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The development of a long-term research program on the neurosecretory hormones of arthropods is described. The purification and full characterization of the first invertebrate neurohormones, the red pigment-concentrating hormone (RPCH) and the distal retinal pigment hormone (DRPH) demonstrated that they are peptides, an octapeptide and an octadecapeptide, respectively. Physiological function studies with the pure hormones and their synthetic preparations showed that the RPCH acts as a general pigment-concentrating hormone (PCH), and that the DRPH, in addition to its light-adaptive function, also constitutes a general pigment-dispersing hormone (PDH). In the regulation of the color-adaptation of the animals, the two hormones act as antagonists. The chromatophorotropic activities are widely distributed within the arthropod neuroendocrine systems. Purification of the pigment-concentrating activities from the locust corpora cardiaca lead to the isolation and characterization of the first insect neurohormones, the adipokinetic hormones (AKH I and AKH II). These two hormones, AKH I being a decapeptide and AKH II being an octapeptide, are close structural analogs to the crustacean PCH, demonstrating a common evolution of arthropod neurohormones. The hormones of this PCH-family all cross-react, but structure-function studies of the hormones show that quite different parts of their structure are involved in their binding to the various receptors.

The effects of either cation removal or ionic channel blockade were determined on the dose-response curve (DRC) to PCH (pigment-concentrating hormone) in Macrobrachium potiuna erythrophores. In sodium-, potassium- and calcium-free salines, the pigment-aggregating responses to PCH were depressed; in the former condition, maximal aggregation was not achieved and the slope of the regression curve determined from the DRC was significantly different from control. Tetrodotoxin, verapamil or tetraethylammonium (TEA) treatments also diminished the pigment-aggregating responses to PCH, and the slopes of the regression curves were different from control in the presence of 10 −6 M verapamil or 10 −6 M TEA. Interestingly, the DRC determined in the absence of both sodium and calcium ions was not significantly different from control. When verapamil was applied in sodium-free conditions, maximal aggregation was prevented. The erythrophore resting membrane potential ranged from −62 mV to −78 mV and did not vary during PCH-induced pigment aggregation as compared to the control. Our results suggest that transient modifications of potassium equilibrium potential may interfere with PCH signal transduction, revealing a more relevant role of potassium in the process, and that a sodium influx and an intracellular calcium mobilization are necessary to maintain a cytosolic balance between the ions for normality of PCH-induced responses.

Purification of the first dipeptidases, glycylleucine dipeptidase (EC 3.4.13.2) and proline dipeptidase (EC 3.4.13.9), from intestine, have shown them to be true dipeptidases, hydrolysing only dipeptides in their laevo form. Although they have quite different specificities they show great similarities in their chemical and physicochemical properties. Specific antibodies against the two dipeptidases have been raised and used in combination with the double-layer immunofluorescent staining technique to study their histological localization in the small intestine. The results conclusively demonstrated that both glycylleucine dipeptidase and proline dipeptidase are exclusively located in the cytosol of the enterocytes. This location fits well with the current idea of transport and hydrolysis of dipeptides in the intestine where the dipeptides are taken by a specific transport mechanism and are hydrolysed intracellularly. Although it is tempting to add a specific role for the dipeptidases of the enterocytes in the final digestion of exogenous proteins, studies so far have shown their identity with the corresponding dipeptidases of other tissues. It is therefore suggested that their role in the digestion process may be based entirely on their abundance in the intestine.

An in vitro crustacean (freshwater shrimp, Macrobrachium potiuna) erythrophore bioassay for chromatophorotropins and other pigment cell agonists is described. The present assay is a quantitative method that determines the pigment responses with the aid of an ocular micrometer. The pigment granules within the erythrophores are dispersed out into the dendritic processes of the cells when the isolated carapace is placed in physiological solution. This bioassay provides, therefore, a method for measuring the response of the pigment cells to aggregating agents such as pigment concentrating hormone (PCH). This bioassay is sensitive to PCH at a concentration as low as 3 x 10(-12) M. Calcium ionophore A23187 mimics the actions of PCH, but, unlike the hormone, the ionophore-induced pigment aggregation is irreversible after physiological solution rinses. Therefore, chromatophorotropic activities of pigment dispersing agents, such as pigment dispersing hormones (PDH), can be determined on ionophore-treated erythrophores. The potencies of alpha-PDH and beta-PDH show a threefold difference (not significant). Because of its convenience and its ability to make an objective determination of the bidirectional pigment movements within erythrophores, this bioassay is a suitable method for further structure-activity studies of the various chromatophorotropins and their analogs.

The cellular system responsible for the transduction of the pigment-concentrating hormone (PCH) signal was investigated in erythrophores of the freshwater shrimp, Macrobrachium potiuna. Dose-response curves to the hormone were determined in the absence and in the presence of several drugs that affect sequential steps of the Ca(2+)-dependent signalling pathway. Additionally, the ability of forskolin to induce pigment dispersion was evaluated. Neomycin sulphate (10(-4) and 10(-3) mol.l-1), trifluoperazine (10(-5) and 10(-4) mol.l-1), 1-(5-isoquinolinesulfonlyl)-2-methylpiperazine (10(-7) and 10(-5) mol.l-1) and okadaic acid (10(-7) mol.l-1) significantly (P0.05) decreased the responses to PCH. However, okadaic acid at low concentration (10(-9) mol.l-1) and cyclosporin A (10(-6) and 10(-5) mol.l-1) did not significantly (P0.05) affect PCH activity. Forskolin (10(-4) mol.l-1) was able to half-maximally reverse the hormone-induced aggregation. Our results suggest that the pigment-concentrating hormone induces pigment aggregation through a Ca(2+)-dependent pathway with a posteriori phosphatase activation, probably the serine/threonine phosphatase 1.

The red pigment-concentrating hormone of the eyestalks of the prawn Leander adspersus has been purified. It is an octapeptide with the following amino acid composition: Asp1, Glu1, Gly1, Leu1, Phe1, Pro1, Ser1, Trp1. The complete identity in purification behavior, in amino acid composition, in electrophoresis, and in partition chromatography with the previously purified red pigment-concentrating hormone from the prawn Pandalus borealis, strongly indicate structural identity between the hormones of the two species.

Purification of prolidase (imidodipeptidase, EC 3.4.3.7) from pig small intestine is described. Two alternative purification procedures have been developed, one of which utilizes the capability of the enzyme to aggregate to high molecular weight complexes and allows purification of 20 mg of prolidase in each preparation. The purity of the prolidase was found to be more than 99% when tested on polyacrylamide electrophoresis, and it showed homogeneity in crossed immunoelectrophoresis. The enzyme has a specific activity of about 200 units of activity per mg protein when determined against l -alanyl- l -proline. The specificity of the enzyme is in accordance with the original definition of an imidodipeptidase, as its activity is limited to dipeptides with the constitution aminoacyl- l -proline or aminoacyl- l -hydroxyproline, and it has no activity against tripeptides.

The red pigment-concentrating hormone (RPCH) from crustacea is an octapeptide with the following structure: pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH 2 , [4-Tyr]RPCH has been synthesized and was 4 times as active as RPCH in the Leander assay. Iodination of the hormone analog using chloramin-T, lactoperoxidase, solid-state lactoperoxidase, and thallium chloride was carried out and the results were compared. 125 I-labeled [4-Tyr]RPCH was prepared using a modificated chloramin-T method and was purified from unlabeled and diiodinated peptide by chromatography on Sephadex LH-20.

A new peptide with both adipokinetic activity in the locust and red pigment-concentrating activity in the shrimp, can be readily separated from the adipokinetic hormone in extracts of corpora cardiaca (CC) from Schistocerca americana gregaria by the use of gel chromatography and the Leander adspersus bioassay. The new peptide accounts for 20% of the total biological activity in locust CC, and it is located principally in the CC glandular lobe. The amino acid composition of the pure peptide is: Asp, Thr, Ser, Glu, Gly, Leu, Phe, Trp. This composition is similar to, but not identical with the red pigment-concentrating hormone of the shrimp.

The crustacean pigment-translocating hormones, the red pigment-concentrating hormone (RPCH), an octapeptide, and the light-adapting distal retinal pigment hormone (DRPH), an octadecapeptide, are the first invertebrate neurohormones to be fully characterized. Studies with both purified and synthetic hormones show that, in certain decapods, RPCH is a general pigment-concentrating hormone (PCH), affecting the pigments of all kinds of chromatophores (erythrophores, xanthophores, leucophores and melanophores); the DRPH seems to serve not only light-adapting function, but also act as a general chromatophore pigment-dispersing hormone (PDH). The two hormones thus function as antagonists when regulating the color-adaptation of the decapod crustaceans. PCH activity is widely distributed within the arthropod endocrine systems. The first characterized insect neurohormones, the locust adipokinetic hormones (AKH I and AKH II), show close structural similarities to the crustacean hormone, indicating a common evolution of some of the arthropod neurohormones. Physiological studies of the three hormones (RPCH, AKH I, and AKH II) and their synthetic analogs show that they crossreact, i.e., they all exhibit pigment-concentrating activity when tested on decapod crustaceans, adipokinetic activity when tested on locusts, and hyperglycemic activity when tested on cockroaches, although each of the hormones is more potent in its own system. Structure-function studies show, however, that quite different binding-site requirements exist for the hormones in activating their receptors on the various target tissues. The physiological specificity in their action therefore seems to depend on a differential evolution of the hormone receptors.

Antibodies were raised in rabbits against glycylleucine dipeptidase (EC 3.4.13.2) and proline dipeptidase (EC 3.4.13.9) purified from the pig. IgG fractions were isolated and purified by affinity chromatography on Sepharose (dipeptidase) columns and then used in histological studies on the small intestine and the liver from pig, using the double layer immunofluorescence-staining technique. In the small intestine the dipeptidases were predominantly localized in the cytoplasm of the enterocytes. A reaction was also seen in the leukocyte cytoplasm in the stroma of the small intestine. The dipeptidases were present in the villi but not in the crypts. In the liver the dipeptidases were localized, in the cytoplasm of the hepatocyte, but strong fluorescence was also obtained in the epithelium of the bile ducts. Glycylleucine dipeptidase and proline dipeptidase have been isolated from liver tissue and they were shown to be immunologically identical with the purified intestinal dipeptidases.

The significance of the C-terminal tryptophan residue of the red pigment-concentrating hormone (RPCH: Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2) regulating the blanching of the crustacean chromatophores has been investigated. RPCH and a number of analogues that differ only in the C-terminal part of the hormone, have been synthesized and assayed for biological activity on the shrimp Leander adspersus. It has been shown that the indole skeleton of tryptophan is an absolute requirement for the biological activity of the hormone. To provide maximum response the tryptophan must be blocked as the amide. The activity of synthetic [Tyr4]RPCH and adipokinetic hormone (AKH) purified from Schistocerca gregaria has been compared with the activity of synthetic RPCH.

Purification of a soluble dipeptidase (EC 3.4.3.2) from pig small intestine is described. 10–20 mg of the purified enzyme were obtained in each preparation (30 m small intestine). It was tested to be at least 99% pure on polyacrylamide electrophoresis and homogenous in crossed immunoelectrophoresis. The specific activity of the enzyme, estimated against glycyl- l -leucine, varied between 1400–1700 units of activity per mg protein in different batches. The enzyme has a wide specificity against dipeptides, although it has no prolidase activity. Among the dipeptides tested, l -alanyl- l -alanine, l -alanyl- l -serine and glycyl- l -leucine were hydrolyzed most rapidly. The enzyme constitutes a true dipeptidase, as it has no tripeptidase or arylamidase activities. It is strictly stereospecific and requires free end-groups of the dipeptides. No activity of the enzyme was found against dipeptides containing β-amino acids.

Five dipeptidase activities in the mucosa of the rat small intestine have been determined and characterized by the aid of a new spectrophotometric assay method.The distribution of the dipeptidase activities along the gastrointestinal tract has been investigated. The activities were low in the stomach, the cecum and the large intestine, while high activities were found along the whole length of the small intestine with the maximum localized to its middle part. The relation of our present findings to previous observations about the rat intestinal dipeptidases has been discussed. Some differences with respect to both their properties and their distribution were observed in com- parison with the dipeptidases of the pig intestinal mucosa.

Summary 1. A study has been made of the distribution of L -alanyl- L -glutamic acid dipeptidase, glycylglycine dipeptidase (EC 3.4.3.1), glycyl- L -leucine dipeptidase (E.C 3.4.3.2) and glycyl- L -valine dipeptidase along the small intestine of adult pig. 2. With the exception of the most proximal part of the duodenum, activities were present along the whole length of the intestine. They all showed a rather wide zone of maximum activity including the distal part of the jejunum and the proximal part of the ileum. 3. The relative activities of the individual enzymes were found to vary greatly. 4. The relation of these findings to protein absorption is discussed.

1. 1. ‘Red-pigment-concentrating hormone’ and distal retinal pigment hormone from aqueous extract of lyophilized eyestalks of Pandalus borealis have been separated on Sephadex G-25. 2. 2. Low activity yields (30%) of the distal retinal pigment hormone were increased by combining the active material with separated inactive material. 3. 3. Final purification of the ‘red-pigment-concentrating hormone’ has been achieved by repeated chromatography on Sephadex LH-20 using different mixtures of water and butanol as solvent. 4. 4. The purified ‘red-pigment-concentrating hormone’ has a specific activity of 1.7·10 6 times that of dry eyestalks. The total recovery of hormone activity was about 35%. 5. 5. A reference standard for the ‘red-pigment-concentrating hormone’ is described and a unit of ‘red-pigment-concentrating hormone’ is defined. 6. 6. The hormone is a peptide containing the eight amino acids, aspartic acid, glutamic acid, glycine, leucine, phenylalanine, proline, serine and tryptophan. 7. 7. No N-terminal amino group has been found. 8. 8. Large and small erythrophores in Palaemon adspersus are both affected by this hormone.

Josefsson, L., O. Noren and H. Sjostrom. Comparison of dipeptidase activity in different tissues of the pig. Acta physiol. scand. 1968. 72. 108–114. A comparative investigation of three dipeptidase activities (L-alanyl-L-glutamic acid. L-alanyl-L-proline and glycyl-L-leucine dipeptidase activity) of the trachea, the tuba uterina, the uterus and the small intestine of the pig has been made to study if the intestinal dipeptidasrs differ in their characteristics from the dipeptidases of other tissues. No indication was obtained supporting the idea that the intestinal dipeptidases would be different and constitute a group of conzymes, particularly adapted for intestinal digestion.

1. 1. A method is described for the purification of a ribonuclease (polyribonucleotide 2-oligonucleotide transferase (cyclizing), EC 2.7.7.16) from mature, unfertilized eggs of the sea urchin Psammechinus miliaris . The enzyme was purified 19 000-fold with a yield of 48% with the aid of chloroform treatment, (NH 4 ) 2 SO 4 fractionation, and by chromatography on Sephadex G-100, on SE-Sephadex C-50 and on CM-Sephadex C-50. 2. 2. The enzyme had a pH-optimum at 5.3–5.5. 3. 3. The enzyme was heat labile, loosing about 50% of its activity in 3 min at 60° and pH 5.3, 70% of its activity in 3 min at 60° and pH 2.1, and 80% of its activity in 3 min at 40° and pH 9.0. 4. 4. The enzyme hydrolyzed yeast RNA completely to 2′,3′-cyclic nucleotides, and finally hydrolyzed the purine cyclic nucleotides slowly to their corresponding 3′-isomers. 5. 5. The enzyme hydrolyzed guanosine 2′-3′-cyclic phosphate about three times faster than adenosine 2′,3′-cyclic phosphate. 6. 6. The purified enzyme was found to be free of deoxyribonuclease and non-specific phosphodiesterase activity. 7. 7. A molecular weight of about 37 000 was estimated for the enzyme by comparative gel filtration experiments.

The blanching hormone of the prawn, Pandalus borealis, is pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH(2). Its structure was settled by a combination of mass spectrometry and Edman-dansyl analysis of a thermolysin fragment. Confirmation of the structure was obtained by chemical synthesis from amino acids. This neurosecreted hormone is active in picogram amounts when tested in shrimps.

Shrimp red pigment-concentrations hormone (RPCH), locust adipokinetic hormone (AKH), and locust Compound I all elevate hemolymph lipid in locusts and concentrate pigment in shrimp erythrophores. In addition, RPCH and AKH both elevate hemolymph carbohydrate in cockroaches. Material with comparable biological activity has been reported in many insects, but not in representatives of the order Hymenoptera. Distilled water head extracts of the honeybe, Apis , exhibited red pigment-concentrating activity in the shrimp Leander and hyperglycemic activity in the cockroach Periplaneta . Chemical characterization of the active substance in Apis extracts showed that like RPCH, AKH, and Compound II, it was heat stable, soluble in butanol and methanol, degraded by proteolytic enzymes, and localized in the cephalic neuroendocrine system. Similarly, like RPCH and Compound II, but unlike AKH, the Apis substance eluted at 1.2 V t during Sephadex G-25 gel filtration. The data thus provide the initial demonstration that the endocrine system of at least one hymenopteran contains a peptide that is chemically and biologically comparable to the RPCH/AKH/Compound II group of hormonally active peptides. Furthermore, the results support the hypothesis that such peptides may be commonly used as hormones in holometabolic insects. In addition, pure Compound II was shonw for the first time to be hyperglycemic in the cockroach.

The significance of the terminal residues of the red pigment-concentrating hormone (RPCH: Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2) for its blanching effect on crustacean chromatophores has been investigated. The chemical synthesis of the following small analogues, starting from the C-terminal amino acid are described: Ac-Trp-NH2, Boc-Gly-Trp-NH2, Ac-Gly-Trp-NH2, Gly-Trp-NH2, Ac-Pro-Gly-Trp-NH2, Glu-Trp-NH2, Glu-Pro-Gly-Trp-NH2, Ac-Pro-Gly and Glu-Pro-Gly-NH2. Assay of the biological activity of the various synthetic compounds in the shrimp Leander adspersus has established that only the C-terminal tryptophan residue is indispensable for the blanching effect of the hormone, although elongation of the chain length improves its potency.

The red pigment-concentrating hormone, purified from Pandalus borealis, as well as the chemically synthesized octapeptide, is effective in concentrating the pigments in both the erythrophores and the leukophores of natantians, when tested on living animals as well as on isolated chromatophores. The existence of a receptor substance for the red pigment-concentrating hormone has been demonstrated. This is extractable from the chromatophores and might be a protein. The use of the synthetic hormone for an affinity chromatographic procedure for the isolation and purification of the receptor substance is envisaged.

INVESTIGATIONS of intestinal mucosal dipeptidases have been hampered in the past by the lack of suitable assay methods. The methods in use at present1 require relatively large amounts of substrate and are inadequate for the examination of dipeptidase activities in crude tissue extracts or partially purified preparations. The new assay method described here has a wide application and is simple and rapid. It is convenient even when a large number of determinations is required.

1. 1. It has been demonstrated that glycyl- dl -serine, l -alanyl- l -serine and l -leucyl- l -serine are transformed to their corresponding O-formyl derivatives when treated with anhydrous formic acid under the same conditions as those used for the reversible inactivation of the protein enzymes. No N,O-acyl shift was observed. The mechanism of the enzymic inactivation is discussed in relation to these findings. 2. 2. The peptidyl O-formylserine derivatives have been crystallized and characterized. 3. 3. N- Formylglycyl- dl -serine and glycy -O- acetyl- dl -serine have been prepared and characterized. 4. 4. The behaviour of these different formyl and acetyl derivatives in dilute aqueous solution has been studied.

Dipeptidases are released very quickly (10–20 sec) from the mucosa cells of the pig small intestine when mucosa scrapings are placed into aqueous solution. The release is somewhat slower from intact pieces of the intestine but the enzymes are obtained in equal yields. Much higher values of specific activity are obtained this way than when the mucosa cells are homogenized. Distribution of the dipeptidases in the subcellular fractions of the mucosa cells has been determined. The activities are located in the supernatant fraction. No conclusion can be made about the intracellular location of the enzymes because of their rapid release into the solvent. The physiological interpretation of these findings is discussed.

DURING the past three decades, investigations of neuro-secretory eye-stalk hormones among crustaceans have revealed a variety of physiological effects produced by eye-stalk ablation as well as by replacement experiments involving injection of extracts of eye-stalk tissue1,2. Resolution of the number of hormones involved in such physiological responses cannot be readily made with the crude extracts so far used. Progress along this line of crustacean endocrinology will evidently depend on isolation and purification of the different hormones, determination of their chemical nature and structure, and testing such purified preparations for the specificity of their physiological effects. Some attempt in this direction with the pigmentary effector hormones indicates that they may be peptide in nature3–5. On the basis of physiological6 and specificity tests7, the light-adapting retinal pigment hormone and the hormone which concentrates pigment in erythrophores appear to be different entities; less-complete separation of retinal pigment hormone and erythrophore-dispersing substance by paper electrophoresis has also been reported8.

KNOWLEDGE about the various types of RNA involved in the biosynthesis of proteins has increased greatly over the past few years. Little is known, however, about the part played by the various ribonucleases (RNases) known to be present in most cells, in the metabolism of the RNA. There has been much speculation on this topic, but few attempts have been made to investigate the physiological action of these substances. This is largely because few of the many RNases identified have been isolated and purified sufficiently to allow them to be characterized. The purification of RNase has great interest at present because of its possible use in the structural analysis of RNA molecules.

IN the course of a study of the N,O-acyl shift at the hydroxyamino-acid residues of proteins (N,O-peptidyl shift) we observed that under certain conditions lysozyme was reversibly inactivated in a way suggestive of a direct dependence on the reversible N,O-peptidyl shift. In earlier work1 on this rearrangement in proteins, concentrated mineral acids were employed, and concomitant, irreversible processes then make it difficult to observe any connexion between the N,O-peptidyl shift and the biological activity. However, a search for milder conditions has revealed that the reaction also takes place in formic acid at ordinary temperature, and apparently without other changes2.