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Dipeptidyl peptidase III (DPP III ; EC 3.4.14.4) is a zinc-exopeptidase which was discovered in extracts of pituitary gland through hydrolysis of Arg-Arg-2-naphthylamide (Arg-Arg-2NA). Until now, DPP III was purified and biochemically characterized from several human and animal tissues, and lower eukaryotes. It is generally found to be a cytosolic protein, but a membrane-associated DPP III has been reported in brain as well. It cleaves dipeptides sequentially from the N-terminus of its substrates, optimally sized from tetra- to octapeptides. In vitro this peptidase displays a broad specificity, and high affinity for angiotensins and enkephalins. A preference for hydrophobic residues at position P1', and proline as a prohibited amino acid at P1 or P1' was postulated fo mammalian DPPs III. The physiological substrates of this metallopeptidase are mostly unknown. Recent data support the role for DPP III in endogenous pain-modulatory system: high concentrations of DPP III were found in the superficial laminae of rat spinal cord dorsal horn where this peptidase co-localizes with opioid peptides enkephalins and endomorphins. In our study of mammalian DPPs III we determined, by activity measurements and Western blotting, a level of this enzyme in the rat tissues, in the brain being among the highest. To investigate further a possible involvement of DPP III in the metabolism of neuropeptides, we examined its affinity (by competitive inhibition of Arg-Arg-2NA hydrolysis) and hydrolytic activity (by TLC) towards several novel opioid peptides. Surprisingly, human DPP III (purified from erythrocytes) cleaved endomorphins, in contrast to the accepted notion that proline imposes a restriction on the hydrolysis of the peptide bond catalysed by this type of enzyme. Furthermore, DPP III hydrolytic activity towards endomorphin-1 (EM-1) was compared to that of dipeptidyl peptidase IV (DPP IV ; EC 3.4.14.5), a well-known post-proline cleaving enzyme. DPP IV was isolated from rat kidney membranes. The enzymatic hydrolysis products of EM-1 were separated and quantified by capillary electrophoresis and kinetic parameters determined. Both DPP III and DPP IV cleaved this tetrapeptide with comparable rate, by liberating the N-terminal Tyr-Pro. In conclusion, our study revealed hydrolytic activity of human DPP III towards representatives of three new groups of opioid peptides: endomorphins, hemorphins and exorphins. These results show for the first time that human DPP III can act as a post-proline-cleaving enzyme.

The dipeptidyl peptidase III (DPP III) is a zinc-exopeptidase and a member of the metallopeptidase family M49 with an implied role in the pain-modulating system and endogenous defense against oxidative stress. The role of the unique fully conserved tyrosine (Tyr318) and tryptophan (Trp300) was investigated in human enzyme by site-directed mutagenesis. The substitution of Tyr318 for Phe decreased kcat by two orders of magnitude without altering the binding affinity of substrate or of a competitive inhibitor. However, replacement of the Trp300 reduced enzyme activity and had a negative effect on the binding of two competitive hydroxamate inhibitors designed to interact with S1 and S2 subsites. The results indicate that the conserved tyrosine could be involved in transition state stabilization during the catalytic action of M49 peptidases, and that conserved tryptophan contributes in maintaining the functional integrity of the enzyme's S2 subsite.

Porodica metalopeptidaza M49 (porodica dipeptidil-peptidaza III) nedavno je prepoznata kao zasebna porodica proteina na temelju značajne sličnosti aminokiselinskih slijedova svojih članova i jedinstvenog strukturnog motiva, HEXXXH, u aktivnom mjestu. Iako fiziološka uloga dipeptidil-peptidaze III (DPP III) još nije potpuno jasna, pretpostavlja se da je povezana s biološkim procesima regulacije boli, stvaranja očne mrene i obrane od oksidacijskog stresa. Unatoč razjašnjenju kristalne strukture ortolognog enzima iz nižeg eukariota, saznanja o katalitičkom mehanizmu ovog tipa proteolitičkih enzima još su uvijek nepotpuna. U svrhu daljnjeg ispitivanja aktivnog mjesta ljudske DPP III, na osnovi bioinformatičkih analiza odabrana su tri ostatka tirozina (Tyr318, Tyr395 i Tyr644) i ispitana je njihova potencijalna uloga u katalizi. U ovom radu prikazujemo rezultate heterologne produkcije i usmjerene mutageneze ljudske DPP III, koji pokazuju funkcionalnu ulogu Tyr318, jedinog potpuno sačuvanog ostatka tirozina u porodici M49. Posljedica zamjene Tyr318 u Phe je smanjenje kcat za dva reda veličine, ali bez utjecaja na afinitet vezanja supstrata ili kompetitivnog hidroksamatnog inhibitora oblikovanog za interakciju sa S1 i S2 podmjestom enzima. Rezultati ukazuju na ulogu Tyr318 u stabilizaciji prijelaznog stanja tijekom katalize.

In recent years the number of deduced amino acid sequences of metallopeptidases has increased dramatically revealing that these enzymes are the most diverse of the four main catalytic types of peptidases, with 51 families identified to date. Peptidase family M49 (dipeptidyl peptidase III family) has been recognized as a distinct group of metallopeptidases based on the significant similarity in primary structures of its members and the unique structural motif, hexapeptide HELLGH, which harbors the predicted active site residues. Dipeptidyl peptidase III (DPP III) was previously biochemically characterized as a cytosolic zinc-exopeptidase involved in the final steps of intracellular protein catabolism of eukaryotes. The regulatory role of DPP III in the metabolism of biologically active peptides (angiotensins and enkephalins) has been suggested. However, 3-D structure, physiological significance, regulation and distribution of this enzyme in the living world still need to be elucidated. Our results indicated that enhanced expression of DPP III might be used as biochemical marker for endometrial and ovarian cancer. We assumed that the new data of genomes sequencing also contain unknown members of this family and we attempted to define its evolutionary conserved amino acid sequence regions through the analysis of their primary structures. By the similarity search and additional manual stringency we have revealed 14 homologous protein sequences (members of family M49), two of them prokaryotic, whose multiple alignment gave five highly conserved regions. Two conserved linear motifs (consensus sequences) harboring four known active site residues of family M49 were defined as stretches of 16 and 6 amino acids located in the third and fourth conserved region. A part of sixteen-amino acid consensus sequence and the complete consensus sequence of six amino acid were predicted to reside in a alpha-helix. In conclusion, the most recent data on complete genome sequences helped us to reveal that metallopeptidase family M49 (DPP III family) is distributed in four kingdoms of organisms (Eubacteria, Protista, Fungi and Animalia), and to define two consensus sequences containing the active site residues. Bacterial homologs have been unexpected and so far confined to the proteins from one human symbiont and one oral pathogen.

Background and Purpose: Peptidase family M49 (dipeptidyl peptidase III family) has been recently recognized among metallopeptidases, based on the unique structural motif, hexapeptide HELLGH, which harbors the predicted active site residues. We assumed that the new data of genomes sequencing also contain unknown members of this family and we attempted to define its evolutionary conserved amino acid sequence regions through the analysis of their primary structures. Methods: Similarity search and the multiple sequence alignment were performed by BLASTP and CLUSTALW program, respectively. PSIPRED method was used for protein secondary structure prediction. Results: The similarity search and additional manual stringency revealed 14 homologous protein sequences (members of family M49), two of them prokaryotic, whose multiple alignment gave five highly conserved regions. Two conserved linear motifs (consensus sequences) harboring four known active site residues of family M49 were defined as stretches of 16 and 6 amino acids located in the third and fourth conserved region. A part of sixteen-amino acid consensus sequence and the complete consensus sequence of six amino acid were predicted to reside in a alpha-helix. Conclusion: The most recent data on complete genome sequences helped to reveal that peptidase family M49 is distributed in four kingdoms of organisms (Eubacteria, Protista, Fungi and Animalia), and to define two consensus sequences containing the active site residues. Bacterial homologs have been unexpected and so far confined to the proteins from one human symbiont and one oral pathogen.

Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopeptidase involved in the final steps of intracellular protein catabolism of eukaryotes. The regulatory role of DPP III in the metabolism of biologically active peptides (angiotensins and enkephalins) has been suggested. Our previous results indicated that enhanced expression of DPP III might be used as clinical marker for endometrial and ovarian cancer. The cDNA encoding the rat and human DPP III has been recently cloned and sequenced, revealing 93% identity of their primary structure. However, 3-D structure, physiological significance and regulation of DPP III still need to be elucidated. We have purified DPP III from human and rat erythrocytes using an identical procedure and determined its physico-chemical properties. The parallel biochemical investigation of the two enzymes revealed pronounced similarities but also significant differences indicating non-identity in their active site topology. The inactivation kinetics by sulphydryl reagent para-hydroxy-mercuribenzoate (pHMB) was monitored and the second order rate constants were calculated, revealing rat DPP III to be hyperreactive. Peptide substrates protected both enzymes from inactivation by pHMB indicating that reactive cysteine residues are part of the substrate-binding site. Because the biological thiols, oxidised glutathione and H2O2 influenced the activity "in vitro", we postulated that reversible redox regulation of zinc-enzyme DPP III could be of physiological importance. The study of modulation of this peptidase level under cellular stress conditions using proteomic approach is ongoing.

Dipeptidyl peptidase III (DPP III) is a cytosolic zinc-exopeptidase involved in the intracellular protein catabolism of eukaryotes. Although inhibition by thiol reagents is a general feature of DPP III originating from various species, the function of activity important sulfhydryl groups is still inadequately understood. The present study of the reactivity of these groups was undertaken in order to clarify their biological significance. The inactivation kinetics of human and rat DPP III by sulfhydryl reagent p-hydroxy-mercuribenzoate (pHMB) was monitored by determination of the enzyme’ s residual activity with fluorimetric detection. Inactivation of this human enzyme exhibited pseudo-first-order kinetics, suggesting that all reactive SH-groups have equivalent reactivity, and the second-order rate constant was calculated to be 3520 M-1min-1. Rat DPP III was hyperreactive to pHMB and showed biphasic kinetics indicating two classes of reactive SH-groups. The second-order rate constants of 3540 M-1s-1 for slower reacting sulfhydryl, and 21, 855 M-1s-1, for faster reacting sulfhydryl were obtained from slopes of linear plots of pseudo-first-order constants versus reagent concentration. Peptide substrates protected both mammalian DPPs III from inactivation by pHMB. Physiological concentrations of biological thiols and H2O2 inactivated the rat DPP III. Human enzyme was resistant to H2O2 attack and less affected by reduced glutathione than the rat homologue. A significantly lower DPP III level, determined by activity measurement and Western blotting, was found in the cytosols of highly oxygenated rat tissues. These results provide kinetic evidence that cysteine residues are involved in substrate binding of mammalian DPPs III.

The aim of this study was to evaluate soluble proteins of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-6 receptor subunit gp80 (sIL-6R gp80), as markers of multiple sclerosis (MS). Paired cerebrospinal fluid (CSF) and serum samples of 20 MS patients and 15 controls suffering from non-inflammatory neurological diseases have been assayed retrospectively using monoclonal antibodies-based ELISAs. While TNF-alpha could not be detected in CSF, it was measurable in 20% of total sera. Interleukin-6 was measurable in 5% of total CSF and in 10% of total sera only. However, soluble IL-6R gp80 protein subunit was readily measurable, showing sera concentration (pg/mL) about 34 times higher and specific content (pg/mg total protein) around five times lower than those in paired CSF, similarly for both group of patients. No significant difference of sIL-6R gp80 level, which could be disease-, gender- or age-related, and no correlation of CSF sIL-6R gp80 content with that of paired serum or with routine clinical data for CSF, have been observed. We have concluded that soluble proteins of TNF-alpha, IL-6 and sIL-6R gp80 assayed by monoclonal antibodies-based ELISAs could not serve as markers of the MS activity.

Tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) are proinflammatory cytokines upregulated in multiple sclerosis (MS). They act via specific receptors. They are also responsible for autoimmune response and demyelination in central nervous system. In order to evaluate TNF-alpha and IL-6 utility in the diagnosis of MS, we assayed these cytokines in 36 paired cerebrospinal fluid (CSF) and serum samples from active MS patients, and from 23 patients with non inflammatory neurological diseases (NIND) serving as a control group. One-step, monoclonal antibody-based "sandwich" ELISA was used to determine the levels of TNF-alpha directly, and the levels of IL-6 indirectly (by measuring his soluble receptor, sIL-6R). CSF contained no measurable amounts of TNF-alpha but it was measurable in some sera. Soluble IL-6R was, however, readily determined showing about 25 to 40 times higher concentrations in serum than in CSF. Linear correlation between sample protein content and sIL-6R concentration has not been demonstrated. Soluble IL-6R constitutes 3 - 6 times higher proportion of total proteins in CSF than those in serum. Regardless of assayed sample, no significant difference relating to the disease status, sex or age of the patient could be observed. It could be concluded that assays of tumor necrosis factor-alpha (TNF-alpha) and soluble interleukin-6 receptor (sIL-6R) in cerebrospinal fluid and/or serum of patients with relapsing-remitting multiple sclerosis are of no value as markers of the disease activity.

Interleukin-6 (IL-6) is a proinflammatory cytokine which mediates cellular responses during inflammation and immune activation. Elevated levels of IL-6 are found in multiple sclerosis (MS) lesions and cerebrospinal fluid (CSF). This cytokine acts through IL-6 receptors. Soluble forms of these receptors are released from the cell surface and can be detected in CSF and serum. The aim of this study was to measure CSF and serum levels of sIL-6R (gp80) in patients with relapsing-remitting MS during exacerbations. We studied 20 patients with relapsing-remitting MS and 19 patients with non-inflammatory neurological diseases (NIND). In MS patients CSF and serum samples were obtained during exacerbations of the disease. Soluble IL-6R (gp80) in CSF and serum was measured by ELISA method, and values (median) were expressed in pg/mL. Low levels of sIL-6R were detected in the CSF of MS (950) and NIND (975) patients and were not significantly different. Serum sIL-6R levels were approximately 25-40 times higher than CSF levels in MS (28750) and NIND (35250) patients and were not significantly different. We found no significant difference in levels of sIL-6R in the CSF and serum between MS and NIND patients. Therefore, analysis of sIL-6R in the CSF and serum of relapsing-remitting MS patients is of limited value as a marker of disease activity.

Heat shock proteins (Hsp) are a family of proteins whose synthesis is induced by heat shock and a wide variety of other stresses (e. g. anoxia, heavy metal ions, amino acid analogs). This induction represents a rapid and highly conserved response to proteotoxic insult. Due to development of drug-resistance, the population of drug-resistant cells could be selected, that would have increased basal level of Hsp70. The altered constitutive and inducible levels of Hsp70 in drug resistant cells may influence their response to combined hyperthermia and anticancer drug treatment. In the present study, the constitutive levels of Hsp70 and induction of these proteins by hyperthermia and two anticancer drugs (used for resistance development) were determined in cervical and laryngeal carcinoma cells. The levels of Hsp70 were quantified by Western blot. Constitutive levels of Hsp70 were similar in parental and drug-resistant cells suggesting that Hsp70 is not involved in drug-resistance. Hyperthermic treatment induced Hsp70 in all examined cell lines but with different kinetics between drug-resistant and parental cells. Following the treatment with anticancer drugs, Hsp70 was induced only in cisplatin-resistant laryngeal cells. In conclusion, kinetics of Hsp70 induction (stress-type and cell-type specific), may be different in drug-resistant cells as compared to parental cells. The observed alterations in Hsp70 induction in drug resistant and parental cells should be taken into account when combined treatments (i. e. hyperthermia and anticancer drugs) are planned

An enzyme test has been worked out for detecting organophosphorus compounds in water. The test is based on the inhibition of cholinesterase. The detection limits for the "nerve gases" are (micrograms per liter): Soman 0.12, VX 5.9, Sarin 9.9 and Tabun 26. The detection limit for the organophosphorus pesticide dichlorvos is 50 micrograms per liter.

U radu su prikazani pouzdani i brzi testovi za kvalitativno određivanje "in situ" alkaloida, cijanida, herbicida i toksičnih kovina u vodi za piće. Testovi su razrađeni od znanstvenih suradnika Instituta "Ruđer Bošković", a "Pliva", Zagreb-Farmaceutika je oblikovala i proizvela odgovarajuće komplete za upotrebu na terenu.

It is general accepted that both B- and T- lymphocyte defects are responsible for frequently observed hypogammaglobulinemia in CLL patients. In this study we have measured the ability of cultivated CLL B-lymphocytes to synthesize IgM or IgG spontaneously or after stimulation with LPS, rIFN-alpha or PWM. Among 30 CLL patients the spontaneous IgM and/or IgG production varied in a wide range from 0 to 500 and 880 ng/ml respectively. LPS alone or in combination with rIFN-alpha stimulated proliferation of B- lymphocytes, accompanied only sporadically by their differentiation into IgM but never into IgG producing cells. On the other hand PWM stimulated both proliferation and differentiation of B- lymphocytes, but again preferentially into IgM secreting cells. These data suggest that CLL B- lymphocytes upon in vitro stimulation might, either proliferate and/or differentiate or do not respond at all depending on the level of their differentiation block. The type of reaction most probably reflects intraclonal heterogeneity among defective CLL B-lymphocytes.

Previous studies from this laboratory have shown that in bacteria E.coli by whitdrawal of ammonium ions from glucose minimal media, an enzymatic system is induced by which exogenous thymine and uracil are catabolised with the release of C-2 as CO2. In the present investigations the response of the catabolic system to various structural analogues and metabolic inhibitors, as well as the control role of glutamine synthetase have been studied in some mutants of E.coli K12 and the following observed: (1) Catabolism of both bases is inhibited by 5-halo uracils, 5-aminouracil, 6-aminouracil, while dihydrouracil and barbituric acid are without effect. (2) The catabolic system is oxygen dependent and sensitive to N-ethylmaleimide, dinitrophenol, KCN and azide. (3) The inhibitory effect of diazosulfanilic acid is less pronounced on uracil degradation than on uracil transport occuring through mediation of uracil phosphoribosyltransferase. (4) Mutants deficient in glutamine synthetase are not able to induce pyrimidine degrading enzyme(s) indicating that glutamine synthetase serves as positive control factor in the synthesis of catabolic enzyme(s).

Bacteria, thallophytes, and seed plants (107 species), supplied with exogenous indole-3-ethanol (tryptophol), formed one or more of the following metabolites: O-acetyl tryptophol, an unknown tryptophol ester (or a set of structurally closely related esters), tryptophol glucoside, tryptophol galactoside, indole-3-acetic acid (IAA), and indole-3-carboxylic acid. The unknown ester was formed by all species examined; O-acetyl tryptophol appeared sporadically in representatives of most major taxonomic groups. Tryptophol galactoside was found in the algae Chlorella, Euglena, and Ochromonas. The glucoside was formed by many eucaryotic plants, but not by bacteria; it was a significant tryptophol metabolite in vascular plants. IAA, if detectable at all, was usually a minor metabolite, as should be expected, if tryptophol oxidase responds to feedback inhibition by IAA. Indole-3-carboxylic acid, formed by a few fungi and mosses, was the only tryptophol metabolite detected which is likely to be formed via IAA.

The present work deals with experiments made to characterise transport of thymine and uracil in E.coli K12S grown in the glucose-mineral medium. Using a membrane filter method and 2-14C labelled bases, total uptake was determined by measuring radioactivity dissapearance from the medium, incorporation, pool formation and evolution of 14CO2. In glucose mineral medium, the majority of 2-14C uracil (0.5 micromolar) was taken up and retained by the cells, while a small portion was degraded to 14CO2. 2-14C thymine under the same conditions, did not enter the cells. With the bacteria preincubated in the medium without ammonium ions, both bases dissapeared from the medium at approximately the same initial rate. The fact that all radioactivity from thymine and a majority from uracil was recovered in 14CO2, indicates that in the absence of ammonium ions transport system for thymine was induced. This was supported by the finding that the presence of chloramphenicol decreased the total uptake of thymine but not that of uracil. In this case however, the majority of radioactivity from uracil remained undegraded within the cells. N-Ethylmaleimide, and to a lesser extent KCN, inhibited the total uptake of both bases, while sodium azide interferred only with incorporation and pool formation of 2-14C uracil and its derivatives. The results suggest a regulatory effect of ammonium ions in the active transport of pyrimidine bases.

Humana serumska holinesteraza (EC 3.1.1.8) je enzim koji hidrolizira estere holina. Sintetizira se u jetri i krvlju raznosi u ostala tkiva i tjelesne tekućine. Fiziološka uloga serumske holinesteraze nije sasvim razjašnjena no snižena aktivnost ovog enzima prati određena patološka stanja organizma izazvana jetrenim i drugim oboljenjima, izvjesnim drogama i hormonima, te trovanjem organo-fosfornim spojevima, i neposredan je uzrok abnormalno produženog zastoja disanja koji se javlja pri anesteziji nekih pacijenata nakon primjene neuromuskularnog relaksanta, sukcinil holina. Idealna metoda za određivanje serumske holinesteraze upotrebljiva u kliničkoj i preventivnoj dijagnostici, toksikologiji i sudskoj medicini, trebala bi biti jednostavna, brza, pouzdana, osjetljiva i specifična. Enzimski imuno testovi svojim karakteristikama najbliži su navedenim zahtjevima, pa je u tom smislu razvijen heterogeni enzim-antigen imuno test (EAIT) za određivanje i karakterizaciju serumske holinesteraze. Princip metode sastoji se u mjerenju signala kojeg proizvodi enzim, koji je ujedno i antigen, kad se veže na specifična poliklonalna ili monoklonalna antitijela adsorbirana na plastičnu podlogu. Imunoglobulinska frakcija s poliklonalnim antitijelima specifičnim za holinesterazu, dobivena je frakcionim taloženjem kunićevog antiseruma s amonijevim sulfatom, te kromatografijom na DEAE-Sephadexu A 50. Monoklonalna antitijela protiv holinesteraze proizvedena su standardnom procedurom fuzije stanica slezene imuniziranih BALB/c miševa i mieloma linije (X63-Ag 8.6.5.3), te imunopurifikacijom ascitne tekućine. Optimizirani test upotrijebljen je za rutinsko određivanje i karakterizaciju humane serumske holinesteraze, a dobiveni rezultati u skladu su s njegovim očekivanim karakteristikama.

Moderne metode dijagnostike i terapije tumora zasnivaju se na interakciji karakterističnih antigena i specifičnih antitijela. U nas je vrlo raširen invazivni duktalni karcinom dojke, pa postoji interes za razvoj odgovarajuće metode za njegovu detekciju. Koristeći ekstrakt tumora kao izvor antigena, a limfoblaste imuniziranih miševa i mijelomske stanice za fuziju, priređen je hibridom CDI 315, koji luči specifična monoklonska antitijela. Monoklonska antitijela su reagirala s ekstraktima karcinoma dojke, a nisu se vezala na zdravo tkivo i dobroćudne tumore dojke. Slaba unakrsna reakcija nađena je s zdravim i tumorskim tkivom rektuma, metastatskim melanomom i liposarkomom. Za antigen je pokazano da je proteinske prirode, a za relativnu molekulsku masu se procjenjuje da je veća od 500 000.

For routine determination of cholinesterase (ChE) in human sera samples, an enzyme-antigen immuno assay was developed. The method is based on selective immuno adsorption of ChE from the serum sample onto (plastic) surface of microtiter plate coated by ChE-specific rabbit polyclonal antibodies, and subsequent measurement of the enzymatic activity of such immobilized antigen by Ellman's reaction.

Prisustvo specifičnih antitijela u serumu svinja bitan je parametar za rano otkrivanje bolesti Aujeszkoga u ovih životinja. U cilju razvoja pouzdane i brže metode za određivanje ovih antitijela, osnovane na enzim-imunološkoj analizi, napravili smo monoklonska antitijela protiv virusa SH1 (Suis Herpesvirus 1), soj B-KAL 58. Nakon spajanja limfoblasta iz slezene imuniziranih miševa i mijelomskih stanica x63 Ag 8.653, odabira hibridoma i rekloniranja, izdvojili smo 4 linije stanica sa stabilnom produkcijom antitijela specifičnih za SH1 B-KAL 68 (SH78, SH125, SH281, SH320). U toku su ispitivanja antigenske specifičnosti, obzirom na pojedine glukoproteine virusnog omotača.

U sintetskoj hranjivoj podlozi opskrbljenoj optimalnom količinom anorganskog dušika (amonijevi ioni), samo timinski auksotrofi bakterija Escherichia coli metaboliziraju izvana dodani timin, koristeći ga za biosintezu timinskih deoksiribonukleotida i DNA. Uklanjanjem amonijevih iona iz medija, u stanicama E.coli inducira se sistem kataboličkih enzima koji razgrađuju egzogeni timin. Uslijed smanjenja raspoloživih količina esencijalnog metabolita izazvanog njegovom degradacijom, timinski auksotrofi gube sposobnost vijabilnosti pokazujući fenomen poznat pod nazivom "smrt zbog nedostatka timina" dok prototrofne stanice rastu brže koristeći timin kao dodatni izvor dušika. Katabolizam timina studiran je in vivo i in vitro na raznim mutantima bakterija E.coli, uz primjenu 2-14C ili metil-14C obilježene timinske molekule. Razgradnja 2-14C timina u stanicama i staničnim ekstraktima manifestira se kvantitativnom pojavom ureidnog CO2. Spomenuti proces karakterizira sve ispitane sojeve osim stanica mutanta K12SG1nA s inaktivnom glutamin sintetazom, što znači da je ovaj enzim na neki način uključen u regulaciju timinske razgradnje. Indukcija kataboličkih enzima može se spriječiti prisustvom kloramfenikola ili amonijevih iona kao i izostavljanjem energetskog izvora (glukoze) iz hranjive podloge. Razgradnja timina ubrzava se dodatkom aminokiselina (posebno glutaminske i asparaginske) u medij za uzgoj bakterija, a rezultati in vivo pokusa pokazuju da je degradativan proces ovisan o respiraciji. Kompeticija uracila te niza njegovih analogona supstituiranih u položaju C5 ili C6 pirimidinskog prstena za sistem enzima koji razgrađuju timin, sugerira da katabolički proces započinje transformacijom tog dijela molekule. Primjenom 2-14C dihidrotimina, 14C ureje i metil-14C malonske kiseline pokazano je da ovi (među)produkti oksidativnog odnosno reduktivnog mehanizma razgradnje opisanih za druge organizme, nisu uključeni u degradaciju timina u E.coli. Kromatografske analize pokazale su da se katabolizam 2-14C timina do ureidnog CO2 odvija bez pojave radioaktivnih intermedijera razgradnog procesa. U rješavanju reakcijskog mehanizma degradacije ispitana je i razgradnja metil-14C timina. Za razliku od staničnih ekstrakata koji ga oksidiraju do 14CO2 bez nastajanja detektabilnih katabolita, stanice kvantitativno transformiraju metil-14C timin u dva, za sada neidentificirana, metabolita izlučujući ih u medij.

Experiments designed to elucidate the mechanism of uracil degradation by E.coli K12S showed that in contrast to uracil, dihydrouracil - the postulated intermediate of a reductive mechanism - did not stimulate the growth of bacteria as additional source of nitrogen, nor it was catabolized to ureido carbon dioxide. However, the chromatographic analysis of dihydrouracil metabolic products, revealed the presence of an enzyme converting dihydrouracil to beta- ureidopropionic acid. Results of growth and biochemical studies indicated that barbituric acid - the postulated intermediate of an oxidative pathway - is not involved in uracil degradation.

Constantly increasing possibilities of proteolytic enzymes application, create a need for the new types of enzymes. A good source to search for them are Streptomyces species known to synthesize a great veriety of proteases. From Streptomyces rimosus culture filtrates waste waters of antibiotic production, a crude enzyme mixture and 4 electrophoretically homogenous enzymes were prepared. Molecular weight, isoelectric point, stability, pH and temperature optimum, preferential substrates and susceptibility to synthetic and natural inhibitors, were determined for the isolated enzymes. Their properties suggest classification as leucine aminopeptidase, chymotrypsin-like, trypsin-like and alkine metallo proteinase. Crude enzyme mixture was used as an active component for the bating agent Encipon, whose performance was compared with that of pancreatic and Bacillus enzymes preparations. Analysis of degradation products and leather quality have shown that S. rimosus enzymes are suitable for hides and skins treatment.

Preparations of N-[3-(hetero)aryl-aminocarbonyl]propanoyl- L-proline derivatives 12, 13, 22 and 23 are described. A novel approach consists of fusing (80-100 °C) N-(hetero)aryl-succinimides (7, 8, 20, 21) with unprotected L-proline and imidazole in the presence of dimethylformamide. In vitro tests for angiotensin converting enzyme (ACE) inhibition showed that all N-(arylaminocarbonyl)- propanoyl-L-prolines (12, 13, 22 and 23) exhibit lower activity than captopril (1), their IC50's ranging from 2.5 X 10-4 to 3.3 X 10-3 M.

In media suboptimally supplied with a nitrogen source, bacteria E.coli catabolise pyrimidines, thymine and uracil, to ureido-CO2. The aim of investigations reported here was to establish which of the two known degradation mechanisms - oxidative via barbituric acids or reductive via dihydropyrimidines, might be operating in E. coli K12S. By combining the growth- and biochemical experimenths, it was shown that clue intermediates of above pathways do not affect the bacterial growth and are not catabolised to ureido-CO2. Since urea only was identified as a catabolite but bacteria showed no urease activity, it was concluded that pyrimidine degradation in E.coli K12S starved for nitrogen source, proceeds by different mechanism.

Although pyrimidine metabolism in E.coli has been intensively investigated, the data on the catabolism of thymine and uracil and its regulation are still lacking. We report the evidence for breakdown of thymine and uracil to CO2, NH3 and urea via respective barbituric acids. In bacteria grown in media optimally supplied with carbon and nitrogen source or in media in which pyrimidines were used as sole carbon source, the activity of degradative enzymes could not be detected. The induction of catabolic enzymes took place in glucose-media supplemented with suboptimal concentrations of NH4+ salts or with different amino acids as source of nitrogen. Since the initiation and the rate of breakdown depended on amino acid used as nitrogen source, it is reasonable to assume that the induction of catabolic enzymes is somehow regulated by the amount of nitrogen metabolically available in the cell.