Processing of the propeptide form of rat renal gamma-glutamyltranspeptidase

The biosynthesis of rat renal gamma-glutamyltranspeptidase (EC 2.3.2.2) was studied by sodium dodecyl sulfate gel electrophoresis and fluorography of specific immunoprecipitates obtained at varying times' postinjection with [35S]methionine. At 20 min postinjection 3 endo-beta-N-acetylglucosaminidase H-sensitive bands were observed representing the propeptide (Mr 75 000) large subunit (Mr 49 500) and small subunit (Mr 29 000) of transpeptidase. The alterations in Mr are consistent with removal of 6 N-linked coreoligosaccharides from the propeptide; 4 from the large subunit and 2 from the small subunit. All 3 bands became more diffuse and less endoglycosidase H-sensitive by 40 min and completely resistant by 60 min postinjection. At 20 h postinjection no propeptide remained. Thus, the primary propeptide cleavage reaction occurs prior to the loss of endoglycosidase H sensitivity while about 30% of the propeptide is processed along with the heterodimer and cleaved at a later time.     

Occurrence and neonatal development of gastrin immunoreactivity in the digestive tract of the rat

Summary The distribution of gastrin immunoreactivity in the rat gut was examined by immunochemical and immunohistochemical techniques. Gastrin occurs predominantly in the antrum proper, but gastrin is found also in the adjacent part of the oxyntic mucosa and in the duodenum. In the remainder of the gut the gastrin concentration is very low. No gastrin cells and very low gastrin concentrations are observed in the antrum at birth. The gastrin concentration as well as the number of gastrin cells increases progressively with age. The antral gastrin concentration reaches adult or near-adult values 30–40 days after birth.This study was supported by the Swedish Medical Research Council (04 X-1007), by Riksföreningen mot Cancer (660-0 IX), Landsforeningen till Kraeftens Bekampelse, Danish Medical Research Council (512-6) and Fonden for Storkobenhavn, Faeroerne og Gronland.     

Studies of isolated and enriched rat antral mucosa gastrin cells

A technique has been developed to obtain viable, isolated and enriched populations of gastrin cells (G-cells) from the rat stomach. Restricted tissue samples from a small area of the pyloric antrum known to be particularly rich in G-cells, were sequentially digested with pronase followed by mechanical agitation, to remove the epithelial cells. This technique resulted in a significant enrichment of G-cells (3-4 fold) since the surface epithelial cells and upper portions of the glands were discarded before the initial G-cell fraction was collected. These cells in suspension were then isolated from each other by gentle pipetting in a DNase containing solution and designated the crude preparation (CP). The G-cells were then purified further by separating the cells according to size by velocity sedimentation. The greatest concentration of G-cells (15-25%) was found in the fraction containing cells with diameters of 10 to 12 micrometer. The effectiveness of the technique was evaluated by counting G-cells as identified by electron microscopy and immunofluorescence and assessing gastrin activity by radioimmunoassay. All three methods indicated that cell separation by gravity velocity sedimentation enriched the G-cell population 15-20 fold over their concentration in the CP. The combined techniques of selective pronase digestion followed by gravity velocity sedimentation resulted in an isolated cell preparation containing a 50-100 fold increase of G-cells over their normal distribution in the intact gastric mucosa. Since these isolated G-cells retain features indicating viability, their usefulness for in vitro studies is suggested.     

Effects of amino acid analogues on protein degradation in isolated rat hepatocytes

Analogues and derivatives of six of the amino acids which most effectively inhibit protein degradation in isolated rat hepatocytes (leucine, asparagine, glutamine, histidine, phenylalanine and tryptophan) were investigated to see if they could antagonize or mimic the effect of the parent compound. No antagonists were found. Amino alcohols and amino acid amides tended to inhibit protein degradation strongly, apparently by a direct lysosomotropic effect as indicated by their ability to cause lysosomal vacuolation. Amino acid alkyl esters and dipeptides inhibited degradation to approximately the same extent as did their parent amino acids, possibly by being converted to free amino acids intracellularly. Of several leucine analogues tested, four (L-norleucine, L-norvaline, D-norleucine and L-allo-isoleucine) were found to be as effective as leucine in inhibiting protein degradation. None of the analogues had any effect on protein synthesis. Since leucine appears to play a unique role as a regulator of bulk autophagy in hepatocytes, the availability of active leucine agonists may help tj elucidate the biochemical mechanism for control of this important process.     

Isolation and characterization of the intact gastrin precursor from a gastrinoma

Antibodies to the extreme C-terminal region of human progastrin have been used to monitor the isolation of high-M_r immunoreactive material in a gastrinoma extract. Microsequence analysis of the product revealed amino acid residues in the first 18 positions corresponding to those predicted from the cDNA sequence for preprogastrin starting at position 22; the sequence and immunochemical data together allow the identification of this material as intact progastrin. Implications for gastrin biosynthesis are discussed.     

Human heart cytochrome c oxidase subunit VIII. Purification and determination of the complete amino acid sequence

Subunit VIII was purified from a preparation of the human heart cytochrome c oxidase and its complete amino acid sequence was determined. The sequence proved to be much more related to that of the bovine liver oxidase subunit VIII than to that found in bovine heart. Our finding of a ‘liver-type’ subunit VIII in the human heart enzyme suggests that either there are no isoforms of human subunit VIII or the human oxidase does not show the type of tissue specificity that has been reported for the oxidase in other mammals.     

The effect on amino acid transport of trypsin treatment of rat renal brush border membranes

Trypsin treatment of isolated rat renal brush border membrane vesicles which preferentially releases l-leucine aminopeptides (EC 3.4.11.2) decreases their ability to take up a variety of amino acids under Na⁺-gradient conditions. Such treatment did not alter the osmotic properties of the vesicles nor affect their fragility. A linear correlation could be demonstrated between the l-leucine aminopeptidase activity of the membranes and the initial rate of uptake of l-leucine and l-proline. Velocity of uptake-concentration dependence studies with these substrates indicate that the major effect of trypsinization is to decrease the maximum velocity (V_max1) of the low-K_m high-affinity system with little effect on the V_max2 of the high-K_m low-affinity transport process and no effect on the apparent Michaelis constants of either. Although the data indicate that l-leucine aminopeptidase activity and uptake of l-leucine and l-proline are affected in parallel, they should not be construed to imply a role of the enzyme in the transport process, especially in view of the global decrease in the uptake of various amino acids and sugars.     

Complete amino acid sequence of human seminal plasma beta-inhibin. Prediction of post Gln-Arg cleavage as a maturation site

The complete sequence of a 94 amino acid human seminal plasma polypeptide exhibiting inhibin-like activity is presented. This molecule, called beta-inhibin, selectively and specifically suppresses the release of pituitary FSH in vivo as well as in vitro. It does not affect the secretion of LH. Such a novel acidic protein contains a very basic C-terminal segment which is easily cleaved by mild tryptic digestion. It is predicted that the FSH inhibiting activity may reside within this region of the molecule. This would imply a post Gln-Arg cleavage to release the basic C-terminal active moiety.     

Prosomatostatin is proteolytically processed at the amino terminal segment by subtilase SKI-1

Processing of prohormones to generate active products typically occurs at basic residues via cleavage by proprotein convertases. A less common type of cleavage is mediated at hydrophobic (L, V, F, N) or small amino acid (A, T, S) residues. Efforts to identify the proteinases responsible for processing precursors at their hydrophobic amino acids has led to the recent cloning of a new type-1 membrane-bound subtilase called SKI-1. The NH₂-terminal region of prosomatostatin, previously shown to contain a sorting signal for the regulated secretory pathways, is processed to generate PSST_[1–10]. The exact cleavage mechanism is unknown, but has been assumed to involve monobasic processing at Lys¹³ followed by carboxypeptidase trimming. We found that K13A mutation did not block PSST_[1–10] production. Since the prosomatostatin sequence R⁸–Q⁹–F¹⁰–L¹¹↓ qualifies as a potential SKI-1 substrate, using a vaccinia virus expression system along with HPLC and radioimmunoassays, we observed that overexpression of recombinant SKI-1 in COS-1 and HEK-293 cells significantly increased the production of PSST_[1–10]. Additionally, in CHO cells lacking SKI-1, there was a significant reduction in PSST_[1–10] production which could be increased upon SKI-1 stimulation. Mutagenesis studies showed that efficient processing of PSST to PSST_[1–10] required the RXRXXL motif. However, this NH₂-terminal cleavage was not a prerequisite for the formation of SST-14 and SST-28.     

Vagally induced release of gastrin, somatostatin and bombesin-like immunoreactivity from perfused rat stomach. Effect of stimulation frequency and cholinergic mechanisms

The isolated stomach of rats was vascularly perfused to measure the secretion of gastrin, somatostatin (SLI) and bombesin-like immunoreactivity (BLI). The gastric lumen was perfused with saline pH 7 or pH 2, and electrical vagal stimulation was performed with 1 ms, 10 V and 2, 5 or 10 Hz, respectively. Atropine was added in concentrations of 10⁻⁹ or 10⁻⁷ M to evaluate the role of cholinergic mechanisms. In control experiments, vagal stimulation during luminal pH 2 elicited a significant increase of BLI secretion only at 10 Hz but not at 2 and 5 Hz. Somatostatin release was inhibited independent of the stimulation frequency employed. Gastrin secretion at 2 Hz was twice the secretion rates observed at 5 and 10 Hz, respectively. At luminal pH 7 BLI rose significantly at 5 and 10 Hz. SLI secrtion was decreased by all frequencies. Gastrin secretion at 2 and 5 Hz was twice as high as during stimulation with 10 Hz. Atropine at doses of 10⁻⁹, 10⁻⁸, 10⁻⁷ and 10⁻⁶ M had no effect on basal secretion of BLI, SLI and gastrin. At luminal pH 2, atropine increased dose-dependently the BLI response at 2 and 5 but not at 10 Hz. The decrease of SLI during 2 and 5 Hz but not 10 Hz was abolished by atropine 10⁻⁹ M. SLI was reversed to stimulation during atropine 10⁻⁷ M at all frequencies. The rise of gastrin at 2 Hz was reduced by 50%. At luminal pH 7, atropine had comparable effects with a few differences: the BLI response at 10 Hz was augmented and the gastrin response to 2 and 5 Hz was reduced. In conclusion the present data demonstrate a frequency and pH-dependent stimulation of BLI and gastrin release. The stimulation of BLI is predominantly due to atropine-insensitive mechanisms while muscarinic cholinergic mechanisms exert an inhibitory effect on BLI release during lower stimulation frequencies (2 and 5 Hz) independent of the intragastric pH and also during higher frequencies at neutral pH. Both, atropine sensitive and insensitive mechanisms are activated frequency dependent. The atropine-sensitive cholinergic mechanisms but not the noncholinergic mechanisms involved in regulation of G-cell function are pH and frequency dependent. Somatostatin is regulated largely independent of stimulation frequency and pH by at least two pathways involving cholinergic mechanisms of different sensitivity to atropine. These data suggest a highly differentiated regulation of BLI, gastrin and SLI secretion and the interaction between these systems awaits further elucidation.     

Evidence for a change in catalytic properties of glyceraldehyde 3-phosphate dehydrogenase monomers upon their association in a tetramer

The complete amino acid sequence of human spleen apoferritin has been determined. It consists of 174 amino acids, corresponding to M_r20017. The sequence is very similar to that of horse spleen apoferritin (14% difference between the two sequences). Some peptides were isolated and sequenced which could not be placed in the sequence but which are homologous with part of the principal sequence. Automatic sequence determination of a large peptide resulting from acid cleavage allows us to establish the presence of two homologous sequences (in the ratio $\text{80}\text{20}$).     

Synergistic action of gastrin and ghrelin on gastric acid secretion in rats

Gastrin and ghrelin are secreted from G cells and X/A-like cells in the stomach, respectively, and respective hormones stimulate gastric acid secretion by acting through histamine and the vagus nerve. In this study, we examined the relationship between gastrin, ghrelin and gastric acid secretion in rats. Intravenous (iv) administration of 3 and 10 nmol of gastrin induced transient increases of ghrelin levels within 10 min in a dose-dependent manner. Double immunostaining for ghrelin and gastrin receptor revealed that a proportion of ghrelin cells possess gastrin receptors. Although (iv) administration of gastrin or ghrelin induced significant gastric acid secretion, simultaneous treatment with both hormones resulted in a synergistic, rather than additive, increase of gastric acid secretion. This synergistic increase was not observed in vagotomized rats.These results suggest that gastrin may directly stimulate ghrelin release from the stomach, and that both hormones may increase gastric acid secretion synergistically.     

Characteristics of gastrin controlled ECL cell specific gene expression

BACKGROUND: The ECL cells are histamine-producing endocrine cells in the oxyntic mucosa that synthesize and secrete proteins and peptides. They are the primary target for gastrin and mediate the control of gastrin on acid secretion and oxyntic mucosal growth. Knowledge of the molecular biology of the ECL cell is therefore important for understanding gastric physiology. Accordingly, we wanted to identify genes that are characteristically expressed in the ECL cells and controlled by gastrin. METHODS: Using Affymetrix GeneChips, RNA expression profiles were generated from ECL cells isolated by counterflow elutriation from hyper- or hypogastrinemic rats. Contamination from non-endocrine cells was eliminated by subtraction of the expression profiles of the fundic and antral mucosa. RESULTS: The expression of 365 genes was ECL cell characteristic. Gastrin was found to control the expression of 120 which could be divided into two major groups depending on the known or anticipated biological function of the encoded protein: genes encoding proteins involved in the secretory process and genes encoding proteins needed to generate energy for secretion. Interestingly, gastrin stimulation also increased ECL cells expression of anti-apoptotic genes. CONCLUSION: The ECL cell specific expression profile is reminiscent of that of neurons and other endocrine cells exhibiting high expression of genes encoding proteins involved in the synthesis, storage and secretion of neuropeptides or peptide hormones. Gastrin regulated the expression of one third of these genes and is thus involved in the control of secretion from the ECL cells.     

High potency of bombesin for stimulation of human gastrin release and gastric acid secretion

Dose-response studies were performed in 6 human volunteer subjects to determine the threshold and optimal doses of intravenous bombesin for stimulation of gastric acid secretion and gastrin release. A significant stimulation of both acid and gastrin was obtained with a very low dose, 3 pmol · kg^?1 · h^?1. Peak stimulation of acid secretion (67% of pentagastrin PAO) was obtained at 12.5 pmol · kg^?1 · h^?1. Serum gastrin response to this dose of bombesinn was similar to that obtained after a high protein meal. Higher doses of bombesin caused further increases in serum gastrin but not in acid secretion. Since very low doses of bombesin, too small to produce detectable increases in immunoreactive serum bombesim, caused parallel increases in gastrin and acid secretion, it is possible that the bombesin-like peptides present in human gastrointestinal tissues contribute to regulation of human gastric secretion.     

Nonrandom amino acid substitution and estimation of the number of nucleotide substitutions in evolution

Summary A method of estimating the number of nucleotide substitutions from amino acid sequence data is developed by using Dayhoff's mutation probability matrix. This method takes into account the effect of nonrandom amino acid substitutions and gives an estimate which is similar to the value obtained by Fitch's counting method, but larger than the estimate obtained under the assumption of random substitutions (Jukes and Cantor's formula). Computer simulations based on Dayhoff's mutation probability matrix have suggested that Jukes and Holmquist's method of estimating the number of nucleotide substitutions gives an overestimate when amino acid substitution is not random and the variance of the estimate is generally very large. It is also shown that when the number of nucleotide substitutions is small, this method tends to give an overestimate even when amino acid substitution is purely at random.     

Developmental gene expression of gastrin receptor in rat stomach

Gastrin, which is present in fetal plasma, may have important roles in the development of gastric mucosa, since it is not only a potent stimulator of gastric acid secretion but also a growth promoting factor. Gastrin regulates various cellular functions via its receptors on cell membrane. Therefore, in order to elucidate a role for gastrin in the development of gastrointestinal system during gestation, Northern blot analysis was performed. The results of the study suggested that gastrin receptor is mainly present on parietal cells. Furthermore, proton pump and gastrin receptor gene expressions in parietal cells were strongly stimulated by the administration of exogenous gastrin. In conclusion, gastrin may be involved in the developmental change of parietal cells through its receptors.     

The complete amino acid sequence of the pediocin-like antimicrobial peptide leucocin C

The pediocin-like antimicrobial peptide leucocin C produced by a strain of Leuconostoc mesenteroides has been purified using a recently developed rapid two-step procedure. The complete and corrected amino acid sequence of the peptide has been determined by Edman degradation of the intact peptide and a C-terminal fragment generated by cleavage with Asp-N endoprotease. Leucocin C contained 43 residues with the following sequence: KNYGNGVHCTKKGCSVDWGYAWTNIANNSVMNGLTGGNAGWHN. The molecular weight of leucocin C as determined by mass spectrometry was 4595, which is consistent with the theoretical molecular weight of 4596 calculated from the sequence. Moreover, the molecular weights of the two fragments generated by cleavage with Asp-N were also consistent with the determined sequence.     

Sequence analysis and feeding responses evoked by the large molecular form of gastrin releasing peptide (GRP) in the rat GRP-29

Microisolation techniques utilizing several reverse phase high performance liquid chromatography (HPLC) steps have resulted in the purification of two rat gastrin releasing peptide (GRP) forms suitable for microsequence and mass spectral analysis. The sequence of the larger form is APVSTGAGGGTVLAKMYPRGSHWAVGHLM-amide and the smaller form is GSHWAVGHLM-amide which is the carboxyl terminal decapeptide of the larger peptide. The peptides were synthesized and their feeding patterns e.g. first meal size (MS), intermeal interval (IMI) and satiety ratio (SR, IMI/MS) were determined in overnight food-, but not water deprived, male Sprague Dawley rats. The peptides were administered in the femoral vein (0, 0.21, 0.41 and 1.03 nmol/kg) immediately before presenting the rats with a 10% sucrose solution. We found that (1) GRP-10 (all doses) and GRP-29 (0.41 nmol/kg) reduced first MS, (2) both peptides prolonged IMI length and (3) both peptides increased the SR to similar extents. In conclusion, GRP-10 and GRP-29 are the two endogenous forms of GRP in the rat intestine and they reduce short term feeding to similar extents when administered intravenously.