Enzymes and inhibitors in leu-enkephalin in metabolism in human plasma

The enzymes degrading leucine enkephalin in human plasma and the inhibitors active on these enzymes were studied by kinetic and chromatographic techniques. Data obtained evidence the existence of complex kinetics of leu-enkephalin hydrolysis and of formation of its hydrolysis byproducts. These appear to originate from the combined effect of further hydrolysis of the enkephalin's fragments after their release and of competition between the different enzymes present in plasma. Chromatographic separation of plasma proteolysis inhibitors indicates the existence of several pools of substances acting on all three enzyme groups that degrade leu-enkephalin. The partial specificity of these substances induces competition effects: consequently, the actual protection over leu-enkephalin is considerably lower that the total inhibitory activity. That notwithstanding, plasma inhibitors control enkephalin hydrolysis to a relevant extent, while they modify only slightly the ratio of hydrolysis between the different enzymes. This latter parameter—and specifically the large prevalence of aminopeptidases over dipeptidylaminopeptidases and dipeptidylcarboxypeptidases—appears controlled mainly by kinetic factors.     

Role of enzymes and inhibitors in leu-enkephalin metabolism in rabbit and human plasma

The hydrolysis of leucine enkephalin by the proteolytic enzymes present in human and rabbit plasma has been studied by kinetic and chromatographic techniques. Data obtained indicate the existence of noticeable intraspecific differences in the kinetics of leu-enkephalin degradation, and of formation of its hydrolysis by-products. The separation of the enzymes active on the substrate and of the inhibitors active on these enzymes evidences the existence of a species specific distribution of both groups of substances. Yet, the dissimilar kinetics of the substrate hydrolysis and of formation of its hydrolysis by-products appear to arise more from diversities in the competition between the enzymes present in plasma and in the role of inhibitors than from the differences in the enkephalin-degrading enzymes. It is suggested that differences observed may be related to the existence of species specific populations of the information-carrying plasma peptides.     

Mechanisms protecting plasma peptides from enzyme hydrolysis: a comparative study

1. The role of the enkephalin-protecting plasma substances in the protection of non-opioid peptides from enzyme hydrolysis has been studied in laboratory animals and in man. 2. The results obtained indicate that all the peptides hydrolyzed by the plasma enzymes are also protected from the hydrolysis by the enkephalin-protecting substances. 3. The protection is fairly uniform in all the species and for all the peptides examined. However, in the human species the protection of leucine enkephalin is considerably higher than the average. These results are discussed in terms of a possible differential inhibition of the different plasma aminopeptidases.     

Enkephalin binding systems in human plasma III: Comparative protection of different peptides

We have investigated the hydrolysis and protection from hydrolysis of several peptides by plasma enzymes and by the plasma components previously described as inhibitors of enkephalins' hydrolysis. The results shown indicate that all the peptides actually hydrolyzed are also partially protected from hydrolysis by the enkephalin-protecting substances. Protection is fairly uniform for all the peptides tested, but considerably higher in the case of leu- and met-enkephalin, suggesting a partial specificity of the protecting substances towards opioid peptides.     

Comparison of leucine enkephalin and adrenocorticotrophin effects on adrenal function in fetal and adult sheep

At Day 120-125 of gestation equimolar amounts of ACTH and leu-enkephalin injected in vivo provoked similar rises in plasma cortisol concentrations in chronically catheterized fetuses. There was no concomitant change in plasma DHEA concentrations, or in maternal cortisol concentrations. At term (Days 135-140) 2 out of 5 animals responded similarly to both leu-enkephalin and ACTH injections with a rise in plasma cortisol concentrations, but the other 3 animals, in which basal cortisol concentrations had already risen, showed no response to either agonist. In adult sheep, ACTH provoked a significant increase in the plasma cortisol concentrations, but equimolar amounts of leu-enkephalin were without effect. There was a significant output of cortisol in response to ACTH administration by collagenase-dispersed adrenal cells from term sheep fetuses in vitro. Leu-enkephalin had no effect on cortisol output from dispersed adrenal cells when added by itself, or with ACTH. We conclude that leu-enkephalin is able to function as a stimulator of pituitary-adrenal function during fetal life. The lack of effect of leu-enkephalin on adrenal cells implies that its action is exerted not directly at the adrenal gland, but indirectly at the level of the hypothalamus or pituitary through stimulation of the release of other corticotrophic substances.     

Peripheral enkephalin hydrolysis in different animal species: A comparative study

Using column and thin layer chromatography, plasma hydrolysis of leu-enkephalin has been studied in man and several laboratory animals. The hydrolysis kinetics determined in the various species examined are considerably different. In addition, also the enzyme forms evidentiated, their molecular weight distribution and relative ratios have been found to vary greatly in the animals under test. Our data suggest that the widely different hydrolysis kinetics reported by various authors are attributable to the differences between species, rather than to differences in the analytical techniques employed.     

Soluble enkephalin-degrading enzymes released by lymphomic and erythroleukaemic cell lines

The possible existence of soluble proteolytic enzymes released by cells of lymphomic (U937 and 1301) and erythroleukaemic (K562) lines was studied measuring the hydrolysis of³H-leucine enkephalin in the presence of cell-free supernatants obtained from these lines. Results indicate that leu-enkephalin is rapidly degraded in the presence of these supernatants, and that enkephalin disappearance is paralleled by the formation of peptides that can be interpreted as its hydrolysis fragments. To characterize the factors involved in leu-enkephalin degradation, cell supernatants were analyzed by ion exchange and by steric exclusion chromatography. Data obtained indicate the presence of three groups of proteins active in leu-enkephalin degradation: aminopeptidases, dypeptidylaminopeptidases and dypeptidylcarboxypeptidases. In all three lines, these enzymes are represented by a considerable number of distinct activities. The sizable number of soluble enzymes identified and the signficant total activity observed suggest a possible role in the regulatory degradation of informational peptides, as proposed by several groups for the membrane-bound proteolytic enzymes of immunocompetent cells.     

Met- and leu-enkephalin immuno- and bio-reactivity in human stomach and pancreas

Acid extracts of human pancreas and gastric corpus and antral mucosa and muscularis were investigated for the presence of met-enkephalin and leu-enkephalin by radioimmunoassay, Sephadex chromatography and radioreceptor assay. As the assays for leu-enkephalin crossreacted with those for met-enkephalin, only cyanogen bromide-treated samples were used for the determination of leu-enkephalin. Met-enkephalin immunoreactivity was destroyed by more than 94% when treated with cyanogen bromide. Serial extract dilutions displaced ¹²⁵ I labelled met-enkephalin and ¹²⁵ I leu-enkephalin in the respective enkephalin radioimmunoassay both roughly parallel to the standard curves. Sephadex chromatography of the extracts resulted in elution of met-enkephalin and leu-enkephalin immunoreactivity similar to the size of ³H-met-enkephalin, and these eluates displaced ³H-met-enkephalin from rat brain membranes in an opioid radioreceptor assay. The highest concentration of met-enkephalin and leu-enkephalin immunoreactivity in tissue obtained at surgery was in the mucosa of the body of the stomach. Met- and leu-enkephalin receptor bioreactivity concentrations exceeded immunoreactivity concentrations. These investigations provide evidence of the presence of met-enkephalin- and leu-enkephalin-like substances in human stomach and pancreas.     

Regulatory effects of opioid peptides on bone marrow hematopoiesis in stress

It was shown that inhibition of bone marrow hyperplasia disappear due to injection of leu-enkephalin and dalargin as well as the increase of plasma leu-enkephalin by means of D-phenylalanine on the immobilized mice. The effect of enkephalin on hematopoietic precursor cells may be realized by inhibition of T-lymphocytes migration to bone marrow. Besides, direct specific influence of dalargin on myelokaryocytes is observed.     

Neuropeptides in the female genital tract: effect on vascular and non-vascular smooth muscle

The presence of vasoactive intestinal polypeptide (VIP), substance P (SP), somatostatin, enkephalin, and avian pancreatic polypeptide (APP) in nerves in the female genital tract raises the question of their physiological significance as neurotransmitter substances. We have examined the effect of these peptides on non-vascular uterine smooth muscle in vivo as well as in vitro, and the effect on blood flow in the genital tract of rabbit and cat. SP caused a dose-dependent increase in mechanical and myoelectrical activity, an action which could be antagonized by VIP. Substance P, leu-enkephalin and VIP induced a concentration related increase in blood flow of the uterus, where VIP seems to be the most potent vasodilator. Neither the effects on vascular nor on non-vascular smooth muscle were inhibited by adrenergic nor cholinergic blocking agents. APP was able to inhibit the VIP-induced vasodilation in rabbits. These findings suggest that several peptides are involved in the local nervous control of both uterine contractions and haemodynamic events.     

Effect of opiate-active substances on pancreatic polypeptide levels in dogs

The present study examines the effect of orally and intravenously administered opiate-active substances on peripheral vein plasma pancreatic polypeptide (PP) levels in conscious dogs. The intragastric instillation of digested gluten stimulated postprandial PP levels significantly which was reduced by the specific opiate-receptor antagonist naloxone. Naloxone had no effect when added to undigested gluten. Similarly, naloxone reduced significantly the postprandial PP response to a test meal of casopeptone which contains the opiate-active β-casomorphins. The addition of synthetic β-casomorphins to a liver extract/sucrose test meal significantly augmented the rise of postprandial PP levels which was also blocked by naloxone. The intravenous infusion of morphine, leu-enkephalin, D-ala²-D-leu⁵-enkephalin, β-casomorphin-5 and β-casomorphin-4 elicited a dose-dependent and naloxone reversible effect on basal PP levels. During a background infusion of glucose and amino acids the same opiate-active substances had either none or a stimulatory effect on PP release in these dogs. The addition of naloxone abolished the stimulatory effect in response to β-casomorphin-5 and β-casomorphin-4 and resulted in an inhibition of PP levels during the infusion of morphine and leu-enkephalin. This latter inhibitory effect was no longer observed when the dose of naloxone was increased ten- and fifty-fold, respectively. The present data suggest that orally ingested opiate-active substances participate in the stimulation of postprandial PP release in dogs via specific opiate-receptor mediated mechanisms. The effect of intravenously administered opiate-active substances on PP levels depends on the metabolic state with regard to the level of circulating nutrients. It is suggested that PP release is stimulated via μ-opiate receptors and inhibited via δ-opiate receptors. An increase of circulating nutrients would “activate” μ-receptor sites which are masked in the basal state when exogenous opiates are administered. However, with regard to endogenous opiates an increase of circulating nutrients, mainly carbohydrates, activates inhibitory effects of endogenous opiates suggesting that exogenous and endogenous opiates act at different target sites.     

Properties and precursors of hordeumin produced from uncooked barley bran by ethanol fermentation

Hordeumin (a purple pigment) formed by the ethanol fermentation of uncooked barley bran had properties partially different from those of conventional anthocyanins and showed high molecular tannin-like properties. When the polyphenol constituents (containing proanthocyanidins) in barley bran and the fermented filtrate were removed, the amount of the pigment formed was reduced. Hordeumin was decomposed to lower molecular weight substances and anthocyanidins (cyanidin and delphinidin) by acid hydrolysis. On the other hand, the anthocyanidin components formed by acid hydrolysis of the polyphenol constituents in barley bran and the fermented filtrate coincided with those of hordeumin. The precursors of hordeumin were proanthocyanidins contained in barley. Hordeumin seems to be a novel pigment with a high molecular weight having the properties of anthocyanin pigment.     

The importance of SH-containing substances for red blood cells in acute myeloic leukemia

Electron spin resonance (ESR) spectra of lyophilized erythrocytes obtained from patients with acute myeloid leukemia (AML) show, in comparison to controls, a characteristic change especially in the low-field region of the spectrum concomitant with a reduction of the spin concentration. This effect can be simulated by addition of SH-containing substances (e.g. reduced glutathione or cysteine) to healthy erythrocytes. S-S containing compounds exhibit no effect. Since SH-containing substances can hardly permeate plasma membranes, the membrane surface seems to be defective in the case of "AML" erythrocytes. Furthermore, it can be concluded that the concentration of SH-containing substances, such as cysteine, is increased in the plasma of AML-patients, which could be confirmed by HPLC-measurements. In the case of a successful treatment of the patients with alexan, daunoblastin, and thioguanine the spin concentration increased again and the resulting ESR spectrum is very similar to the control spectrum. It should be pointed out, that the ascorbic acid concentration is very low in both plasma and erythrocytes of AML patients.     

Produktion oder übernahme von schutzstoffen als ursache des Nesselschutzes von Anemonenfischen?

Investigations concerning the protection of anemone fishes have been conducted on reefs near Shimoni (Kenya). Acclimated Amphiprion xanthurus Cuv. & Val. when placed in perforated plastic boxes in the midst of anemones do not lose their protection against stinging. Unacclimated A. xanthurus became protected in this manner. Protected A. xanthurus lost their protection and unprotected ones could not acquire this protection when they were placed within a distance of 5–7 cm or more from actinians. In order to maintain the protection, the intimacy of anemones is absolutely necessary. The quality of protection acquired by the fishes depends on the species of anemone, being either good against only one species or good against several species simultaneously. The fact that the fishes remain protected only if they live in the midst of anemones or very close to them and that the degree of protection is determined by the anemone indicates the importance of the anemones for the acquisition of this protection. These results confirm the suggested mechanism of protection formulated previously by Schlichter, namely that the fishes acquire anemone-substances (‘protective substances’) during the process of acclimation. After becoming protected they bear the same chemical impregnation on their surface as, e.g., tentacles, which do not sting each other. The transmission of protecting substances to unprotected fishes by incubation with extracts from anemones was found to be impossible. After incubation in pepsin, acclimated fishes lost their protection. It seems as though anemone fishes exploit for their protection a principle which the actinians have for their own use. This mechanism exists quite independently of the symbiosis with fishes. The anemones themselves require such ‘protective substances’ for several purposes.     

The effect of leucine-enkephalin on the peristaltic reflex of the isolated guinea-pig ileum

Leucine (leu)-enkephalin depresses or inhibits the peristaltic reflex of the isolated guinea-pig ileum. Opiate antagonists (naloxone and nalorphine), choline esters (acetylcholine, methacholine and carbachol), cholinomimetics (muscarine and arecoline) and polypeptides which stimulate peristalsis (eledoisin and angiotensin) antagonize the peristaltic block caused by leu-enkephalin. On the other hand, nicotinic ganglionic stimulants (nicotine and dimethylphenylpiperazine) as well as muscarinic ganglionic stimulants (McN-A-343 and AHR-602) do not restore the peristaltic reflex abolished by leu-enkephalin. Thus the inhibitory effect of leu-enkephalin is due mainly to an action on myenteric ganglia as well as on axon terminals of the myenteric plexus subserving the peristaltic reflex. The inhibitory action of leu-enkephalin may be ascribed to the opiate as well as to the cholinoceptive sites in the nervous elements in the myenteric plexus. The blocking action of leu-enkephalin is not associated with ganglionic muscarinic M-1 receptors as well as with ganglionic nicotinic receptors in the myenteric plexus of the guinea-pig isolated ileum.     

Direct membrane effects of morphine and endorphins on Amoeba proteus.

Morphine, leu-enkephalinamide, met-enkephalin, alpha-neoendorphin and its Arg8 1-8 fragment increase contractile vacuole output in the freshwater Amoeba proteus at 18 microM. Significant effects of leu-enkephalin and naloxone are obtained at 180 microM. All compounds have reached their maximal activity at 720 microM. Alpha-neoendorphin and leu-enkephalin are inactive in the presence of isotonic, non-penetration sucrose, hence these compounds increase plasma membrane permeability to water. Results from molecular modeling show a clear correlation of activity with amphiphilicity, charge distribution and general flexibility of molecules. We conclude that, like previously-studied vasopressin analogues and non-hormonal amphiphilic peptides, active opioids embed themselves into the Amoeba plasma membrane, disrupting the lipid bilayer and increasing its permeability. In our Amoeba system, naloxone, a general morphine-like inhibitor, blocks active opioids as well as a vasopressin analogue. Naloxone, being less active than other tested amphiphiles, acts as a membrane stabilizer, protecting the lipid bilayer against the disruption action of more active compounds.     

Enkephalin-degrading enzymes and their inhibitors in human saliva

The possible presence of enzymes able to hydrolyze leucine enkephalin has been investigated in human saliva. The data obtained indicate that, in the presence of saliva, Leu-enkephalin is partially hydrolyzed. The disappearance of the substrate is paired with the formation of hydrolysis byproducts whose composition indicates the presence of all three classes of enzymes known to hydrolyze enkephalins: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases. The presence of low molecular weight substances with inhibitory activity on proteolytic enzymes has also been detected. These substances are active on all three classes of enkephalin-degrading enzymes, although the inhibition is more evident on dipeptidylpeptidases than on aminopeptidases. Substrate degradation was found to be higher in male than in female saliva: this seems to be caused by the activities both of enzymes and low molecular weight inhibitors that are different in the two sexes.     

The phenolic compounds in <Emphasis Type="Italic">Cladonia</Emphasis> lichens are not antimicrobial in soils

According to classic text books on lichen biology, the phenolic secondary chemicals in lichens have antibiotic effects on soil microorganisms and mycorrhizal fungi in ecosystems. However, the experimental evidence for this under natural conditions is still relatively scarce. We examined some of the assumptions behind the concept of antimicrobial effects of lichen secondary substances: (1) the secondary substances of Cladonia stellaris, usnic and perlatolic acids, are leached out from the lichens by rainwater; (2) these substances inhibit the microbial activity of soil, and; (3) since they are extremely resistant to microbial decomposition, the soil underneath a continuous lichen mat is enriched in usnic and perlatolic acids. Our results did not support any of these assumptions. The evidence for the antimicrobial activity of lichen secondary substances seems to be weak in comparison to other suggested functions such as light filtering and herbivore protection. We suggest that it is time to re-evaluate the evidence for the antimicrobial ecological role of lichen secondary substances in natural systems.     

Biodegradation of coumaphos, chlorferon, and diethylthiophosphate using bacteria immobilized in Ca-alginate gel beads

Calcium-alginate immobilized cell systems were developed for the detoxification and biodegradation of coumaphos, an organophosphate insecticide, and its hydrolysis products, chlorferon and diethlythiophosphate (DETP). Optimum bead loadings for bioreactor operation were found to be 200 g-beads/L for chlorferon degradation and 300 g-beads/L for DETP degradation. Using waste cattle dip (UCD) solution as substrate, the degradation rate for an immobilized consortium of chlorferon-degrading bacteria was five times greater than that for freely suspended cells, and hydrolysis of coumaphos by immobilized OPH(+)Escherichia coli was 2.5 times greater. The enhanced degradation of immobilized cells was due primarily to protection of the cells from inhibitory substances present in the UCD solution. In addition, physiological changes of the cells caused by Ca-alginate immobilization may have contributed to increased reaction rates. Degradation rates for repeated operations increased for successive batches indicating that cells became better adapted to the reaction conditions over time.     

Low molecular weight humic substances stimulate H+-ATPase activity of plasma membrane vesicles isolated from oat (Avena sativa L.) roots

The effect of <5 KDa (low molecular weight, LMW) and >5 KDa (high molecular weight, HMW) humic fractions on transport activities of isolated plasma membrane vesicles was studied. The K⁺-stimulated component of the ATP-hydrolyzing activity was considerably increased by LMW humic substances at concentrations ranging from 0.075 mg org CL^-1 to 1 mg org CL^-1. The stimulation was still evident when the detergent Brij-35 was added in the assay mixture, indicating a direct effect of LMW humic substances on plasma membrane ATPase activity. The LMW humic fraction stimulated ATP-dependent intravesicular H⁺-accumulation with a pattern similar to that recorded for ATP hydrolysis. LMW humic substances induced also an increase in passive membrane permeability to protons, as revealed by following the dissipation of an artificially imposed pH gradient. Membrane permeability to anions, as measured by the anion-dependent active proton accumulation was affected by LMW humic substances. In the presence of NO₃ ^- these molecules clearly enhanced proton transport, while Cl^--dependent activity was almost unaffected, thus suggesting a specific action of LMW humic fraction on transmembrane NO₃ ^- fluxes. On the other hand, HMW humic substances decreased the passive permeability to protons and reduced the anion-dependent intravesicular H⁺-accumulation. The results suggest that the stimulatory effect of soil humic substances on plant nutrition and growth might be, at least in part, explained on the basis of both direct action of LMW humic molecules on plasma membrane H⁺-ATPase and specific modification of cell membrane permeability.