Modulation of KB and A431 cell adhesion by epidermal growth inhibitory pentapeptide.

A pentapaptide, pyroGlu-Glu-Asp-Ser-GlyOH (EPP), isolated from mouse epidermis, inhibits mitoses and enhances differentiation in primary cultures and in transformed mouse epidermal cells in vitro (Elgjo et al. 1986 b). The present work demonstrates that EPP also modulates the adhesiveness of two human tumour cell lines (KB and A431) of epidermal origin to uncoated plastic and to plastic coated with fibrinogen or collagen type 1. The adhesion modulatory effect of EPP was observed over a broad range of concentrations (10(-12)-10(-6) M), and depended on the substrate the cells were growing on. Thus, when cells were seeded on plastic or collagen, the attachment to the substrate was suppressed at the highest concentrations of EPP, and stimulated at the lowest ones. The opposite concentration-response pattern was observed when fibrinogen was used as substrate.     

Identification and partial characterisation of a chitinase from Nile tilapia, Oreochromis niloticus

Measurement of chitinase activity in extracts from stomach, intestine, and serum of Nile tilapia with the artificial substrates 4-methylumbelliferil beta-D-N,N'-diacetylchitobioside and 4-methylumbelliferil beta-D-N,N'N"-triacetylchitotrioside (4MU[GlcNAc](2,3)) showed that an endochitinase was involved in the liberation of the fluorophore 4-methylumbelliferone (MU). Enzymes were isolated from tilapia serum by a combination of gel filtration, ion exchange, and reverse-phase chromatography. The molecular mass of the enzyme was estimated to be 75 kDa by SDS-PAGE, suggesting that the enzyme occurs as a monomer. The partially purified enzyme showed maximal activity at pH 7.0 when assayed with 4MU[GlcNAc](2) and lost its activity below pH 5.0 and above pH 8.0. The optimal pH of the purified enzyme toward the substrate 4MU[GlcNAc](3) was pH 9.0 and activity was lost below pH 8.0 and above pH 9.0. Our study has revealed the presence of a chitinolytic enzyme in the gastrointestinal tract and serum that may play a role in digestion and/or defense.     

Angiotensin converting enzyme inhibitory pentapeptide isolated from supernatant of pig plasma treated by trichloroacetic acid

Summary Angiotensin converting enzyme (ACE) inhibitory peptide was isolated from supernatant of trichloroacetic acid treated pig plasma using gel filtration chromatography and reverse phase high pressure liquid chromatography. Protein sequencing showed that the peptide is a pentapeptide of Gly-Val-His-Gly-Val with 2 M as IC₅₀ value.     

Identification of enzyme inhibitory mechanisms from steady-state kinetics

Enzyme inhibitors are used in many areas of the life sciences, ranging from basic research to the combat of disease in the clinic. Inhibitors are traditionally characterized by how they affect the steady-state kinetics of enzymes, commonly analyzed on the assumption that enzyme-bound and free substrate molecules are in equilibrium. This assumption, implying that an enzyme-bound substrate molecule has near zero probability to form a product rather than dissociate, is valid only for very inefficient enzymes. When it is relaxed, more complex but also more information-rich steady-state kinetics emerges. Although solutions to the general steady-state kinetics problem exist, they are opaque and have been of limited help to experimentalists.Here we reformulate the steady-state kinetics of enzyme inhibition in terms of new parameters. These allow for assessment of ambiguities of interpretation due to kinetic scheme degeneracy and provide an intuitively simple way to analyze experimental data. We illustrate the method by concrete examples of how to assess scheme degeneracy and obtain experimental estimates of all available rate and equilibrium constants. We suggest simple, complementary experiments that can remove ambiguities and greatly enhance the accuracy of parameter estimation.     

Isolation and structure of an epidermal mitosis inhibiting pentapeptide

A mitosis inhibiting peptide pyroGlu-Glu-Asp-Ser-GlyOH has been isolated from mouse skin extracts. Both the biological and a synthetic peptide with the same structure reversibly inhibit epidermal mitoses in a curvilinear fashion after intraperitoneal injection. The two compounds are chromatographically identical.     

Presence and partial characterisation of a major proteolytic enzyme in the larval gut of Callosobruchus maculatus

Protease activity in the gut of larvae of the bruchid beetle, Callosobruchus maculatus (a storage pest of cowpea seeds), has been investigated to help clarify nutritional mechanisms in view of reports that these insects carry out little or no proteolysis (Applebaum, 1964). Larval gut homogenates showed protease activity against a variety of different protein substrates, but did not hydrolyse a synthetic trypsin substrate. The proteolytic activity had a pH optimum of 5.4. It was not inhibited by serine protease inhibitors, but was inhibited by reagents reactive against-SH groups. Protein trypsin inhibitors from legume seeds which are not hosts to C. maculatus (soybean, limabean) were not effective inhibitors of the larval proteolytic activity but a cowpea protease inhibitor preparation and aprotinin partially inhibited proteolysis. The latter two inhibitors also inhibited the plant thiol protease papain. It is suggested that C. maculatus has replaced the normal insect proteases with an enzyme similar to plant proteases to evade the antimetabolic effects of trypsin/chymotrypsin inhibitors in seeds. Besides trypsin/chymotrypsin inhibitors, cowpea seeds also contain proteins which inhibit papain; these inhibitors were purified and were shown to be effective inhibitors of C. maculatus larval protease.

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Mise en évidence et caractérisation partielle d'une enzyme protéolytique importante du tube digestif des larves de Callosobruchus maculatus

Résumé Callosobruchus maculatus attaque les graines stockées particulièrement de Vigna unguiculata. L'activité protéolytique du tube digestif des larves a été examinée. Aucune hydrolyse n'a été observée contre la N-benzoyl arginine p-nitroanilide (substrat de la trypsine synthétique), mais la protéolyse a été mise en évidence en utilisant la viciline de V. unguiculata comme substrat naturel ou la myoglobine comme substrat artificiel, le p.H optimum est de 5,4. Les inhibiteurs chimiques des protéases de la sérine n'ont pas altéré l'activité enzymatique, mais les réactifs du groupe SH l'ont inhibée. Nous suggérons que cette protéase est une thiol protéase, c'est à dire avec un groupe actif semblable à celui de la papaïne, protéase végétale. En accord avec cette hypothèse, les inhibiteurs de protéine efficaces seulement contre la trypsine, c'est à dire les inhibiteurs de la trypsine de Glycine max et de la trypsine de Phaseolus lunatus n'affectent pas cette protéase larvaire, tandis que les protéines avec une faible action inhibitrice contre la papaïne (préparation inhibitrice de la protéase de V. unguiculata, aprotinine) inhibent partiellement la protéolyse par des extraits de tube digestif larvaire.Des inhibiteurs protéiques de papaïne extraits de graines de V. unguiculata sont des inhibiteurs très efficaces de la protéase larvaire. On peut penser que C. maculatus contient une protéase semblable aux protéases végétales, plutôt qu'aux protéases classiques, d'insectes, ce qui permet d'éviter les effets antimétaboliques directs des inhibiteurs de protéase (inhibiteurs de trypsine et de chymotrypsine) trouvés en quantité relativement importantes dans les graines de légumineuses.

     

Identification and partial purification of a novel milk clotting enzyme from Onopordum turcicum

Summary Seeds, flowers and leaves of Onopordum turcicum were found to contain proteolytic enzymes able to coagulate milk. Extraction, concentration and identification of the operational parameters affecting the activity of the enzyme complex were followed by partial purification steps involving gel-filtration and ion-exchange chromatography. Milk clotting activity of the enzyme complex was tested in several steps of its purification and an increase of almost 200 fold was obtained. Molecular weight of the proteolytic enzyme fraction having the maximum activity was determined to be about 19000–24000. Isoelectric point (pI) of the enzyme complex with maximum activity was estimated to be in the range 3.3–3.7.     

Inhibitory epidermal pentapeptide modulates proliferation and differentiation of transformed mouse epidermal cells in vitro.

A transformed mouse epidermal cell line ("308 cells") and nontransformed rat tongue squamous epithelial cells ("RT10 cells") were treated 3 times weekly for a period of two weeks with relatively large doses (150 micrograms/ml) of a synthetic inhibitory epidermal pentapeptide; pyroGlu-Glu-Asp-Ser-GlyOH. The peptide was recently isolated from mouse skin extracts and inhibits normal epidermal cells in vivo and in vitro at a restricted and low dose level. Repeated treatments with the large dose was followed by a 30-40% reduction in the number of 308 cells per well, starting as early as day 1. The number of RT10 cells was reduced about 20% only at termination of the experiment on day 14. In contrast to this, the number of unattached cornified envelopes on day 10 in the RT10 cells was increased by 85%, while the number of cornified, unattached 308 cells was similar to that in the controls. The effects of the pentapeptide thus seem to affect differentiation stronger than proliferation in the nontransformed cell line. Bivariate BrdUrd/DNA flow cytometry analysis on day 10 indicated that the reduced number of 308 cells was mainly due to a slower rate of cell proliferation and not to a increased sloughing off of keratinized cells. This analysis also demonstrated that an inhibition of DNA synthesis in the RT10 cells could be detected prior to a reduction of the cell number per well.     

Identification, characterisation and specificity of a cell wall lytic enzyme from Lactobacillus fermentum BR11

Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Phiadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.     

Transepithelial transport and stability in blood serum of angiotensin-I-converting enzyme inhibitory dipeptides

The dipeptides Ala-Trp, Val-Phe, and Val-Tyr inhibit the angiotensin-I-converting enzyme. They are encrypted within the primary sequences of different food proteins, e.g. milk proteins. The angiotensin-I-converting enzyme inhibitory potency of these synthetic dipeptides was quantified using a spectrophotometric assay. The dipeptides showed no adverse effects on differentiated Caco-2 cells (model for human intestinal epithelium), as confirmed by transepithelial electrical resistance, microscopy and the activity of the brush-border enzyme dipeptidyl aminopeptidase IV. Furthermore, the transport of these bioactive dipeptides through intact Caco-2 monolayers and their stability to incubation in human blood serum has been demonstrated for the first time. Low molecular mass peptides represent the minimal structures required for angiotensin-I-converting enzyme inhibition which have a high potential bioavailability. Therefore, they may act as target peptides in enriched hydrolysates for the preparation of an angiotensin-I-converting enzyme inhibitory peptide and for the use in special formulations as functional foods/foods of specified health use.     

Acid nucleoside triphosphatase: partial purification and characterization of a new enzyme from human serum

Acid nucleoside triphosphatase (Acid NTPase), an enzyme which catalyzes the hydrolysis of all nucleoside triphosphates to the corresponding diphosphates was purified from human serum with a purification factor of 190 and a recovery of 31%. The molecular weight was 75,000 as estimated by gel filtration. Gel-electrophoresis revealed an Rf-value of 0.11, and the isoelectric point was determined at pH 4.4. It exhibited a temperature optimum of 44 degrees C and the activation energy was estimated to be 41.6 kJ/mol. The enzyme was active in the absence of divalent cations, since activity was not inhibited by EDTA. The presence of this chelator reduced the Km-value from 70 to 40 microM. Inhibitor experiments revealed that tartrate was a weak mixed-type noncompetitive inhibitor, Ki = 88 mM. The enzyme was specific for the hydrolysis of nucleoside triphosphates. P-nitrophenyl phosphate was not accepted as a substrate. The enzyme revealed optimum activity at the exceptionally acid pH of 3.0. These unique characteristics indicate the presence of a novel enzyme.     

Identification and partial purification of a thiol endothelin-converting enzyme from porcine aortic endothelial cells.

Endothelin is a potent peptide vasoconstrictor. The final step in the processing of endothelin has been postulated to be the cleavage of the Trp21-Val22 peptide bond in proendothelin by a putative endothelin-converting enzyme. A soluble extract of primary porcine aortic endothelial cells was found to contain an enzyme activity that converted proendothelin-1 (proET-1) to an endothelin-1 (ET-1)-like peptide as determined by the rabbit aortic ring contraction assay. This enzyme was partially purified by DE52 ion-exchange chromatography. Incubation of proET-1 with the partially purified enzyme generated a product which had a retention time on HPLC identical to that of authentic ET-1. Further analysis of the product showed that it caused contraction of rabbit aortic rings, had a molecular weight identical to ET-1 as measured by fast atom bombardment mass spectrometry, and competed for [125I]ET-1 binding in an RIA using specific antibodies which recognize the carboxy terminal tryptophan of ET-1. The enzyme activity could be inhibited by thiol protease inhibitors such as Z-phe-pheCHN2 and p-hydroxymercuribenzoate, but not by serine- or metalloprotease inhibitors. The optimal pH for the enzymatic activity was between 7.0 and 7.5, and no activity was detected at pH 4.0. These results demonstrate that this thiol protease is a potential endothelin-converting enzyme.     

Cyclic hexa- and pentapeptide somatostatin analogues with reduced gastric inhibitory activity

The gastric inhibitory activity of cyclic hexa- and pentapeptide analogues of somatostatin was investigated in conscious cats with gastric fistulae. Gastric acid and pepsin secretions were stimulated by pentagastrin. Cyclo(Phe-Phe-D-Trp-Lys-Thr-Phe) showed no inhibition of acid secretion at molar doses up to 50-times the ID50 for somatostatin. This peptide inhibited pepsin secretion at the highest dose (50 micrograms kg-1 hr-1), and its potency is approximately 0.005 compared with somatostatin (1.0). Cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) inhibited acid (approximately 50%) and pepsin (approximately 85%) secretions, but the inhibition was not dose-related being similar with doses of 10 to 50 micrograms kg-1 hr-1. The cyclic pentapeptide, cyclo(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr), was inactive in the dose range studied, with a potency less than 0.01. Cyclo[7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl)] has been described as a somatostatin antagonist with respect to inhibition of growth hormone, insulin and glucagon release in rats [2]. Up to 60-fold molar excesses of this peptide failed to antagonise the inhibitory activity of somatostatin in the stomach. The results demonstrate that residues outside the central 6-11 region of somatostatin are very important for its gastric activity. The lack of gastric antagonistic activity of the pentapeptide antagonist indicates that these residues are likely to be involved in receptor recognition/binding.     

Identification and partial characterisation of metalloproteases secreted by a Mesanophrys-like ciliate parasite of the Norway lobster Nephrops norvegicus

A ciliate parasite, tentatively identified as Mesanophrys sp. of Norway lobsters Nephrops norvegicus, is demonstrated to secrete several proteases into the culture medium (modified Nephrops saline). Analyses using substrate-impregnated sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed 12 activity bands differing greatly in mobility in the gels. The complete inhibition of proteolytic activity by 1,10-phenanthroline indicated that the proteases are of the metallo class. The proteases were active at the physiological temperature (8 degrees C) and haemolymph pH (7.8) of the host. The proteases were selective in the degradation of several host proteins, including the myosin heavy chain, which is a major structural component of lobster muscle. Consequently, these proteases may have important roles in several aspects of the host-parasite interaction including invasion, nutrient uptake by the ciliate, and pathogenesis.     

Identification and partial characterisation of the non-collagenous amino- and carboxyl-terminal extension peptides of cartilage procollagen.

Procollagen secreted by embryonic chick cartilage cells has been partially purified by (NH₄)₂SO₄ precipitation and DEAE-cellulose chromatography. Analyses of the products of digestion by human rheumatoid synovial collagenase and bacterial collagenase indicated the presence of non-collagenous peptide sequences at the N- and C-termini. Both regions were found to incorporate [³⁵S]cystine but inter-chain disulphide bonds were restricted to a C-terminal location. Electrophoretic analysis gave apparent molecular weights of 13000 and 36000 daltons for the respective N- and C-terminal extensions.