences being most marked in comparisons of material from the ‘stem’ region. Therefore, subsequent work focused on a detailed comparison between DBA and C57 strains and used fractions obtained by zonal ultracentrifugation in an approximately exponential sucrose gradient. This fractionation technique has been developed and standardized in our laboratory with cerebral cortex of rat brains; only minor mod& cations were made of the published techniques (COTMAN, MAHLER and ANDERSON, 1968; MCBRIDE, MAHLER, MOORE and WHITE, 1971). Optical density profiles of the zonal effluents as a function of sucrose density are shown in Fig. 1 for the brain stem membrane fractions P2L (lysed P2). The subsequent comparisons between electrophoretic patterns were made with samples from identical narrow ranges of sucrose concentration, which should, therefore, represent subcellular particulates of nearly identical isopyknic densities. Electron microscopic examination of such pairs of samples indicated that they were indistinguishable in gross morphology as well as in distribution of subcellular organelles and membraneous entities. The gradient fractions at isopyknic densities between 1.165 g and 1.185 g ~ m ~ contain mainly mitochondria. The electrophoretic patterns obtained on four fractions taken at the same isopyknic density within the mitochondrial range from two zonal preparations, each on 10 DBA and 10 C57 animals, are always virtually identical. From such data, we can conclude that neither qualitative nor quantitative differences are indicated between the mitochondria1 proteins of the two strains on the basis of the methods of examination employed. The electrophoretic separation, in the acidic, strongly denaturing medium used, depends upon both charge and size of the migrating entities and the observed bands may be due to association and/or dissociation products as well as undegraded proteins. Although one cannot interpret these patterns in terms of individual polypeptide units of welldefined mol. wt. differences and congruences in the patterns are sensitive indicators of the distributions of proteins and polypeptide components in the fractions examined.

Structure-function relationships of S-carboxymethyl methionine27 glucagon

Carboxymethylation of glucagon and subsequent purification of the hormone has provided a derivative modified by the addition of bulk to the methionine at position 27 without a net charge alteration in the side chain. Unreacted glucagon was removed after methylation of the methionine which provides a positively charged chromatographic handle. The derivative has a half-maximum concentration for binding of 5.3 nM and is a full agonist. These findings along with those provided by methylation of the methionine indicate that a positive charge rather than bulk on the methionine side chain disrupts the binding of hormone to its receptor. The S-carboxymethyl derivative lacks the concentration-dependent aggregation characteristic of glucagon at pH 10.2 as does the S-methyl derivative but increases its helical content in 30% 2-chloroethanol to the same extent as native and S-methyl hormone. Full activity of the S-carboxymethyl methionine27 glucagon does not favor the existence of the globular structure proposed by Korn and Ottensmeyer [(1983) J. Theor. Biol. 105, 403] as the binding species whereas multiple considerations do favor a flexible hormone with nucleation followed by conformational changes for complete binding and activation. Isotopic enrichment using labeled iodoacetate is feasible and can provide more definitive structural information.

Nα-Malto-glucagon and Nα-malto, S-methyl methionine27-glucagon: Preparation and characterization of two partial agonists☆

Abstract N α -Maltoglucagon was prepared by demethylation of N α -malto, S -methyl methionine 27 glucagon, and the two derivatives were purified to > 99% and 99.7%, respectively. S -Methylation of glucagon lowers the reactivity of Lys-12 and provides an alternative strategy to ϵ-amino protection for directing glycosylation of glucagon to the α-amino group. Both derivatives are partial agonists, with their adenylate cyclase activation and binding reduced in parallel. N α -Maltoglucagon produces 70% and N α -malto, S -methyl methionine 27 glucagon 40% of the maximum activity of native hormone. N α -Maltoglucagon binds equivalently to N α -biotinyl, N ϵ -acetimidoglucagon whose maximum activity is near 35%, but a p K shift of the imidazole moiety cannot account for the difference in their abilities to produce transduction. Both glycosylated derivatives bind noncooperatively and both inhibit adenylate cyclase at high concentrations. The presence of a maltose residue on the amino terminal of glucagon may be required but, alone provides insufficient structural complementarity for concanavalin A binding to occur. The glycosylated derivatives are resistant to aminopeptidase degradation, are more soluble, and the maltose residue is unlikely to cause toxicity with in vivo use. Such attributes may be advantageous in the development of other analogs.