Isolation and characterization of cathepsin B from bovine brain

Cathepsin B (EC 3.4.22.1) was purified 746-fold with a 21% recovery from bovine brain by autolysis, fractional precipitation with acetone, carboxy-methyl-Sephadex chromatography, affinity chromatography on a cystamine containing column and gel filtration chromatography. The purified cathepsin B eluted on gel filtration with an apparent molecular weight of 27,000 but was resolved into three bands of 30,000, 25,000 and 5,000 molecular weight by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Antibodies to cathepsin B, raised against the 30,000 dalton band, were shown by immunoblots to react with both the 30,000 and 25,000 dalton proteins with results suggesting that the former predominated as the immunoreactive form in bovine brain homogenates. Isoelectric focusing demonstrated multiple bands, ranging from pH 4.75–5.2 with the major band at pH 5.1–5.2, all of which were capable of degrading N-carbobenzoxy-l-arginyl-l-arginine 4-methoxy--naphthylamide. The cathepsin B activity against N-benzoyl-dl-arginine -naphthylamide (BANA) and bovine myelin basic protein (MBP) had a pH optimum of pH 6.0. The Km for the degradation of BANA was 1.0 mM and 5.1 mM when assayed in the presence of 1% and 2.5% dimethylsulfoxide, respectively. Cathepsin B from bovine brain has many properties similar to cathepsin B isolated from other organs. The degradative effect of cathepsin B on MBP suggests a role for this proteinase in inflammatory demyelination.     

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)     

Isolation and characterization of bovine brain myelin distribution of 5′-nucleotidase

Myelin was isolated from bovine brain by several published procedures and modifications of these procedures. High activity of the myelin marker (2,3-cyclic nucleotide 3-phosphohydrolase) and low activity of contaminants markers in white matter homogenates in respect to cerebral cortex showed the white matter to be better than the cerebral cortex or the whole brain for myelin isolation. A procedure is described for the preparation of purified myelin from bovine white matter which yielded a content of protein (40%), myelin marker (51%), and 5-nucleotidase (25%) in purified myelin higher than by any used method. Acetylcholinesterase or succinate dehydrogenase was lower than 7% of its activity in the white matter homogenate, and monoamine oxidase and NADPH: cytochrome c reductase were not recovered in myelin fraction. Morphologically, myelin fraction was shown to mainly consist of multilamellar membranes of different sizes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of myelin fraction showed a characteristic protein pattern of myelin. When our procedure was applied to frozen white matter, lower protein (32%) and myelin marker (34%) and similar 5-nucleotidase activity (24%) were recovered in myelin, increasing its recovery in denser fractions of white matter.     

The effect of thyroid hormone on mitochondrial biogenesis and cellular hyperplasia

The purpose of this investigation was to study the effects of thyroid hormone treatment on the levels of DNA, RNA, and protein in hepatocytes and hepatocyte mitochondria. A preliminary investigation was conducted to establish an effective dosage of thyroid hormone. Male Sprague-Dawley rats were given daily subcutaneous injections ofl-thyroxine (20, 40, or 60 µg/100 g body weight) and the following determinations made over a 14-day period: (1) body weight; (2) total body respiration; and (3) the activities of the mitochondrial enzymes, succinate dehydrogenase and -glycerophosphate dehydrogenase. Dosages of 20 and 40 µgl-thyroxine/100 g body weight produced significant stimulation of (a) total body respiration and (b) succinate dehydrogenase and -glycerophosphate dehydrogenase activities without any inhibitory effects on normal weight gain of the animals. Injections of 40 µgl-thyroxine/100 g body weight were utilized for subsequent studies. Hepatic DNA levels of treated animals were greater than age-paired control values by 28% on day 7 and 43% by day 14. Total liver RNA levels of thyroid-treated animals were 17% greater than those of controls by day 7 and 47% greater by day 14. Analyses were also performed on mitochondria quantitatively collected by rate zonal centrifugation. Total liver mitochondrial DNA levels in thyroid-treated animals were greater than age-paired controls by 79% at 7 days but only 67% at 14 days since a small gain occurred in control animals and no further increase occurred in treated rats during the second week. Mitochondrial RNA and protein from treated livers were 26% and 16% higher, respectively, than age-paired controls at day 7 and 40% and 58% higher, respectively, at day 14. The results of this study indicated that thyroid hormone treatment produces hyperplasia and an increase in mitochondrial number and mass in rat liver.     

Molecular cloning and expression of multiple cellulase genes of Ruminococcus flavefaciens strain 186 in Escherichia coli

Summary Cellulase genes of the ruminant micro-organism Ruminococcus flavefaciens strain 186 have been cloned and expressed in Escherichia coli using the bacteriophage vector NM1149. Twenty-six clones showed expression of endo--1,4-d-glucanases and were divided into four groups according to their insert sizes of approximately 2, 3, 4 or 9 kilobases (kb). Two of the clones with 4 kb inserts also showed exo--1,4-d glucanase activity while two clones with 9 kb inserts showed -glucosidase activity. One of the clones with 9 kb inserts (M903) showed the activities of all three cellulase activities. In addition, two of the 4 kb-insert clones and one 9 kb-insert clone degraded Avicel (PH101).     

Localization and some properties of lysosomal dipeptidases in rat liver

1. 1. The rates of hydrolysis of 26 synthetic depeptides by extracts from highly purified lysosomal fractions from rat liver at pH 5.0 and by whole liver homogenates at pH 7.4 have been determined. Extracts from the lysosomal fractions hydrolysed most peptides at a lower rate per mg protein than the homogenates, and some peptides not at all.

2. 2. Properties of two dipeptidases present in the extracts from the lysosomal fractions, splitting Ile-Glu and Leu-Gly, respectively, were studied in greater detail. The enzyme that hydrolysed Ile-Glu was strongly activated by dithiothreitol, showed optimal activity at pH 4.5 and had a molecular weight of about 120 000. Leu-Gly dipeptidase did apparently not contain an essential thiol group and had a molecular weight of approx. 90 000. It showed maximal activity at pH 6.5.

3. 3. After differential centrifugation of liver homogenates, Ile-Glu and Leu-Gly-splitting activities were determined in the fractions, under the optimal conditions mentioned above. The Ile-Glu-hydrolysing enzyme activity showed about the same distribution as the lysosomal marker enzyme acid phosphatase. Leu-Gly-splitting activity, however, was largely present in the cytosol fraction, with only a small peak in the lysosomal fraction. We obtained evidence that the activities present in the lysosomal fraction and in the cytosol fraction were due to different enzymes, and that one of these enzymes was localized exclusively in lysosomes.

4. 4. It is concluded that some dipeptides originating from intralysosomal proteolysis might be split by lysosomal dipeptidases, whereas others are probably hydrolysed only in the extra-lysosomal compartment of the cell.

Alterations in the activity of phospholipases A2 in postmortem white matter from patients with multiple sclerosis

Activities toward arachidonyl-labelled phospholipase A₂ substrates were assayed in fractions of white matter and cerebral cortex from control subjects and in fractions of demyelinated plaque, normal-appearing white matter and cerebral cortex from subjects who died with multiple sclerosis. Membranous activity at pH 8.6 in the presence of Ca²⁺, characteristic of 14 kDa secretory phospholipase A₂, in either multiple sclerosis white matter or cortex did not differ from controls, whereas membranous activity at pH 4.5 in the absence of added Ca²⁺, characteristic of lysosomal enzymes was increased over controls in both plaque and normal-appearing white matter but not cerebral cortex. Activity in the cytosol fraction, at pH 8.6 in the presence of Ca²⁺ and glycerol characteristic of the cytosolic 85 kDa enzyme was decreased by greater than 50% in both white matter and cortex samples from multiple sclerosis subjects. Immuno-precipitation and-blotting confirmed that the deficient activity was largely attributable to the 85 kDa enzyme although the enzyme protein was not similarly reduced.Special issue dedicated to Dr. Leon S. Wolfe.     

A quantitative microdensitometric and autoradiographic study of the effect of 4''-demethyl-epipodophyllotoxin-beta-D-thenylidene glucoside (VM-26) on the cell cycle of cultured fibroblasts

Synopsis The effect of 4-demethyl-epipodophyllotoxin--d-thenylidene glucoside (VM-26), a semi-synthetic derivative of podophyllotoxin, on the cell cycle was studied with chick embryo fibroblasts cultivatedin vitro. DNA, RNA and protein content, as well as NADH-diaphorase activity were determined by quantitative microdensitometry and cytofluorometry. The incorporation of [³H]thymidine and [³H]leucine into DNA and proteins were analysed by autoradiography. These metabolic data correlated with morphological observation showed that VM-26 blocks the cell cycle at different moments of its kinetics depending on both the dose and the time exposure. NADH-diaphorase activity is the first to be affected, then biochemical changes (involving the metabolism of RNA and proteins) and morphological alterations (especially of mitochondria) follow. This suggests that VM-26 may act primarily upon the mechanism of respiration of the cell.Dedicated to Professor G. Conti on the occasion of his 60th birthday.     

Studies of γ-glutamyltransferase activity in the brain tissue post mortem

Our experiments showed that the activity of -glutamyltransferase (-GT) did not remarkably change in homogenates of mouse, rat, and bovine brains during the first four days post mortem. In the course of that period, the brain microvessels also retained their -GT activity. -GT of microvessels from bovine brain cortex, solubilized with sodium deoxycholate, was eluted in the void volume V_o when chromatographed on a Sephadex G-200 column with the detergent Triton X-100. In human post mortem brains, the specific activity of -GT in choroid plexi was found to be about five times higher than that in the cerebral cortex, white matter, basal ganglia, pons, and cerebellum but about four times lower than that in the microvessels obtained from the studied brain regions. Our findings suggest that it is possible to study the components of the blood-brain barrier on material from deceased subjects.     

Cilastatin-sensitive dehydropeptidase I enzymes from three sources all catalyze carbapenem hydrolysis and conversion of leukotriene D4 to leukotriene E4

The dipeptidase, dehydropeptidase I (EC 3.4.13.11), was purified to homogeneity from rat lung, rat kidney, and hog kidney. Analysis of physical parameters (subunit molecular weights, Km values for glycyldehydrophenylalanine, Ki values for dehydropeptidase I inhibitors, and immunoreactivity) showed the rat dipeptidases to be similar to each other but different from the hog dipeptidase. However, all three enzymes hydrolyzed imipenem and converted leukotriene D4 to leukotriene E4, and these activities were inhibited by cilastatin.     

DNA and RNA and the cytoarchitecture of human frontal cortex

DNA and RNA were studied in the layers of human prefrontal cortex by quantitative microchemical analyses on microtome-prepared serial frozen sections. Eleven cortical specimens from six autopsy brains were assayed. The mean total number of cells per mm³ of fresh cortex was estimated from DNA values. The number in layer I (61,000) was falsely high because of the inclusion of cells from the pia mater. From a plateau of 82,000-86,000 in layers II, IIIa and IIIb, the number of cells rose to 91,000 and 113,000 in layers IIIc and IV, respectively. The mean number was slightly lower in layer V, then gradually rose through layer VI to 127,000 cells in white matter. Per unit dry weight, DNA and cells varied much less than per unit volume; values averaged 14 per cent higher in layers II and IV than in neighbouring layers and in white matter were 20 per cent lower than in layer I. Intracortical patterns of RNA and RNA/cell reflected chiefly the distribution of neuronal cell bodies. Per unit fresh volume, RNA roughly paralleled DNA in layers I-V; through layer VI and into white matter RNA declined as DNA rose, reflecting the decline in neurons and increasing predominance of glial cells of lower RNA content. Per unit dry weight, RNA rose 50 per cent from layer I to layer II; a plateau of high values extended through layers II-V, then RNA declined rapidly through layer VI to a level in white matter that was 28 per cent of the value in layer II. Mean RNA/cell in cortex was 9-8 pg, with a maximum in layer IIIb (11.4 pg); in subcortical white matter it was 5.3 pg.     

Glycyl-l-leucine hydrolase, a versatile `master' dipeptidase from monkey small intestine

1. A highly active and electrophoretically homogeneous dipeptidase was purified from the soluble extracts of monkey small-intestinal mucosa. 2. By gel-filtration studies the molecular weight of the enzyme was found to be 107000. It is composed of two identical, subunits of molecular weight 54000. 3. A paper-chromatographic method of dipeptidase assay was developed to overcome some of the difficulties encountered in the generally used spectrophotometric procedure. By using this method, the K_m and k₀ values of a few substrates were determined. 4. The substrate specificity of the enzyme was investigated in great detail with substrates of a wide range of possible structural types. The enzyme hydrolyses a very large proportion of the range of dipeptides tested. This enzyme, which exhibits such a wide range of action, has been termed the `master' dipeptidase of the intestine. Glycylglycine, glycyl-l-proline, glycyl-l-histidine, l-prolylglycine and some of the arginine- and aspartic acid-containing dipeptides were not substrates and are possibly hydrolysed by other peptidases. These results therefore suggest that in the intestine the number of dipeptidases is rather limited. 5. In the light of these findings, the implications on the role of dipeptidases in intestinal peptide transport are discussed.     

Das unterschiedliche Verhalten der Exopeptidasen des Hepatopankreas und Magensaftes vom Flußkrebs Astacus astacus (L.) und Cambarus affinis (Say) bei der Gelfiltration auf Sephadex sowie gegenüber Effektoren

Zusammenfassung Das Hepatopankreas (HP) der Flußkrebse Astacus astacus (L.) und Cambarus affinis (Say) enthält eine hochmolekulare Carboxypeptidase A-Wirkung (Substrat: Carbobenzoxyglycyl-l-phenylalanin) (K _d-Wert auf Sephadex G-200=0,04) und eine Arylamidase-Wirkung [Substrat: l-Leucin--naphthyl-amid·HCl (LNA)], sowie Dipeptidase-Wirkung (Substrat: Glycyl-l-leucin) (K _d-Werte auf Sephadex G-200=0,46 bzw. 0,39). Carboxypeptidase B (Substrat: Hippuryl-l-arginin)-Aktivität wurde im HP nicht gefunden. Im Gegensatz zum HP ist die Carboxypeptidase A des Magensaftes (MS) niedriger molekular (K _d-Wert auf Sephadex G-150=0,62; Molekulargewicht: ca. 30000), die LNA-ase des MS höher molekular (K _d-Wert auf Sephadex G-150=0,26). Außerdem enthält der MS eine hochaktive Carboxypeptidase B-ähnliche Wirkung, die sich auf Sephadexgel wie die Carboxypeptidase A verhält. Chelatbildner (,-Dipyridyl, o-Phenanthrolin) hemmen die Hippurylarginin-Wirkung nicht. Die Carboxypeptidase A des HP wird durch EDTA und Hydrozimtsäure deutlich, durch p-Chlormercuribenzoat gering aktiviert und durch 2-Mercaptoäthanol (10^–3 m und höhere Konzentrationen) stark gehemmt.

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The differential behaviour of the exopeptidases from hepatopancreas and gastric juice of the crayfish Astacus astacus (L.) and Cambarus affinis (Say) during gelfiltration on sephadex and towards effectors

Summary Hepatopancreas (HP) of the crayfishes Astacus astacus (L.) and Cambarus affinis (Say) contains a high molecular carboxypeptidase A like action (substrate: carbobenzoxyglycyl-l-phenylalanine) (K _d-value on Sephadex G-200 =0,04), an arylamidase like action (substrate: l-leucine- -naphthylamide·HCl; LNA), and a dipeptidase (substrate: glycyl-l-leucine) (K _d-values on Sephadex G-200 0,46 and 0,39 respectively). Carboxypeptidase B (substrate: hippuryl-l-arginine) activity was absent in HP. Contrary to the exopeptidases of HP the carboxypeptidase A of the gastric juice is of lower molecular weight (K _d-value on Sephadex G-150=0,62; molecular weight approx. 30.000), and the arylamidase of the gastric juice is of higher molecular weight (K _d-value on Sephadex G-150=0,26). Moreover gastric juice contains a highly active carboxypeptidase like activity, with identical behaviour on Sephadexgel as carboxypeptidase A. ,-dipyridyl and o-phenanthroline are without effect on the hippurylarginine splitting activity. Carboxypeptidase A of HP is significantly activated by EDTA and hydrocinnamic acid, and slightly activated by p-chloromercuribenzoate. 2-mercaptoethanol (10^–3 molar and higher concentrations) inhibits strongly the carboxypeptidase A of HP.

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Herrn Prof. Dr. Hanson möchte ich für sein Interesse an dieser Arbeit sowie für die kritische Durchsicht des Manuskriptes vielmals danken. — Der medizinisch-technischen Assistentin Frau Johanna Scheel danke ich für ihre wertvolle Mit-arbeit bei den Versuchen.     

Postnatal Changes in Cathepsin D in Rat Neural Tissue

Cathepsin D, an aspartyl endopeptidase, was analyzed in cortex from forebrain and cerebellum, spinal cord, and optic and sciatic nerves, and in the liver of rats from 1 to 120 days of age. Cathepsin D was quantitated in tissue extracts by measurement of enzyme specific activity on a substrate of [methyl-¹⁴C]-methylated hemoglobin and by radioimmunoassay. Immunocytochemistry was used to ascertain the identity of the mixed cell types that contributed to the cathepsin D detected. As quantitated by radioimmunoassay, immunoreactive cathepsin D varied between 0.2 and 1 ng/μg of total protein. Maximum activity occurred at approximately the 15th postnatal day; the least amount of immunoreactive cathepsin D was found at 30 or 60 days of age. A subsequent increase of varying magnitude occurred at postnatal day 120. There was good correspondence between immunoreactive enzyme and enzyme specific activity, which ranged from 1 to 4 ng/μg of total protein, and the activities determined by the two methods provided similar, but not identical, developmental profiles. Cathepsin D was demonstrated by immunocytochemistry to be present in most neurons, in all choroid plexus epithelium, and in certain oligodendrocytes from the first postnatal day. Cathepsin D was present in oligodendrocytes in cord lateral funiculi and optic nerve by the first postnatal day, and by the sixth postnatal day many oligodendrocytes were abundantly stained. In contrast, oligodendrocytes in the corpus callosum and in the cerebellar white matter did not contain demonstrable cathepsin D until postnatal days 10 and 15, respectively. These results indicate a role for cathepsin D during the postnatal development of rat CNS and suggest that this proteinase may be involved in the steps of myelination.     

Regulation of β-xylosidase formation by xylose in Trichoderma reesei

The soft-rot fungus Trichoderma reesei forms -xylosidase (EC 3.2.1.37) activity during cultivation on xylan and xylose, but not on glucose. When mycelia precultivated on glycerol were washed and transferred to fresh medium without a carbon and nitrogen source, -xylosidase formation was induced by xylan, xylobiose and xylose. A supply of 4 mm xylose and a pH of 2.5 provided optimal conditions for induction. -Xylosidase accounted for the major portion of total extracellular protein under these conditions, and could be purified to physical homogeneity by a single anion exchange chromatography step. A recombinant strain of T. reesei that carries multiple copies of the homologous xylanase II-encoding gene has a six-fold increased xylanase activity, but forms comparable -xylosidase activities. This shows that the rate of xylan hydrolysis has no effect on the induction of -xylosidase. Methyl--d-xyloside inhibited -xylosidase competitively and was a weak -xylosidase inducer. The induction by xylobiose and xylan was strongly enhanced by the simultaneous addition of methyl--d-xylosidese and xylan or xylobiose. The results suggest that a slow supply of xylose is a trigger for -xylosidase induction.     

Experimental phenylketonuria: Metabolic studies in rat liver

Summary The in vivo effects of L-phenylalanine on the gluconeogenic pathway in the liver of fasted rats with experimentally induced phenylketonurialike characteristics have been investigated. Significant increases of the fructose 6-phosphate, glucose 6-phosphate and glucose concentrations were observed. The study of the effect of L-phenylalanine on the cytoplasmic and mitochondrial redox state and energy charge showed an increase in the mitochondrial NAD⁺/NADH ratio while the energy charge was virtually unchanged.The effects of phenylalanine and its metabolic derivatives (phenylacetate, phenylethylamine, phenyl-lactate, o-hydroxyphenylacetate and phenylpyruvate) on the activity of lactate de-hydrogenase (EC 1.1.1.27), malate dehydrogenase (EC 1.1.1.37) and 3-hydroxybutyrate de-hydrogenase (EC 1.1.1.30) in rat liver have been also investigated. Phenylpyruvate inhibited the lactate dehydrogenase activity with a Ki of 5.3mm. Phenylpyruvate also inhibited both the mitochondrial (Ki = 4mm) and cytoplasmic (Ki = 5mm) malate dehydrogenase activities. Phenyl-pyruvate, phenylacetate and o-hydroxyphenylacetate inhibited the 3-hydroxybutyrate dehydrogenase activity with Ki values of 0.7, 6.0 and 9.5mm respectively.     

Synthesis of macromolecules and rubratoxin by Penicillium rubrum

The relationship between primary metabolism and biosynthesis of rubratoxin was studied with replacement cultures of Penicillium rubrum 3290. Synthesis of protein and RNA was measured by determining incorporation of [U¹⁴C]L-leucine and [2¹⁴C]-uridine into the respective components of the fungal biomass. Rubratoxin formation was measured by determining incorporation of [1¹⁴C]acetate into the toxin. Both protein and RNA were synthesized rapidly with synthesis increasing during 108 h of incubation and then decreasing rapidly. Rubratoxin formation increased up to 72 h, declined through 96 h, became maximal at 108 h, and then decreased rapidly. Cycloheximide, at 100 g/ml, moderately blocked accumulation of dry weight and protein synthesis by the mold; at 150 g/ml, cycloheximide completely blocked in vivo synthesis of protein. When cycloheximide was added to cultures after synthesis of toxin had begun, protein synthesis, but not toxin formation, was blocked. Inhibition of protein synthesis by cycloheximide was reversed by washing the drug out of mold cultures. Rubratoxin was formed throughout the incubation; a transitional phase, characteristic of secondary biosynthesis, was not observed.     

Production,purification and characterization of an α-l-arabinofuranosidase from Bacteroides xylanolyticus X5-1

Cell-free extracts of L-arabinose- and d-xylose-grown cells of the mesophilic anaerobic bacterium Bacteroides xylanolyticus X5-1 contained high activities [2 units (U)/mg] of an -l-arabinofuranosidase (EC 3.2.1.55). The enzyme was also produced during growth on xylan, but not during growth on glucose or cellobiose. The enzyme was mainly extracellularly attached to the cell when the organism was grown on xylan and was not released into the medium. The enzyme was purified 41-fold to apparent homogeneity. The native enzyme had an apparent molecular mass of 364 kDa and was composed of six polypeptide subunits of 61 kDa. The enzyme displayed a pH optimum of 5.5–6.0, and a pH stability of 5.5–9.0. The temperature optimum was 50° C and the enzyme was stable up to 50° C. Thiol groups were essential for activity, but the enzyme activity was not dependent on divalent cations. The Michaelisconstant (K_m) and maximal reaction velocity (V_max) for p-nitrophenyl--l-arabinofuranoside were 0.5 mm and 155 U/mg protein, respectively. The enzyme was specific for the -linked arabinoside in the furanoside configuration. The enzyme displayed activity with arabinose-containing xylo-oligosaccharides with a polymerization degree of 2–5, but not with the polymeric substrates oat-spelt xylan or arabinogalactan. The enzyme belongs to the Streptomyces purpurascens-type of -l-arabinofuranosidase.     

Studies on the nature of the Li locus in Trifolium repens L. II. The effect of genotype on enzyme activity and properties

Various reagents which prevent enzyme inhibition by phenolic compounds were tested in attempts to improve the medium used to extract the Li-controlled enzyme activities from white clover leaves. The addition of 50 mm diethyldithiocarbamate to the extraction medium gave a fivefold increase in the enzyme activity of LiLi white clover extracts against p-nitrophenyl -d-glucoside, linamarin-lotaustralin, and p-nitrophenyl -d-galactoside. These three substrates were used in tests on the effect of genotype on enzyme activity. An absence of dominance at the Li locus was demonstrated, with a dosage effect of Li alleles on enzyme activity. A new Li allele was identified in the Lili clone, C11, which had low levels of enzyme activity. In crosses with two lili clones, Li(C11)li progeny were produced with activity levels similar to those of the C11 parent. Inhibition and heat-inactivation tests suggest that the lili clone, D4, produces an altered form of -glucosidase which may also be present in LiLi plants. The nature of the Li locus is discussed.     

Uptake of glycine froml-alanylglycine into renal brush border vesicles

Summary Isolated renal brush border microvilli vesicles were employed to study the uptake of radiolabel froml-Ala · [³H]Gly andd-Ala · [³H]Gly as well as to determine the presence of dipeptidase activity. Microvilli vesicles were prepared from porcine kidney cortex by differential centrifugation through hypotonic Tris buffer containing Mg²⁺. The microvilli vesicles transiently accumulated radiolabel froml-Ala · [³H]Gly to higher levels than were initially present in the incubation medium (overshoot phenomenon). This accumulation was dependent on the presence of an inward-directed (extravesicular > intravesicular) Na⁺ gradient and was osmotically sensitive and linear with respect to microvilli protein concentration. Analysis of intravesicular contents revealed that all³H uptake froml-Ala · [³H]Gly appeared as free glycine. Hydrolysis studies demonstrated the rate ofl-ala · [³H]Gly hydrolysis to free alanine and [³H] glycine by the microvilli to be greatly in excess of their rate of radiolabel uptake from this dipeptide. In addition, the uptake profiles and kinetic constants for vesicular uptake of radiolabel froml-Ala · [³H]Gly and free glycine were demonstrated to be identical when measured by double-labeling techniques in the same experiments. These results indicate thatl-Ala · [³H]Gly is hydrolyzed at the external surface of the microvilli with the [³H]glycine released being transported into the vesicles by a Na⁺ gradient-dependent system identical to that employed for free glycine.Microvilli vesicle uptake of radiolabel fromd-Ala · [³H]Gly exhibited no Na⁺ dependent overshoot effect.d-Ala · [³H]Gly was completely resistant to microvilli-catalyzed hydrolysis.Analysis of the microvilli for renal dipeptidase, an enzyme with hydrolytic activity against a wide range ofl-dipeptides, revealed this enzyme to be enriched in the microvilli vesicles to a degree equivalent to that observed for marker enzymes for renal microvilli.Renal dipeptidase catalyzed hydrolysis ofl-Ala · Gly but notd-Ala · Gly, as was the case with microvilli-catalyzed hydrolysis of these dipeptides.With its location in the renal brush border microvilli and its hydrolytic action againstl-dipeptides, renal dipeptidase may act at the luminal surface of the proximal tubule cell to hydrolyzel-dipeptides present in the glomerular filtrate, with the resultant free amino acids transported across the brush border microvilli by Na⁺ gradient-dependent processes.