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A rapid filter paper disk assay for cyclic AMP based on the activation of a cyclic AMP dependent protein kinase from rabbit skeletal muscle is described. This procedure permits determination of as little as 0.2 picomoles of cyclic AMP. In addition this procedure employs ATP-γ-33P instead of ATP- γ-32P.

ConclusionBy use of 30% Amberlite IRC 50 (W.A. 1) resin-exchange paper, it is possible to separate both insulin and glucagon independently from their mechanical mixtures with non-specific serum or gamma globulin.

The amino acid sequence of the porcine vasoactive intestinal octacosapeptide is His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-L ys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2. Its amino acid residues 1, 2, 6 and 7, counted from the N-terminus, are identical to those in the corresponding positions in both porcine glucagon and secretin. The residues in positions 3, 12, 13, 14 and 23 are identical to those in secretin, but not in glucagon, and position 10 is occupied by a tyrosyl and position 28 by an asparaginyl residue, like in glucagon but not in secretin. If in addition to identical amino acid residues also chemically similar residues, such as isoleucine in position 26 of the octacosapeptide, as compared to leucine in secretin and glucagon, are taken into consideration then the similarity between these three polypeptides is still more evident. At a more remote level there is some structural resemblance also between these peptides and the other four peptides from the intestinal wall, cholecystokinin-pancreozymin, motilin, the gastric inhibitory peptide and substance P, the structures of which are known. The elucidation of the structure of the octacosapeptide was facilitated by the finding that pancreatic kallikrein preferentially cleaved only one of the three bonds in its N-terminal cyanogen bromide heptadecapeptide that are susceptible to cleavage with trypsin.

The specificity of penicillopepsin was investigated with a number of different substrates. In agreement with earlier work (1), no action was observed on di- and tripeptide and ester substrates. However, the pepsin substrate carbobenzoxyglycylglycylphenylalanylphenylalanine 3-(4-pyridyl)propyl-1 ester was hydrolyzed between the two phenylalanine residues. No cleavage of homopolymers of glycine, alanine, and glutamic acid and the random copolymer of glutamic acid and tyrosine was observed. Polymers of lysine, lysine and tyrosine (2:1), and lysine, glutamic acid, and tyrosine (13:20:1) were, however, hydrolyzed giving mainly tripeptides to pentapeptides. Polylysine hydrolysis showed a pH dependence centering about a group of pK between 3 and 4. In glucagon and the S-sulfo B-chain of insulin, penicillopepsin hydrolyzed the same peptide bonds as the other acidic proteases, including pepsin and rennin, with very few exceptions. There are, however, distinct differences between the action of penicillopepsin and that of other proteases of low specificity. Peptide bonds which have a hydrophobic amino acid in the P1′ position (as defined by Berger and Schechter, Ref. 2) are preferentially cleaved by penicillopepsin. A kinetic study of the hydrolysis of bovine serum albumin showed very high rate constants for the initial cleavages. The present study shows the requirement for an extended binding site in penicillopepsin.

Summary Nineteen principal peptides of the proteolytic digest of glucagon by papain were isolated on columns of Bio-Rad AG 50 W X 2, Bio-Gel P 2, by paper chromatography and high tension electrophoresis. The localization of the peptides and the extent to which the various peptide bonds in the chain of glucagon had been split indicate that the three peptide bonds Thr 7 -Ser 8 , Leu 14 -Asp 15 and Gln 24 -Trp 25 are very sensitive to the proteolytic action of papain ; the peptide bonds Thr 5 -Phe 6 , Ser 11 -Lys 12 , Arg 17 -Arg 18 , Arg 18 -Ala 19 , Gln 20 -Asp 21 and Trp 25 -Leu 26 are less sensitive. These results confirm the studies of Berger and Schechter [2] according to which the bonds of the type A -X ↓ - Y (A = phenylalanine, valine or leucine) are frequently attacked by papain. Our results prove that tyrosine displays the same role when in the site A. Furthermore, our results confirm that peptide bonds in which lysine or arginine is involved by its carboxyl group are also split.

THE essential principle of competition for a given amount of anti-hormone between standard or unknown quantities of hormone and a fixed amount of labelled hormone in Yalow and Berson's1 radio-immunoassay has been maintained in all modifications. The modifications have simplified the apparatus and have achieved a more rapid and complete separation of free and antibody-bound radioactive hormone after the immunological reaction has taken place. This has been achieved best by double-antibody techniques2,3. The introduction of a second antibody has made these immunoassays sensitive to factors in serum and plasma4,5 which may give rise to spuriously high, or spuriously low, or even “negative” plasma values6. Another drawback is the difficulty in evaluating and correcting for the increased denaturation in serum or plasma of labelled hormone. This is inherent in modifications where free radioactive hormone cannot easily be isolated. Yalow and Berson1 called this phenomenon “incubation damage” and estimated the percentage breakdown in individual sera by incubation without anti-hormone. Their chromato-electrophoresis, as does all paper chromatography, allows for a correction, because free polypeptide hormone is isolated at the site of application. The evaluation of breakdown is important in assay of peptide hormones, particularly glucagon, because pancreatic glucagon is very rapidly destroyed in serum or plasma unless special precautions are taken.

A radioimmunoassay has been developed for glucagon. The antibodies raised in rabbits were sensitive to 0.01 ng of glueagon in a volume of 100 μl. The free and the antibody-bound125I-glucagon were separated by addition of ethanol or by paper chromatography on Whatman 3 MM. Two types of antisera were used: one that did not react, with gut GLI (glucagon-like immunoreactivity), and another one that reacted with both the pancreatic glucagon and the gut GLI, giving identical dilution curves for the two substances. Exogenous pan-creatic glueagon and gut GLI experienced a high degree of degradation during the preparation of plasma unless trasylol was added. A degradation of endogenous pancreatic and gut GLI could also be shown in the absence of trasylol. The damage to125I-glueagon occurring during incubation in plasma was eliminated by using an easy plasma extraction method yielding an approximately 80% recovery of pancreatic glueagon and gut GLI. Normal human fasting plasma extracts contained 0.26 ± 0.024 ng equiv, of pancreatic GLI and 0.38±0.048 ng equiv. of gut GLI (mean of 21 samples ± s.e.m.). During OGTT the pancreatic GLI decreased while the gut GLI increased. The specificity and validity of glueagon determinations are discussed.

Rat liver β-hydroxy-β-methylglutaryl coenzyme A reductase activity and the amplitude of the diurnal variation of this enzyme are progressively reduced to very low levels within 1 week after the onset of diabetes induced by streptozotocin. Daily insulin therapy to 7-day diabetic rats restores the activity and the amplitude of this diurnal variation in enzyme activity to near-normal levels within 4 days. Insulin also produces a rapid 2-hr stimulation of the reductase activity in diabetic rats to the level found in normal animals at that time of day regardless of the duration of diabetes. Hence, insulin is required for the diurnal rise of reductase activity in rat liver. Glucagon, dibutyryl cyclic AMP, and hydrocortisone, in contrast, markedly inhibit the diurnal rise of reductase activity in normal rats. Therefore, the relative concentrations of insulin, glucagon, and glucocorticoids are important in the regulation of the diurnal variation of hepatic reductase activity.

Chymotrypsin, trypsin, and elastase were isolated from pancreas glands of the moose (Alces alces) by acid extraction, (NH4)2SO4 fractionation, and affinity chromatography conducted sequentially on 4-phenylbutylamine (PBA) – Sepharose and lima-bean protease inhibitor (LBI) – Sepharose. The removal of chymotrypsin on PBA–Sepharose led to the coabsorption of trypsin and elastase on to LBI–Sepharose. The binding site of elastase was identified as the 'chymotrypsin-site' of LBI. Elastase and trypsin were each selectively desorbed from LBI–Sepharose. These proteases exhibited identical elution profiles during carboxymethylcellulose chromatography and had similar migration rates on polyacrylamide disc gel electrophoresis. Moose elastase contained some trypsin activity whereas the trypsin preparation was pure based on comparison of ionograms of peptides derived from the proteolytic digestion of glucagon by moose chymotrypsin, trypsin, elastase, bovine trypsin, and porcine elastase.

Biosynthesis of glucagon was studied using isolated pigeon islets of Langerhans. H3-leucine and H3-tryptophan were found to be actively incorporated into glucagon and a large molecular weight glucagon-related component. Incorporation into both components was inhibited by cycloheximide and significantly reduced by glucose. The possibility of a precursor molecule for the biosynthesis of glucagon was suggested.

Chloride and sulfhydryl activators in glucagol degradation and secretion by purified rat liver dipeptidyl aminopeptidase I /Cathepsin C/

The incorporation of phosphate into the phospholipids of rat-liver slices incubated in vitro is stimulated by adrenalin; the 3 main types of phospholipids, phosphatidylcholine, phosphatidylethanolamine and inositolphosphatides, are all influenced in the same way. This action is not obtained with glucagon. The incorporation of amines (choline and serine) is either slightly decreased or not affected by the 2 hormones. The incorporation of inositol is considerably stimulated by adrenalin but not by glucagon or by Ado-3',5'- P . These results show that not all of the metabolic actions of adrenalin on the liver are shared by glucagon nor, probably, mediated by Ado-3',5'- P .

In solutions at high pH, amino acid residues of glucagon undergo racemization without cleavage of the polypeptide backbone. This racemization process is accompanied by an unfolding of the molecule with the loss of both its antigenicity and its ability to stimulate adenyl cyclase from rat liver.

The radioimmuno assay for glucagon using two-antibody system was described in detail. The value of sensitivity ranged 0.06 to 0.60mμg on the standard curves obtained with crystalline glucagon (0 to 14mμg per ml) in buffer solution. The precision of the standard curves was 0.8 to 1.5 in per cent precipitated radioactivity for 0 to 14mμg per ml range of glucagon. Glucagon added to human plasma was recovered at the rate of 74.6 to 116.3 per cent. The glucagon level in fasting human plasma measured by this method showed a wide range of variety and even high values (2.2 to 7.1mμg/ml) were obtained in non-diabetics than in diabetics who gave the values of 1.5 to 5.6 mμg/ml. Human plasma globulin reduced the percentage of precipitated radioactivity, whereas human plasma albumin did not appear to interfere with the immunological reaction. Since human plasma globulin did not exhibit any effect upon the rate of bound radioactivity in paper chromatography, it is suggested that the human plasma globulin may inhibit the immunological reaction at the second reaction. The cross reaction between antirabbit serum goat serum (ARGS) and human plasma or plasma globulin was observed on the immunoelectrophoresis and this was regarded as one of the inhibitors. EDTA failed to reduce the inhibition. The inhibition could be minimized by the extraction of plasma glucagon using acid-ethanol acetone method, by absorption of cross reactant in ARGS by a small amount of human globulin and by the use of anti-rabbit serum globulin goat serum. Even in the assay using anti-rabbit serum globulin goat serum as the second antibody, however, the rate of precipitated radioactivity in the assay of the standard glucagon added to the pooled human plasma with the minimun glucagon was lower than expected. This suggests the presence of another inhibitor (s) in human plasma and the necessity of the use of glucagon-free human plasma in the preparation of the standard glucagon solution.

The specificity of a cathepsin D-like enzyme from bovine fibroblasts (dental pulp) has been examined using porcine glucagon, the insulin A chain ( S- sulfo ), and the insulin B chain ( S- sulfo ) as substrates. Peptides obtained from exhaustive digests were separated by chromatography and high-voltage electrophoresis and, after elution and hydrolysis, identified by quantitative amino acid analysis. The character of susceptible peptide bonds and the relative rate of their hydrolysis led to the following conclusions: 1. 1. The residues most favorable for hydrolysis are large and hydrophobic. 2. 2. Fully charged amino acids were never found as carboxyl or amino donors of the hydrolyzed bond. 3. 3. Susceptible bonds were at least two residues removed from the N- or C-terminal end of the substrate peptide. 4. 4. Some bonds possessing the above characteristics were not cleaved suggesting hindrance by “residual conformation”.

SummaryGlucagon treatment for 10 days induced rat liver phenylalanine:pyruvate amino-transferase approximately eightfold but did not result in increased urinary phenylpyruvate excretion by rats administered a phenylalanine load after they had been injected with an amount of glucagon sufficient to induce the enzyme substantially. In fact, an inverse correlation was observed between phenylpyruvate excretion and aminotransferase activity. Hepatic phenylalanine hydroxylase was not induced in rats treated for 10 days with glucagon. It is suggested that hepatic phenylalanhe:pyruvate aminotransferase does not function as such in vivo.

We have developed a sensitive double-isotope assay to measure the enzymatic synthesis of S-adenosylhomocysteine from adenosine and l-homocysteine. The enzyme is present in all rat tissues except the small intestinal mucosa. In the liver, the enzyme is found in the postmicrosomal supernatant. We studied some of the kinetic properties of the liver enzyme. At high concentration, both substrates inhibit product formation. S-Adenosylmethionine also inhibits the reaction but other metabolites of methiouine do not. The specific activity of the enzyme in liver increases in animals fed a high protein ration. Treatment with hydrocortisone and estradiol also resulted in an increase in the specific activity while administration of thyroxine led to a decrease.

Growth hormone release inhibiting hormone (GH-RIH) was infused at a rate of 1.3 mug/min for 28 hours into four patients with acromegaly, two of whom also had clinical diabetes mellitus. Growth hormone and glucagon were suppressed throughout the infusion though delayed secretion of insulin occurred in association with both meals and an oral glucose load. Glucose tolerance was improved in one diabetic patient who was taking chlorpropamide while the other required much less insulin than usual. Secretion of endogenous thyroid-stimulating hormone was lowered in one euthyroid patient on carbimazole. Luteinizing hormone, follicle-stimulating hormone, ACTH, and prolactin were not affected. Serum somatomedin levels were reduced in one patient. There was a rapid rebound of all the suppressed hormones when the infusions stopped. Longer-acting analogues of GH-RIH will be needed before long-term therapy of acromegaly or diabetes mellitus becomes possible, but such preparations should be available soon for clinical trial.

Wick chromatography has been used for assessment of iodination damage as well as for the separation of free from the antibody bound fraction of five polypeptides (insulin, A and B chains of insulin, glucagon and gastrin). The study shows that this technique should be destricted to basic polypeptides (insulin, B-chain of insulin and glucagon) and that the method cannot be applied to the radioimmunoassay of acidic polypeptides (gastrin and A-chain of insulin).

Glucagon given intravenously to 20 patients with acute diverticulitis was followed by symptomatic relief within an average of 12 hours and obviated the need for therapy with analgesics or other antispasmodics. In a similar group of patients receiving conventional treatment symptoms persisted for an average of 96 hours. The administration of glucagon had no apparent effect on abdominal symptoms which mimicked those of diverticulitis but were caused by other conditions. There was no clinically apparent difference in effectiveness between continuous infusion and intermittent injection of glucagon.

1. The permeability of human chorion laeve in vitro to 125I-labelled glucagon was investigated. The problem caused by degradation of 125I-labelled glucagon in the preparation and a means to prevent this are described. 2. The human chorion laeve was found to be impermeable to iodinated glucagon.

Twenty-three patients with obstructive jaundice were given a 50-g oral glucose tolerance test (G.T.T.) and an insulin stimulation test with oral glucose and intravenous tolbutamide and glucagon. An abnormal glucose response was as common in patients with carcinoma of the pancreas (seven out of 12) as in patients with “other” causes of obstructive jaundice (six out of 11). Though both groups had a low and delayed insulin response the insulin levels were significantly lower in patients with carcinoma of the pancreas. After the insulin stimulation test patients with obstructive jaundice who did not have carcinoma of the pancreas had a greater and significantly different insulin response from patients with pancreatic cancer. There was, however, considerable overlapping between the results of individual patients in the two groups.

The pancreatic hormone, glucagon, stimulates the net uptake of inorganic 32 P in vivo into rat liver and its incorporation into proteins of microsomes, mitochondrial membranes, and lysosomes. Incorporation into cytosolic proteins was enhanced only slightly by glucagon. More than 95% of the protein-bound phosphate is present as phosphoserine. Both the radioactivity and the total amount of protein-bound phosphate are increased after injection of glucagon. Glucagon treatment enhanced 32 P incorporation into the alcohol-ether soluble lipids of mitochondria but did not alter the relative distribution of 32 P in various phospholipid species.

The recovery of protein in preparative polyacrylamide gel electrophoresis (PAGE) is a function of protein load, buffer system (pH), and gel concentration. Recovery can be substantially improved (to about 90%) by polymerization of the gel in the presence of an extraneous protein (e.g., BSA, bacitracin) but not by addition of formamide, reducing agent, sucrose, or polyaminoacids to the gel.