Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Summary Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed and increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and -N-acetyl-d-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and -glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the German Research Foundation (Sfb 174)     

Tetrazoliummethoden zum histochemischen Hydrolasennachweis

Zusammenfassung An gefriergetrockneten und Schnitten von nativem oder aldehyd-fixiertem Material wird die Eignung von Nitro BT (NBT), Tetranitro BT (TNBT), Distyryl Nitro BT (DS-NBT), Thiocarbamyl Nitro BT (TC-NBT) und Benzothiazolylstyrylphthalhydrazidyltetrazoliumcholorid (BSPT) als Hilfsreagentien zur Lokalisation von Glykosidasen, Phosphatasen und unspezifischen Esterasen mit Indoxylsubstraten an Rattenorganen geprüft.Mit NBT und TNBT lassen sich die saure -d-Galactosidase, -d-N-Acetylglucosaminidase, saure Phosphatase, Neuraminidase und unspezifische Esterase höchstens auf Zellebene nachweisen; präziser kann die Lactase--d-glucosidase im intestinalen Bürstensaum dargestellt werden. Die besten Resultate liefert die Untersuchung der alkalischen Phosphatase; mit TNBT ist das Tetrazoliumverfahren unter allen bisher beschriebenen Reaktionen die Methode der Wahl für die lichtmikroskopische Darstellung dieses Enzyms.Mit BSPT als Tetrazoliumsalz und anschließender Osmierung seines Formazans lassen sich alle sauren und neutralen Hydrolasen exakt in Lysosomen, Sekretgranula, Cytoplasma und/oder Mikrovilli fassen; gleichzeitig sind damit ultracytochemische Studien möglich. Die alkalische Phosphatase reagiert mit BSPT nur an hochaktiven Stellen positiv.-DS- und TC-NBT sind NBT, TNBT und BSPT unterlegen.

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Tetrazolium methods for the histochemical investigation of hydrolases

Summary Using freeze-dried or sections from fresh-frozen or aldehyde-fixed material nitro BT (NBT), tetranitro BT (TNBT), distyryl nitro BT (DS-NBT), thiocarbamyl nitro BT (TC-NBT) or benzothiazolylstyrylphthalhydrazidyl tetrazolium chloride (BSPT) were tested as auxiliary reagents for the localization of glycosidases, phosphatases and non-specific esterases with indoxyl substrates in rat tissues.By means of NBT or TNBT as a tetrazolium salt acid -d-galactosidase, -d-N-acetylglucosaminidase, acid phosphatase, neuraminidase and non-specific esterase can only be localized at the cellular level; a more precise localization is possible for lactase--d-glucosidase in the intestinal brush border, and the best results are obtained in the demonstration of alkaline phosphatase; among all methods described previously the tetrazolium procedure with TNBT is the method of choice for the light microscopic localization of this enzyme.Reverse data are observed with BSPT as a tetrazolium salt; then, all acid and neutral hydrolases can be exactly localized in lysosomes, secretion granules, cytoplasm and/or microvilli of many cells and tissues provided BSPT-formazan is stabilized by osmification. Furthermore, this procedure enables the reliable ultracytochemical demonstration of these enzymes. However, in the case of alkaline phosphatase only sites with high enzyme activity reveal a positive reaction.-DS- and TC-NBT are inferior to NBT, TNBT or BSPT.

     

Glycoside hydrolases of rumen bacteria and protozoa

Sixteen strains of rumen bacteria and 21 protozoal preparations were screened for glycoside hydrolase and phosphatase activity, using 22 nitrophenyl glycoside substrates. The range and level of bacterial enzyme activities were species dependent, although, the glycosidases associated with plant cell wall breakdown were most active in the cellulolytic and hemicellulolytic species. Alkaline phosphatase occurred widely in the organisms examined, but was most active in the twoBacteroides ruminicola strains.A wide range of enzyme activities was also detected in the holotrich and Entodiniomorphid ciliates isolated from the rumen or cultured in vitro. The glycosidases involved in cellulose and hemicellulose breakdown were detected in all of the protozoa examined, and, with the exception ofEntodinium spp., were most active in the Entodiniomorphid protozoa; -l-arabinofuranosidase, an essential hemicellulolytic glycoside hydrolase, was particularly active in this latter group of ciliates.     

The distribution of acid phosphatases and esterases in differentiating roots of Vicia faba

Summary Lateral roots of Vicia faba have been examined cytochemically to determine the distribution of naphthol AS esterases, bromo-indoxyl esterases, acid -glycerophosphatases and acid naphthol AS phosphatases in the various tissues during differentiation. Attempts have been made to correlate the observed differences in the distribution of the hydrolases with respect to physiological and to structural differentiation processes in cells and tissues.     

Intracellular and extracellular CM-cellulase and β-glucosidase activity during germination of Polysphondylium pallidum microcysts

Germinating microcysts of Polysphondylium pallidum possess the ability to digest carboxymethyl cellulose to its glucose subunits. This CM-cellulase activity, which is cycloheximide-sensitive, increases several-fold intracellularly during germination and is excreted into the extracellular medium. Augmentation of the extracellular level of cellulase enhances emergence of amoebae from microcysts, suggesting that this activity is critical for germination. The inhibition of -glucosidase activity with d-gluconic acid lactone also inhibits emergence, implicating this enzyme in germination as well.     

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

Background and Aims

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.

Methods

The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.

Key Results

Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.

Conclusions

In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.     

Enzymatic and morphological response of the thymus to drugs in normal and zinc-deficient pregnant rats and their fetuses

Summary In the thymus of normally fed pregnant rats the plasma membrane enzymes dipeptidyl peptidase IV (DPP IV) and alkaline phosphatase (alP) were found in cortical and medullary lymphocytes (thymocytes). Plasma membrane aminopeptidase A (APA) and adenosine monophosphate hydrolysing phosphatase (AMPP) were present in cortical reticular cells. In medullary reticular cells, aminopeptidase M (APM), -glutamyl transferase (GGT), adenosine triphosphate (ATPP) and thiamine pyrophosphate (TPPP) cleaving phosphatases were detected. Medullary reticular cells did not contain APA. Lysosomal DPP I and II, acid phosphatase, acid -d-galactosidase, -d-N-acetylglucosaminidase, -d-glucuronidase and non-specific esterases occurred especially in macrophages at the corticomedullary junction. The 21-day-old fetal thymus showed a similar reaction pattern as the maternal organ except for APA which was absent before birth.—After treatment of the pregnant rats with valproic acid (VPA), salicylic acid (SA), streptozotocin (ST) and retinoic acid (RA) APA showed an increase in activity in the thymic cortex. In addition, ST and RA induced AMPP, ATPP and TPPP activity in cortical reticular cells up to the same pattern as in medullary reticular cells. After ethanol (ET) administration severe damages occurred. The thymic cortex was free of DPP IV-positive lymphocytes; the medullary reticular cells showed reduced or no GGT and occasionally an increased APM activity. Dexamethasone (DEXA) given to normal or zinc-deficient rats produced the most severe lesions; thymocytes with DPP IV activity were completely absent in the cortex and medulla. In Zn-deficient pregnant rats similar alterations were observed as after ET. When the drugs were applied to Zn-deficient pregnant rats, the alterations resembled those observed after drug treatment alone. In all cases of severe thymus degeneration, i.e. ET and DEXA treatment and Zn-deficiency, the number of macrophages and activities of lysosomal hydrolases in macrophages and reticular cells were increased; the lysosomal hydrolases were often homogeneously distributed over the cortex. Cell contacts between reticular cells and lymphocytes were reduced. Vacuoles occurred within the reticular cells.—The fetal thymus was reduced in size and the number of macrophages and the activities of their lysosomal enzymes were increased after Zn-deficiency, DEXA treatment and Zn-deficiency combined with ET administration.Supported by the Deutsche Forschungsgemeinschaft (Sfb 174)     

Localization of glycosidases with naphthyl substrates

Synopsis Unsubstituted naphthyl substrates were found to be superior to substituted naphthyl, indolyl and hydroxyquinoline substrates for the histochemical demonstration of -mannosidase, -galactosidase, hetero--glycosidase, glucoamylase and sucrase-isomaltase, equivalent for -N-acetylglucosaminidase and lactase--glucosidase, and inferior for -glucuronidase and acid -galactosidase. Aldehyde fixation is necessary for the localization of lysosomal glycosidases with naphthyl substrates.i-naphthyl substrates are suitable for the detection of acid glycosidases in lysosomes and hetero--glycosidase in the cytoplasm of animal cells, and 2-naphthyl substrates can be employed for the demonstration of microvillous glycosidases and for the evaluation of the total activity of soluble glycosidases with semipermeable membranes. When naphthyl substrates are used coupling should be carried out simultaneously and hexazotized pararosaniline is the coupling reagent of choice.     

Species variation in organellar location and activity ofl-pipecolic acid oxidation in mammals

Summary The oxidation ofl-pipecolic acid to -aminoadipic acid was studied in eight species of mammals using an assay system more sensitive than those previously employed. After percoll-gradient fractionation, activity was localized to the mitochondrial-enriched fractions in tissues from rabbit, guinea pig, pig, dog, and sheep, with guinea pig kidney cortex showing greatest specific activity. These results contrast with the peroxisomal oxidation ofl-pipecolic acid observed in macaques and man (Mihalik and Rhead 1989; Mihalik et al. 1989). Rats and mice had undetectable levels of both peroxisomal and mitochondriall-pipecolic acid oxidation. In the rat, peroxisomal oxidation activity was not induced by feeding with either clofibrate or clofibrate andl-pipecolic acid. Thus, among mammals, both the ability to oxidizel-pipecolic acid and the organellar location of this oxidation is species dependent.     

Ultrastructural demonstration of acetylcholinesterase activity of motor endplates via osmiophilic diazothioethers

Summary The ultrastructural chemical localization of acetylcholinesterase of motor endplates of rat intercostal muscle has been studied with three new esterase substrates. These substrates, although not specific for acetylcholinesterase, have differential affinities for various types of esterases; two of them (NTA and TAB) are hydrolyzed preferentially by acetyl esterase enzymes, and the third (TPB) is a propionic acid ester and is hydrolyzed preferentially by pseudocholinesterase and other esterases. The end-product of the enzymatic reaction is converted to a diazothioether (droplet form) and upon osmication this is converted to a coordination polymer of osmium which has ideal properties for electron microscopy.Although this study supports previous observations that enzymatic activity can be found primarily on the post-synaptic membranes of the motor endplate, no enzymatic activity was noted on the pre-synaptic membrane, within the synaptic cleft, or on the basement membrane unless incubation was prolonged, resulting in overstaining. Neither was enzyme activity seen on membrane-free ribosomes and the ribosome-studded sarcoplasmic reticulum. Axonal vesicles also failed to exhibit enzymatic activity which had been noted with the method using thiolacetic acid and lead. A correlation of esterase activity with ultrastructural localization, using the substrate TPB, suggests that a buffer zone of nonspecific esterase activity is present beneath the subneural apparatus which limits the aberrant, accidental, or abnormal distribution of acetylcholine within a clearly defined area of sarcoplasm in the vicinity of the motor endplate of the muscle fiber.This investigation was supported by research grants from the National Cancer Institute (CA-2078 and CA-02478) and National Institute of Neurological Diseases and Blindness (NB 04096). Acknowledgement for technical assistence is due Miss Julia Silhan.This investigation was carried out during the tenure of a Public Health Service research career program award NB 5820 from the N.I.N.D.B.     

Enzymhistotopochemische Studien an inaktiven Salzdrüsen von Hausenten (Anas platyrhynchus)

Zusammenfassung In der sog. inaktiven Salzdrüse von Hausenten wurden die Glykosidasen -d-Glucuronidase, -d-Glucosaminidase, -Glucosidase und -d-Galaktosidase sowie die Hydrolasen unspezifische saure Phosphatase, unspezifische alkalische Phosphatase, Esterase, ATPase und Leucinaminopeptidase mit enzymhistochemischen Methoden untersucht. Die Drüsenzellen zeigen deutliche, wenn auch quantitativ unterschiedliche Esteraseaktivitäten. Besonders auffallend ist die hohe Aktivität der lysosomal lokalisierten sauren Phosphatase in den Epithelzellen der Zentralkanäle und Ausführungsgänge. Die Bedeutung der Befunde in bezug auf sekretorische und resorptive Leistungen der Salzdrüse wird diskutiert.

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Enzyme histochemical studies on the salt gland of ducks (Anas platyrhynchus)II. Cytochemical localization of some hydrolases

Summary Studying the site of hydrolytic enzymes in the salt gland of domestic ducks the glycosidases -d-glucuronidase, -d-glucosammidase, -glucosidase and -d-galactosidase as well as the hydrolases non-specific phosphatases, esterases, ATPase and leucinaminopeptidase have been investigated with histochemical techniques. The epithelia of the salt gland show distinct activities of non-specific esterases, that differ quantitatively. Furthermore we noticed a strong granular activity of non-specific acid phosphatase in the central canal and in the glandular duct system. The meaning of these findings in relation to secretion and absorption is discussed.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft (Ku-210/2).     

Ultrastructural localization of some phosphatases in the prothoracic gland of the insect Leucophaea Maderae

Summary The cytochemical localization of acid phosphatase and thiamine pyrophosphatase activity was studied by light and electron microscopy in prothoracic gland cells of the cockroach Leucophaea moderae. Nymphal and young adult animals were used.Prominent sites of acid phosphatase activity included large membrane-bounded dense bodies or lysosomes, and certain cisternae of the Golgi apparatus. The results suggest a possible difference in the enzymatic activity toward glycerophosphate and aromatic phosphates as substrates.Thiamine pyrophosphatase activity was localized in elements of the Golgi apparatus and endoplasmic reticulum, and in lysosome-like dense bodies. This latter activity was abolished by sodium fluoride treatment, whereas the phosphatase activity in the Golgi apparatus and endoplasmic reticulum is unaffected by such inhibition.The cytochemical results confirm through direct evidence the suggestions of Scharrer (1964), that the large dense bodies present in the prothoracic gland cells are lysosomes, and that their activity may be related to stages in the life history of the glands. Furthermore, the lysosomes or their derivative structures may play an essential role in the autolysis of the prothoracic glands toward the end of their active period.The enzymatic activity of the endoplasmic reticulum may indicate the involvement of this organelle in the metabolism of steroid-like precursor materials necessary for the synthesis of ecdysone.This study was supported by U.S.P.H.S. grants 5 T1-MH-6418 and NB-05219, and grant RO 1-AM-3984 to Dr. Berta Scharrer. I would like to express my appreciation to Dr. Scharrer for her encouragement and assistance during this study. I also wish to thank Mrs. Sarah Wurzelmann for her competent technical aid.     

Improvement of production of l-aspartic acid using immobilized microbial cells

Summary In our laboratory, EAPc-7 a strain having higher aspartase activity was derived from Escherichia coli ATCC 11303. For the improvement of l-aspartic acid productivity using EAPc-7 cells immobilized in -carrageenan, it was necessary to eliminate the fumarase activity which converts fumaric acid to l-malic acid. Several treatments for specifically eliminating fumarase activity from EAPc-7 cells were tested and it was found that when EAPc-7 cells were treated in a culture broth (pH 4.9) containing 50 mM l-aspartic acid at 45° C for 1 h, fumarase activity was almost completely eliminated without inactivation of the aspartase.The treated cells, immobilized in -carrageenan, were used for continuous production of l-aspartic acid from ammonium fumarate. The formation of l-malic acid was negligible and the half-life of the immobilized preparation was 126 days.Productivity of immobilized preparation of treated EAPc-7 cells in l-aspartic acid production was six times of that of the parent cell preparation.     

Endogenous elemental sulfur production froml-cysteine in dormant α-spores ofPhomopsis viticola

The enzymatic production of sulfur froml-cysteine was studied in young dormant -spores ofPhomopsis viticola. Cysteine aminotransferase (CAT) and mercaptopyruvate sulfurtransferase (MST) activities could be responsible for the production of endogenous elemental sulfur (S⁰) in -spores.l-Cysteine was first deaminated, with production of -mercaptopyruvate, by the CAT. The -mercaptopyruvate produced is successively desulfurated by the MST with production of sulfur and pyruvate. Deaminase activity was recovered principally in the cytoplasmic fraction, whereas desulfurase activity was recovered mainly in the mitochondrial fraction.l-Cysteine and S⁰ sharply affected the respiratory activity, the ATP content, and suppressed germination of -spores. In contrast, reduced glutathione did not affect these metabolic parameters. Production of S⁰ by enzymatic degradation ofl-cysteine could be responsible for the inhibitory action of this amino acid. We suggest that CAT and MST, by their capacity to produce sulfur or S⁰, plays a key role in regulation of morphogenetic processes ofPhomopsis viticola.     

Iron requirement for and effects of promoters and inhibitors of ethylene action on stimulation of Fe(III)-chelate reductase in roots of strategy I species

Stimulation of root Fe(III) reductase activity by iron additions to iron-deficient growth media may be the result of iron activation of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase required for ethylene biosynthesis. Two different ethylene inhibitors, aminooxyacetic acid (AOA) (20 m; ACC synthase inhibitor) and cobalt (3 m CoCl₂; ACC oxidase inhibitor), were used to study the effects of iron supply and cobalt inhibition on ethylene action in controlling the activity of Fe(III)-chelate reductase in pea (Pisum sativum L.) roots. Supplying 20 gm m Fe(III)-N,N-ethylenebis[2-(2-hydroxypheyl)-glycine [Fe(III)-EDDHA] to either cobalt-treated, iron-deficient Sparkle (normal parent) or E107 (brz mutant genotype) pea seedlings reversed the negative effects of cobalt on root Fe(III)-reductase activity. Re-supplying 20 m Fe(III)-EDDHA to iron-deficient, AOA-treated seedlings did not enhance root Fe(III)-reductase. Apparently, cobalt competes with iron for the active site in ACC oxidase during ethylene synthesis. Inhibition of root reductase activity by cobalt treatment lowered manganese, zinc, magnesium and potassium content of mutant E107 pea seedlings. In contrast, iron enhancement of root reductase activity in iron-deficient, cobalt-treated E107 seedlings resulted in higher seedling accumulations of manganese, zinc, magnesium and potassium. These results support the hypothesis that root cell plasma membrane reductase activity plays a role in cation uptake by root cells.     

In situ heterogeneity of peroxisomal oxidase activities: An update

Summary Oxidases are a widespread group of enzymes. They are present in numerous organisms and organs and in various tissues, cells, and subcellular compartments, such as mitochondria. An important source of oxidases, which is investigated and discussed in this study, are the (micro)peroxisomes. Oxidases share the ability to reduce molecular oxygen during oxidation of their substrate, yielding an oxidized product and hydrogen peroxide. Besides the hydrogen peroxide-catabolizing enzyme catalase, peroxisomes contain one or more hydrogen peroxide-generating oxidases, which participate in different metabolic pathways. During the last four decades, various methods have been developed and elaborated for the histochemical localization of the activities of these oxidases. These methods are based either on the reduction of soluble electron acceptors by oxidase activity or on the capture of hydrogen peroxide. Both methods yield a coloured and/or electron dense precipitate. The most reliable technique in peroxisomal oxidase histochemistry is the cerium salt capture method. This method is based on the direct capture of hydrogen peroxide by cerium ions to form a fine crystalline, insoluble, electron dense reaction product, cerium perhydroxide, which can be visualized for light microscopy with diaminobenzidine. With the use of this technique, it became clear that oxidase activities not only vary between different organisms, organs, and tissues, but that heterogeneity also exists between different cells and within cells, i.e. between individual peroxisomes. A literature review, and recent studies performed in our laboratory, show that peroxisomes are highly differentiated organelles with respect to the presence of active enzymes. This study gives an overview of thein situ distribution and heterogeneity of peroxisomal enzyme activities as detected by histochemical assays of the activities of catalase, and the peroxisomal oxidasesd-amino acid oxidase,l--hydroxy acid oxidase, polyamine oxidase and uric acid oxidase.     

Ferric reductases of Legionella pneumophila

Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (K _m = 7.0 m) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (K _m = 15.3 m). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells.     

d-Alanine metabolism in the lucinid clam Lucinoma aequizonata

The chemoautotrophic symbiont-bearing clam Lucinoma aequizonata contains very high levels of free d-alanine in all tissues. The possible sources for this amino acid and its involvement in the clams' metabolism were investigated. Very low levels of d-alanine (generally below 1 mol·l^-1) were measured in the sediment porewaters from the habitat of the clams. Experiments with ¹⁴C-labeled tracers demonstrate an active metabolism of d-alanine in the clams rather than a role as inert waste product. d-alanine is metabolized at about 0.12 mol·g fw^-1·h^-1. Label from aspartate, but not glucose and CO₂, is incorporated into d-alanine. Incubation with labeled d-alanine did not result in formation of radioactive l-alanine. Tests for alanine racemase (EC 5.1.1.1) and d-amino acid oxidase (EC 1.4.3.3.) did not show activity in either gill, i.e. symbiont and host, or foot tissue. d-Alanine amino transferase (EC 2.6.1.b.) was demonstrated in gill and foot tissues. Two sources for d-alanine are proposed: a degradation of cell walls of symbiotic bacteria and production by the host using a d-specific alanine transaminase.Abbreviations aa amino acid(s) - fw fresh weight - HPLC high-performance liquid chromatography - MBH methyl benzethonium hydroxyde - NAC N-acetyl-l-cysteine - OPA ortho-phthaldialdehyde - TCA tricarbonic acid     

Ionic and non-ionic compounds in the germination of spores of bacillus megaterium texas

Summary Germination requirements of suspensions of spores of Bacillus megaterium, Texas strain, an l-alanine-inosine type, have been examined employing a decrease in optical density as the criterion of germination. In deionized water, l-alanine and inosine were devoid of germinative powers. They were effective only in conjunction with any one of a large variety of salts. Data are given for germination by the monovalent and divalent alkali metal chlorides. The potassium halides were germinative; potassium fluoride was the best. Salts of organic acids, including fatty acids and polycarboxylic acids, were germinative. The need for inosine could be bypassed by various salts, e.g., ammonium propionate or salts of dipicolinic acid. Also, l-alanine was replaceable by a variety of amino acids, provided suitable ions were present. In the presence of magnesium chloride, sodium dipicolinate could substitute for either inosine or l-alanine, but not both. Salts of n-hexylamine and n-heptylamine bypassed the need for both l-alanine and inosine. A primary role for ions in germination is proposed and a secondary, augmentative action is attributed to l-alanine and inosine.     

Lysine metabolism in the human and the monkey: Demonstration of pipecolic acid formation in the brain and other organs

Metabolism ofl-[U-¹⁴C]lysine was studied in the human autopsy tissues and the intact monkeys through intracerebroventricular and intravenous injections. The human tissues were more active in the metabolism ofl-[¹⁴C]lysine to [¹⁴C]pipecolate than the rat tissues previously reported. This metabolism was equally active in the phosphate (pH 7) and the glycyl-glycine (pH 8.6) buffers with the brain and the kidney having higher activity than the liver. Besides [¹⁴C]pipecolate, traces of [¹⁴C]saccharopine and -[¹⁴C]aminoadipate were also detected in the liver incubation. Twenty-four hr after intraventricular injection ofl-[¹⁴C]lysine to the monkey, substantial labeling of pipecolate and -aminoadipate was observed in the brain and spinal cord, with the kidney, liver and the plasma having much reduced levels. Radioactivity levels of these two compounds were found low in the organs and plasma of the intravenously injected monkey. The urine of both monkeys contained only traces of [¹⁴C]pipecolate, even though it contained high levels ofl-[¹⁴C]lysine and -[¹⁴C]aminoadipate. It was concluded thatl-lysine is actively metabolized to pipecolate and -aminoadipate in the human and the monkey, that this reaction is most active in the brain whenl-lysine is intraventricularly administered, and that in contrast to the rat, the monkey may have an effective renal reabsorption for pipecolate which is similar to the human.