Lipoperoxidation rates and drug-oxidizing enzyme activities in the liver and placenta of some mammal species during the perinatal period

Summary Lipoperoxidation and drug-metabolizing enzymes were measured in livers and placentas of different mammal species during the perinatal period. In placentas and fetal livers of rat, rabbit and guinea-pig, cofactor-supported lipoperoxidation was negligible, as were the activities of drug-oxidizing enzymes. Human fetal liver contained an intact drug-oxidizing electron transport chain, and lipoperoxidation activity was accordingly observed. It is suggested that lesions mediated by lipoperoxidation may be possible in human fetus, but they are less probable in animal fetuses.The skillful technical assistance of Ms.Liisa Tuhkanen and Ms.Vuokko Väisänen is gratefully acknowledged.     

Debriding ability of a novel multi-enzyme preparation isolated from Antarctic krill (Euphausia superba)

Summary The wound-debriding activity of various types of proteolytic enzymes and proteases from Antarctic krill (multi-enzyme system consisting of both endo- and exopeptidases) was evaluated. The results, based on the enzymatically acieved weight reduction of a necrotic animal material (excised rat skin) in vitro, clearly showed that the multi-enzyme system (krill) had a higher degrading activity than the single enzyme preparation, or that with only a few enzymes. The debriding effect of the krill enzymes was markedly related to the enzyme concentration, resulting in 70–100% substrate degradation after 24 h. The digesting capacity of trypsin reached about 50%, but an increase in concentration of this enzyme did not substantially influence its overall activity. The effect of streptokinase-streptodornase, collagenase and plasmin-desoxyribonuclease was weak (10–20% digested).     

Presence of a partial urea cycle in the leech, Poecilobdella granulosa.

Ornithine carbamoyltransferase (OCT) and arginase, but not arginine synthetase (AS), were detected in the body wall and gut tissues of the leech. The activities of these enzymes were not altered by starvation. The high activity of arginase in body wall is probably due to the association of the latter with botryoidal tissue. Hirudineans, which evolved from oligochaete ancestors, appear to have lost the citrulline-arginine segment of the urea cycle due to their ammonotelic mode of nitrogen excretion.     

Presence of a partial urea cycle in the leech,Poecilobdella granulosa

Ornithine carbamoyltransferase (OCT) and arginase, but not arginine synthetase (AS), were detected in the body wall and gut tissues of the leech. The activities of these enzymes were not altered by starvation. The high activity of arginase in body wall is probably due to the association of the latter with botryoidal tissue. Hirudineans, which evolved from oligochaete ancestors, appear to have lost the citrulline-arginine segment of the urea cycle due to their ammonotelic mode of nitrogen excretion.S. N. and B. J. thank the Council of Scientific & Industrial Research and the Department of Ocean Development, Government of India, respectively for the award of research fellowships.     

Diversity in enoyl-acyl carrier protein reductases

The enoyl-acyl carrier protein reductase (ENR) is the last enzyme in the fatty acid elongation cycle. Unlike most enzymes in this essential pathway, ENR displays an unusual diversity among organisms. The growing interest in ENRs is mainly due to the fact that a variety of both synthetic and natural antibacterial compounds are shown to specifically target their activity. The primary anti-tuberculosis drug, isoniazid, and the broadly used antibacterial compound, triclosan, both target this enzyme. In this review, we discuss the diversity of ENRs, and their inhibitors in the light of current research progress. Received 3 November 2008; received after revision 5 December 2008; accepted 8 December 2008     

Relationship between the renal kallikrein activity and the urinary excretion of kallikrein in rats

Summary Adrenalectomy reduces, and sodium depletion increases, both the daily urinary excretion of kallikrein and the kallikrein activity in the renal cortex. These 2 variables were found to correlate significantly in normal, sodium depleted and adrenalectomized rats, thus supporting the view that kallikrein excretion reflects the activity of the enzyme in the kidney.Acknowledgments. This word was supported by the Deutsche Forschungsgemeinschaft. The technical assistance of Mrs G. Breypohl and Ms J. Kopatsch is gratefully acknowledged. Dr M. Marin-Grez was a fellow of the Alexander von Humbodt Foundation and Dr G. Bönner of the Deutsche Forschungsgemeinschaft.     

On the distribution of plasma L-asparaginase

Summary The guinea-pig,Cavia porcellus, is unusual in possessing plasma L-asparaginase, an enzyme with anti-tumor activity. 21 additional species have been examined as to the presence of this enzyme: the results confirm and extend its remarkably limited species distribution.This work was supported in part by grant GM/CA 25378 from the National Institute of General Medical Sciences, National Institutes of Health.We are grateful to dr Hanna Hensch, Philadelphia Zoological Garden, and Ms Joan Whitla and Dr Richard J. Montali, National Zoological Park/Smithsonian Institution, for samples of frozen serum.To whom correspondence should be addressed.     

Intragenic complementation and the structure and function of argininosuccinate lyase

Argininosuccinate lyase (ASL) catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate, a reaction important for the detoxification of ammonia via the urea cycle and for arginine biosynthesis. ASL belongs to a superfamily of structurally related enzymes, all of which function as tetramers and catalyze similar reactions in which fumarate is one of the products. Genetic defects in the ASL gene result in the autosomal recessive disorder argininosuccinic aciduria. This disorder has considerable clinical and genetic heterogeneity and also exhibits extensive intragenic complementation. Intragenic complementation is a phenomenon that occurs when a multimeric protein is formed from subunits produced by different mutant alleles of a gene. The resulting hybrid protein exhibits greater enzymatic activity than is found in either of the homomeric mutant proteins. This review describes the structure and function of ASL and its homologue delta crystallin, the genetic defects associated with argininosuccinic aciduria and current theories regarding complementation in this protein.     

Enzymic basis for the nutritional requirement of arginine in insects

Summary The fat body of the cockroach, Blaberus cranifera, and the silkmoth, Hyalophora gloveri, has been tested for some enzymes of the urea cycle. While ornithine transcarbamoylase and argininosuccinase activities could not be detected, arginase is present in the fat body of these insects. These results explain the essentiality of arginine in the diet of insects.This work was supported by grants from the National Science Foundation (GB-4524 and GB-8172) and the U. S. Public Health Service (AI 05006).Recipient of a Fulbright Travel Grant from the USEFI (New Delhi)     

Mitochondrial abnormalities in fibroblast line GM3093 defective in oxidative metabolism

Fibroblast line GM3093 deficient in the activity of the pyruvate dehydrogenase complex, was derived from a patient reported to have an inherited defect affecting the tricarboxylic acid cycle. Our results suggest a generalized defect consisting of few and abnormal mitochondria and low activities of all mitochondrial enzymes examined.     

Inactivation of human glutamate dehydrogenase by aluminum

Aluminum inactivated glutamate dehydrogenase (GDH) by a pseudo-first-order reaction at micromolar concentrations. A double-reciprocal plot gave a straight line with a k_inact of 2.7 min^-1 and indicated the presence of a binding step prior to inactivation. The inactivation was strictly pH dependent and a marked increase in sensitivity to aluminum was observed as the pH decreased. At a pH higher than 8.5, no inactivation was observed. The completely inactivated GDH contained 2 mol of aluminum per mole of enzyme subunit monomer. When preincubated with enzyme, several chelators such as citrate, NaF, N-(2-hydroxyethyl) ethylenediaminetriacetic acid or ethylenediaminetriacetic acid efficiently protected the enzyme against the aluminum inactivation. In a related experiment, only citrate and NaF released the aluminum from the completely inactivated aluminum-enzyme complex and fully recovered the enzyme activity. Ferritin, NADP⁺, or nerve growth factor did not show any effects on the recovery of the aluminum-inactivated GDH activity. The dissociation constant for the aluminum-enzyme complex was calculated to be 5.3 M. Although aluminum has been known to form a complex with nucleotides, no such effects were observed in the inactivation of GDH by aluminum as determined using GDHs mutated at the ADP-binding site, NAD⁺-binding site or GTP-binding site. Circular dichroism studies showed that the binding of aluminum to the enzyme induced a decrease in helices and sheets and an increase in random coil. Therefore, inactivation of GDH by aluminum is suggested to be due to the conformational change induced by aluminum binding. These results suggest a possibility that aluminum-induced alterations in enzymes of the glutamate system may be one of the causes of aluminum-induced neurotoxicity.Received 25 July 2003; received after revision 27 August 2003; accepted 15 September 2003     

Tubulin pools in human erythrocytes: altered distribution in hypertensive patients affects Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase activity

The presence of tubulin in human erythrocytes was demonstrated using five different antibodies. Tubulin was distributed among three operationally distinguishable pools: membrane, sedimentable structure and soluble fraction. It is known that in erythrocytes from hypertensive subjects (HS), the Na⁺, K⁺-ATPase (NKA) activity is partially inhibited as compared with erythrocytes from normal subjects (NS). In erythrocytes from HS the membrane tubulin pool is increased by ~150%. NKA was found to be forming a complex with acetylated tubulin that results in inhibition of enzymes. This complex was also increased in erythrocytes from HS. Treatment of erythrocytes from HS with nocodazol caused a decrease of acetylated tubulin in the membrane and stimulation of NKA activity, whereas taxol treatment on erythrocytes from NS had the opposite effect. These results suggest that, in erythrocytes from HS, tubulin was translocated to the membrane, where it associated with NKA with the consequent enzyme inhibition.     

Comparative study of oxalate oxidase in three genotypes ofSorghum vulgare

Summary The presence of an oxalate oxidase (EC 1.2.3.4) has been demonstrated in 15,000×g supernatants prepared from 10-day-old seedlings of three genotypes ofSorghum vulgare: grain sorghum hybrid (CSH-5), grain-cum-forage sorghum (PC-6) and forage sorghum (PC-1). The specific activity of the enzyme in the different tissues of seedlings was found to be present in the order leaves > stems > roots in PC-6 and PC-1, but this order was reversed in CSH-5. A comparison of the different properties of the leaf enzyme of these three genotypes of sorghum revealed that the enzyme has maximum activity in the acidic pH range from 4.0 to 5.0 and in the temperature range from 37°C to 40°C. The enzyme was stimulated by Cu²⁺ and Fe²⁺. The rate of H₂O₂ formation in the enzyme reaction was linear up to 5 min and was stoichiometrically related to oxalate consumption. The enzyme is unaffected by Na⁺ at physiological concentration (0.15 M). The superiority of this enzyme over moss and other plant enzymes for enzymic determination of urinary oxalate is discussed.     

Mitochondrial abnormalities in fibroblast line GM3093 defective in oxidative metabolism

Summary Fibroblast line GM3093 deficient in the activity of the pyruvate dehydrogenase complex, was derived from a patient reported to have an inherited defect affecting the tricarboxylic acid cycle. Our results suggest a generalized defect consisting of few and abnormal mitochondria and low activities of all mitochondrial enzymes examined.Preliminary reports of aspects of this work appeared as abstracts in Trans. Am. Soc. Neurochem.15 (1984) 178, and Soc. Neurosci.10 (1984) 996.Developmental and Metabolic Neurology Branch.Surgical Neurology Branch.     

Resistance of purified cholera toxin to enzymatic treatment with pancreatic elastase and papain

Summary Treatment of Cholera toxin with pancreatic elastase and papain in vitro shoed a high resistance of the toxin molecule to these enzymes, under non-denaturing conditions or in the presence of 2M urea. These experiments support the hypothesis of a particularly stable molecular structure of the toxin, as an explanation of its activity in the intestinal lumen where the pancreatic proteases are active.     

Snake venom action: are enzymes involved in it?

Enzymes were the first clearly recognized components of snake venoms. When several more were discovered, attempts were made to correlate venom action with enzymic functions. The last few years have seen most successful efforts in the identification, isolation and structrual elucidation of highly toxic polypeptides present in snake venoms, in particular of 'neurotoxins' and membrane-active toxins. Following this development the polypeptides were called the true toxic components and the enzymes lost their previous central position in venom pharmacology. The time, therefore, has come re-evaluate the role of enzymes in the complex interaction between snake and prey. While highly active polypeptides indeed dominate the actionof hydrophiid venoms, they appear to play a lesser role in crotalid venom action as compared with enzyme components. Enzymes are involved in many levels of venom action, e.g. by serving as spreading factors, of by producing very active agents, such as bradykinin and lysolecithins in tissues of preys or predators. Some toxins, e.g. the membrane-active polypeptides appear to participate in the interaction between membrane phospholipids and venom phospholipases. The classical neurotoxin, beta-bungarotoxin, has been recognized as a powerful phospholipase. Several instances are known which indicate that some enzymes potentiate the toxic action of others; the analysis of a single enzyme may, therefore, not fully reveal its biofunction. For 3 enzymes,ophidian L-amino acid oxicase, ATPpyrophosphatase, and acetylcholinesterase, some of the problems pertaining to venom toxicity are discussed.     

Proteasomes in apoptosis: villains or guardians?

The proteasome (multicatalytic proteinase complex, prosome) is a major cytoplasmic proteolytic enzyme, responsible for degradation of the vast majority of intracellular proteins. Proteins degraded by the proteasome are usually tagged with multiple ubiquitin moieties, conjugated to the substrates by a complicated cascade of enzymes. Over the last years, evidence has accumulated that changes in the expression and activity of the different components of the ubiquitin-proteasome system occur during apoptosis. Proteasome inhibitors have been used to induce apoptosis in various cell types, whereas in others, these compounds were able to prevent apoptosis induced by different stimuli. The proteasome mediated step(s) in apoptosis is located upstream of mitochondrial changes and caspase activation, and can involve in different systems Bcl-2, Jun N-terminal kinase, heat shock proteins, Myc, p53, polyamines and other factors.     

Photon emission of phagocytes in relation to stress and disease

Phagocytes, the first-line cells of the body's defence mechanisms against invading pathogens, kill microorganisms by means of lysosomal degradative enzymes and highly toxic reactive oxygen intermediates. The reactive oxygen compounds are produced, in a process called the ‘respiratory burst’, by the NADPH oxidase complex in plasma membranes, and by myeloperoxidase in phagolysosomes after degranulation. These processes generate electronically excited states which, on relaxation, emit photons, giving rise to phagocyte chemiluminescence (CL). This paper describes the conditions for the measurement of CL, and reviews the activity of phagocytes from individuals undergoing stress or disease. The capability of phagocytes to emit photons reflects remarkably well the pathophysiological state of the host. In many cases even the magnitude of the stress, the presence of a pathogen in the body, or the activity of the disease can be estimated. Physiological changes, e.g. in the reproductive cycle, can also be predicted.     

Epigenetic mechanisms in mammals

DNA and histone methylation are linked and subjected to mitotic inheritance in mammals. Yet how methylation is propagated and maintained between successive cell divisions is not fully understood. A series of enzyme families that can add methylation marks to cytosine nucleobases, and lysine and arginine amino acid residues has been discovered. Apart from methyltransferases, there are also histone modification enzymes and accessory proteins, which can facilitate and/or target epigenetic marks. Several lysine and arginine demethylases have been discovered recently, and the presence of an active DNA demethylase is speculated in mammalian cells. A mammalian methyl DNA binding protein MBD2 and de novo DNA methyltransferase DNMT3A and DNMT3B are shown experimentally to possess DNA demethylase activity. Thus, complex mammalian epigenetic mechanisms appear to be dynamic yet reversible along with a well-choreographed set of events that take place during mammalian development.     

A thiol protease of peritoneal macrophages in the guinea pig

Proteolytic enzymes of the guinea pig peritoneal exudate macrophages were investigated using synthetic fluorogenic peptide substrates. Among several enzymes, t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide cleaving enzymes had the highest activity, and the activity in exudate macrophages was about 3 times stronger than that in resident macrophages. The molecular weight of the enzyme was around 35,000 and optimal pH around 6.5-7.0. It was inhibited by thiol-blocking reagents, suggesting a thiol protease.