Distribution of acid phosphatase and β-glucuronidase in the hypoplastic cerebellum of jaundiced Gunn rats

Summary Activities of acid phosphatase and -glucuronidase in the cerebella of young jaundiced (j/j) and non-jaundiced (j/+; control) Gunn rats were studied with the enzyme histochemical method. The cerebellum of j/+ rats showed high acid phosphatase activities in Purkinje cells and neurons in the cerebellar nuclei. In j/j rats, a number of neurons were lost and numerous microglialike cells with a high acid phosphatase activity appeared in the hypoplastic cerebellum. Although -glucuronidase activity was rarely detected in the control cerebellum, a high enzyme activity was observed associated with microglialike cells in j/j rats. The present results provide a cytological basis for the reported differential increase in the activities of these lysosomal enzymes in the j/j rat cerebellum.     

Quantitative enzyme histochemistry of rat foetal brain and trigeminal ganglion

Summary The increasing concern and the efforts in determining neurological effects in offsprings resulting from maternal exposure to xenobiotics are faced with several difficulties in monitoring damage to the central nervous system. In this paper, the efficiency of several enzyme histochemical reactions for analysing the forebrain and the trigeminal ganglia of rat foetuses are reported. Brains of 20-day-old Sprague-Dawley rat foetuses were frozen and analysed for 18 enzymes that had previously been used to monitor initial injury caused by toxic compounds in liver and other organs. Eight enzymes appeared suitable as histochemical markers for the functional integrity of different areas in brain and ganglia of rats exposed to xenobiotics. They were lactate, malate, glycerophosphate (NAD-linked), succinate, aldehyde and glucose 6-phosphate dehydrogenases, -glycerophosphate-menadione oxidoreductase and cytochromec oxidase. The activities of the enzymes were determined by microphotometry and the arrangement of absorbances of the enzyme final reaction products into appropriate analytical tables is proposed as an efficient procedure for data analysis.Abbreviations AcChE acetylcholinesterase - AldDH aldehyde dehydrogenase - ALKPase alkaline phosphatase - 5AMPase adenosine monophosphatase - ATPase Mg²⁺ dependent adenosine triphosphatase - CytOx cytochromec oxidase - GAPDH glyceraldehyde phosphate dehydrogenase - GIDH glutamate dehydrogenase - GLPDH glycerophosphate: NAD oxidoreductase - CPODH glycerophosphate:menadione oxidoreductase - G6Pase glucose-6-phosphatase - G6PDH glucose-6-phosphate dehydrogenase - IDH lactate dehydrogenase - MaDH malate dehydrogenase - MAO monoamine oxidase - NADPH, DH, NADPH tetrazolium oxidoreductase - SuDH succinate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase     

Histochemical study of enzyme patterns in the human submaxillary gland

Summary The histochemical distribution of various enzymes, such as alkaline phosphatase, acid phosphatase, esterase, -glycosidase, aminopeptidase, succinic dehydrogenese and TPN diaphorase, in human submaxillary glands has been determined.Acini and ducts of human submaxillary gland were devoid of alkaline phosphatase activity, but this enzyme was observed in capillaries and somewhat in myoepithelium.Activities of acid phosphatase, esterase, -glucuronidase and -galactosidase were generally observed in the entire cytoplasm of serous acini; but the cytoplasm of mucous acini was either negative or showed only trace amounts.Aminopeptidase reaction of both acini and ducts was generally negative.Succinic dehydrogenase and TPN diaphorase activities were strongly active in intralobuler ducts. Serous acini exhibited less activity with these enzymes; and mucous cells showed still less and were almost negative. In serous acini, there was much greater activity of TPN diaphorase than of succinic dehydrogenase.With 7 Figures in the Text     

Structure and histochemistry of the normal intestine of the fowl. I. The mature absorptive cell

Synopsis The fine structure and cytochemistry of the intestinal epithelial cell of the fowl have been investigated. The fine structure of the mature absorptive cell of the fowl duodenum was very similar to that described for man and other mammals. Minor differences were the thinner microvillous glycocalyx, the unusual length of the cells and their microvilli, and the wide distribution of lysosomal bodies. The membrane-associated enzymes alkaline phosphatase, ATPase (pH 7.2) and leucine naphthylamidase were mainly associated with the brush border; this organelle also gave positive reactions for mucopolysaccharides and phospholipids. No enzyme activities were found in the terminal web.The distribution of lysosomes between the terminal web and the Golgi apparatus was correlated with the granular localization of the lysosomal enzymes acid phosphatase, -glucuronidase and non-specific esterase. The mitochondrial enzyme succinate dehydrogenase was seen to be localized in rod-like dots which marked the distribution of mitochondria in the absorptive cell. The localization of mitochondrial ATPase (pH 9.4) was not clearly demonstrated because of diffusion artifacts. The region of the Golgi apparatus gave a strong reaction for thiamine pyrophosphatase, together with weak reactions for acid and alkaline phosphatases after extensive overincubation.The endoplasmic reticulum-associated enzymes glucose-6-phosphatase and nonspecific esterase were distributed throughout the absorptive cell, with a maximum activity apical to the Golgi apparatus. Additionally, the jejunal absorptive cells showed endoplasmic reticulum-as well as lysosomal-associated -glucuronidase.     

Enzymhistochemische Untersuchungen an der Harderschen Drüse des Kaninchens

Zusammenfassung In der Harderschen Drüse von Kaninchen wurden mit der Tetrazoliumtechnik die Enzyme NADH-Tetrazoliumreduktase (NADH-T-Red), NADPH-Tetrazoliumreduktase (NADPH-T-Red), NAD-spezifische Isocitrat-Dehydrogenase (NAD-IDH), Succinat-Dehydrogenase (SDH), NAD-spezifische Malat-Dehydrogenase (NAD-MDH), Laktat-Dehydrogenase (LDH), Glucose-6-phosphat-Dehydrogenase (G-6-PDH),-Glycerophosphat-Dehydrogenase (GPDH) und-Hydroxybuttersäure-Dehydrogenase (HBDH) histochemisch nachgewiesen und ihr Verteilungsbild in den beiden Drüsenlappen studiert. Darüber hinaus wurden histochemische Reaktionen auf folgende Enzyme durchgeführt: Cytochromoxydase (nachBurston), Leucinaminopeptidase (nachNachlas,Crawford undSeligman), alkalische Phosphatase (nachGomori), ATPase (nachPadykula undHerman). Fette wurden mit Scharlachrot und Sudan Schwarz B gefärbt. Die histochemisch faßbare Aktivitätsverteilung der Enzyme geht aus der Tabelle 1 hervor. Die Befunde werden diskutiert.

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Summary A study was conducted to investigate the distribution pattern of various enzymes in both lobes of the Harder gland in rabbits by means of the tetrazolium technique [NADH-tetrazoliumreductase (NADH-T-Red), NADPH-tetrazolium-reductase (NADPH-T-Red), NAD-specific isocitrate-dehydrogenase (NAD-IDH), succinate-dehydrogenase (SDH), NAD-specific malate-dehydrogenase (NAD-MDH), lactate-dehydrogenase (LDH), glucose-6-phosphate-dehydrogenase (G-6-PDH),-glycerophosphate-dehydrogenase (GPDH), and-hydroxy-butyrate-dehydrogenase (HBDH)]. In addition histochemical tests for the determination of cytochrome-oxidase (Burston), leucine-amino-peptidase (Nachlas,Crawford andSeligman), alkaline phosphatase (Gomori), and ATP-ase (Padykula andHerman) were carried out. Fets were stained with Scharlach-red and Sudan black B. The results of the histochemically determinable enzyme activities are listed in table 1. The findings are discussed.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.     

The enzyme histochemistry of developing brown fat in the fetal rat

Summary Histochemically demonstrable enzymes, glycogen, and lipids were studied during prenatal histogenesis of brown fat in fetal rats. SDH, MDH, LDH, DPN and TPN diaphorases, - and -naphthyl esterases were demonstrable in brown fat cells on day 17 of gestation. Glycogen, lipid, G-6-PDH, cytochrome oxidase, indoxyl esterase and alkaline phosphatase were detected on day 17.5. The progressive increase in MDH, LDH, SDH, DPN diaphorase and cytochrome oxidase content paralleled increases in mitochondrial population during differentiation. MAO and esterase content increased only slightly during the prenatal period studied. Alkaline phosphatase appeared in morula stage cells. These were rare at 17.5 days, but increased in number until, at day 20, they were the predominant cells in the dorsal suprascapular brown fat lobules. Further maturation resulted in the loss of alkaline phosphatase which was present in only occasional cells at birth. Reactions for acid phosphatase, ATPase, naphthol-AS esterase, leucine aminopeptidase, -glutamyl transpeptidase, cholesterol and plasmalogens were negative in prenatal and neonatal brown fat cells.Supported in part by Grant No. 375, Nutrition Foundation, Inc.Presented in part at the annual meeting of the Histochemical Society, Atlantic City, N. J., April, 1969.     

Enzyme histochemistry of the mesenteric and dorsal root ganglion cells of cat and squirrel monkey

Summary A detailed histochemical study has been made on the mesenteric ganglia of the cat, and dorsal root ganglia of the squirrel monkey by the use of appropriate histochemical techniques accompanied by appropriate controls for phosphatases, esterases, and oxidative enzymes. The different neurons of a particular ganglion show varied amounts of enzyme activity at a particular time depending upon the functional state of the neurons. SDH, CYO and LDH reaction is prominent in the cytoplasm of the neurons, gliocytes and satellite cells, whereas the MAO preparations generally show a weak reaction. The AK is prominent in the neuropil, cell membranes and peripheral part of cytoplasm, whereas ATPase activity has been observed in blood vessels as well. In AC preparations the area of lipofuscin concentration shows more intense reaction than the rest of the cytoplasm. The activity of AChE and BChE varies from mild, to moderate to strong. The TPPase preparations show morphologically different types and amounts of TPPase positive Golgi material even in the adjoining cells. The relationship between the TPPase Golgi material and various oxidative and dephosphorylating enzymes has been briefly discussed.Abbreviations used AC Acid phosphatase - AChE Acetyl-cholinesterase (specific) - AK Alkaline phosphatase - AMPase Adenosine monophosphatase (5-nucleotidase) - ATPase Adenosine triphosphatase - BChE Butyryl-cholinesterase (nonspecific) - CYO Cytochrome oxidase - DPN-D DPN-diaphorase - G6P Glucose-6-phosphatase - LDH Lactic dehydrogenase - MAO Monoamine oxidase - MDH Malic dehydrogenase - NAD-D NAD-diaphorase - SDH Succinic dehydrogenase - SE Simple esterase - TPPase thiamine pyrophosphatase T. R. Shanthaveerappa in previous publications.     

Genetical investigations into β-glucan content in barley

Summary Random inbred lines produced by doubled haploidy (DH) and single seed descent (SSD) have been used to investigate the genetics of -glucan (gum) content in barley (Hordeum vulgare). Genetical analyses indicated that gum content is controlled by a simple additive genetic system. Significant negative genetic correlations were observed between -glucan content, thousand grain weight and height in the DH samples. These correlations were much reduced in the SSD samples and would suggest linkage of the genes controlling these characters. The presence of repulsion linkages could be exploited in a barley breeding programme by producing F1 derived DH to generate recombinants with high thousand grain weight and low -glucan content. Genetical parameters estimated from DH and F3 samples have successfully been used to predict the number of inbred lines transgressing the parental range for -glucan content and bivariate combinations involving -glucan.     

Histochemistry of some acid hydrolases in striated muscles of the rat

Summary The distribution of acid phosphatase, -n-acetylglucosaminidase, -glucuronidase, and acid -galactosidase was studied in mm. extensor digitorum longus, soleus, and diaphragm of rats. Using the technic of semipermeable membranes activities of these enzymes were demonstrated beside cells of the interstitial tissue in muscle fibers themselves as well. Acid phosphatase displayed the highest activity which appeared in many small dots dispersed in the fiber. The activity of acid phosphatase was about 1.2 x higher in the m. soleus than in the m. extensor digitorum longus. In the latter muscle a somewhat higher activity was often found in muscle fibers displaying a higher staining for NADH tetrazolium reductase. The activity of -n-acetylglucosaminidase was slightly lower, that of -glucuronidase very weak but still discernible. The activity of acid -galactosidase was not ascertained in the majority of fibers. The ratio of activities measured in an area of the same size in cells of the interstitial tissue and in muscle fibers amounted in average to 2.6: 1 in the case of acid phosphatase, 2.5:1 in the case of -n-acetylglucosaminidase, 5.7: 1 in the case of -glucuronidase, and 44.3:1 in the case of acid -galactosidase. The importance of the histochemical technic in studies concerned with acid hydrolases in striated muscle fibers in normal and pathological conditions is pointed out.     

Lysosomal membrane adenosine triphosphatase; solubilization and partial characterization

Membranes prepared from Triton WR-1339-filled lysosomes (tritosomes) contained ATPase activity with a pH optimum of 5–8. These membranes also showed adenosine diphosphatase, adenosine monophosphatase, acid β-glycerol phosphatase, and acid pyrophosphatase activities. The soluble (nonmembrane) fraction of the tritosomes also contained these activities, but the properties of the soluble adenine nucleotide phosphatase activities were different from the membrane-associated enzymes. The pH optimum of tritosomal membrane ATPase changed to 5 after solubilization with Triton X-100, but ADPase and AMPase optima remained at 6–7. The pH optimum of intact membrane ATPase was also 5 when the substrate was α,β-methylene-ATP. Thus, tritosomal membrane ATPase apparently exhibits a pH 8 optimum only when acting in concert with ADPase and AMPase in intact membranes. Rates of ATP hydrolysis to adenosine were also significantly greater in intact membranes than in Triton X-100-solubilized fraction. Centrifugation of Triton X-100-solubilized tritosomal membranes in sucrose density gradients showed that ATPase and ADPase activities sedimented to one peak, and that AMPase, acid phosphatase, and pyrophosphatase were grouped in another peak. Thus, tritosomal membrane ATPase activity was not due to the latter enzymes. The resulting purification was about fourfold for ATPase. The Mr for ATPase and ADPase was estimated to be about 65,000 and for AMPase, acid phosphatase, and pyrophosphatase about 200,000.     

Glutaraldehyde-fixation of yeast cells: Kinetics of a model system for enzyme cytochemistry

The kinetics of glutaraldehyde inactivation of a protoplasmic (-fructofuranosidase) and an extracytoplasmic (acid phosphatase) enzyme inSaccharomyces rouxii cells were studied at pH 5.5 and 30°C. The effects of glutaraldehyde concentration (0.5–3%), pH value, and temperature were surveyed by varying the fixation conditions. Cells from 1- to 10-day cultures retained 50–75% of their acid phosphatase activity and 15–24% of their -fructofuranosidase activity after 1-h exposures to 0.5% glutaraldehyde. The surviving -fructofuranosidase activity remained physically cryptic and was revealed only after further membrane perturbation with ethyl acetate. This crypticity barrier disappeared after overnight incubation of the treated cells at 4°C, with or without added glutaraldehyde, during which time the enzyme was resistant to further inactivation. The velocity ratio for raffinose versus sucrose, as substrate, decreased in treated cells, and changes inV _max andK _m were indicative of frank destruction of some enzyme molecules as well as modification of survivors. A comparable set of changes was also generated by treating cell-free extract with glutaraldehyde. Glutaraldehyde (0.5%) killed all yeast cells at 30°C within 5 min; at 4°C survival rates were quite high—81% after 15 min and 65% after 1 h. The bearing of these examples of enzyme inactivation, permeability barrier abolition, and structural stabilization on the general problems of yeast cytochemistry is discussed.     

Isozymic patterns and functional states of cell lines ofDrosophila melanogaster cultured in vitro. II

Summary Our previous isoenzyme investigation ofDrosophila melanogaster cell lines in vitro has been completed with twelve further enzyme systems. The enzyme profiles seem to be in good agreement with a previous hypothesis concerning the precise origin of these cell lines (probably from imaginal discs or nervous tissues). Our results have been summarized with reference to the biochemical genetic map ofDrosophila melanogaster in order to consider a possible functional organization of the genome.Abbreviations NAD nicotine adenine nucleotide - NADP nicotine adenine nucleotide phosphate - NBT nitroblue tetrazolium - PMS phenazine methosulfate - EDTA ethylene diamine tetraacetic acid - GOT Glutamate-oxaloacetate transaminase - PGK Phosphoglycerate kinase - GPDH -glycerophosphate dehydrogenase - MDH Malate dehydrogenase - PGM Phosphoglucomutase - Aph Alkaline phosphatase - MDH-NADP Malic enzyme - Lap Leucine Amino-Peptidase - LDH Lactate dehydrogenase - -1-OHDH L-3-hydroxyacid dehydrogenase - ADH Alcohol dehydrogenase - Aldox Aldehyde oxydase - 6PGD 6 Phosphogluconate dehydrogenase - G6PD Glucose-6-Phosphate dehydrogenase - Hex3 Fructokinase - IDH Isocitrate dehydrogenase - Est 6 Esterase 6 - Est C Esterase C - ODH Octanol dehydrogenase - XDH Xanthine dehydrogenase - AcPh Acid Phosphatase 1     

Purification and partial characterization of ad(−)-lactate dehydrogenase fromDesulfovibrio desulfuricans (ATCC 7757)

Summary A membrane-boundd(–)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genusDesulfovibrio, was solubilized from the membrane fraction ofDesulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced/min/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography withN-octylamine Sepharose 4B. The enzyme eluted off a Sephacryl S-300 column as a single peak with a molecular weight of 400 000±40 000 Da. Denaturing gel electrophoresis showed it to be composed of 5 protein bands. The oxidized and dithionite reduced spectra of LDH resembles the spectra ofc-type cytochromes found inDesulfovibrio species. The addition of lactate to LDH resulted in a partially reduced spectrum. The flavin/cytochromec/non-heme iron content per 400 000 Da LDH molecular weight was found to be 11.64.5. The LDH activity was specific ford(–)-lactate and had aK _m ford(–)-lactate of 4.3×10^–4 M. The pH optimum was between 6.5 and 8.5.     

Efficacy of Citrus reticulata and Mirazid in treatment of Schistosoma mansoni

This work has been carried out to investigate the effect of Schistosoma mansoni infection on mice livers after treatment with the ethanolic extract of Citrus reticulata root or the oleo-resin extract from Myrrh of Commiphora molmol tree (Mirazid), as a new antishistosomal drug. Marker enzymes for different cell organelles were measured; succinate dehydrogenase (SDH); lactate dehydrogenase (LDH) and its isoenzymes; glucose-6-phosphatase (G-6-Pase); acid phosphatase (AP) and 5'- nucleotidase. Liver function enzymes; aspartate aminotransferase (AST); alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were also estimated. Parasitological studies through ova count and worm burden will also be taken into consideration. The results showed a marked reduction in SDH, LDH, AST, and ALT enzyme activities and a significant increase in G-6-Pase, AP, 5'- nucleotidase, and ALP after S. mansoni infection. A noticeable alteration in LDH subunits were also noticed. Treatment with C. reticulata or Mirazid improved all the previous enzyme activities with a noticeable reduction in ova count and worm burden.     

Partial purification and characterization of β-hydroxybutyric acid dehydrogenase of a methylotrophic bacterium,Pseudomonas 135

Summary An intracellular enzyme, d(—)--hydroxybutyric acid dehydrogenase involved in an intracellular poly-d(—)--hydroxybutyric acid degredation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of d(—)--hydroxybutyric acid (D-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5–6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at — 20° C. The K _m values for oxidation and reduction reactions were determined as 1.84 mm for D-HB, 0.244 mm for NAD⁺, 0.319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that d-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mm for d-lactate, 0.196 mm for NADH and 1.82 mm for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive. Correspondence to: J. M. Lebeault     

Die postnatale Entwicklung der Harderschen Drüse der weißen Ratte

Zusammenfassung Im Anschluß an eine lichtmikroskopisch-morphologische Studie (I. Mitteilung) wurden die Harderschen Drüsen neugeborener bis 13 Wochen alter Ratten mit fermenthistochemischen Methoden (ATPase, alkalische und saure Pase, LAP, LDH, G-6-PDH, -GPDH, -HBDH, SDH, MDH, IDH, Cyt-Ox, NADH-T-Red, NADPH-T-Red) untersucht. Dabei wurden folgende Ergebnisse erzielt:Während der ersten Tage nach der Geburt fallen sämtliche Enzymreaktionen nur schwach positiv aus. Mit beginnender Sekretproduktion am 6. Lebenstag nimmt die Dehydrogenasen-und Diaphorasen-Aktivität in den Drüsenzellen zu und steigt bis gegen Ende der 3. Lebenswoche weiter kontinuierlich an.Von den Hydrolasen sind die saure Phosphatase nur in der 1., die Leucinaminopeptidase nur während der 1.–3. Woche post partum nachweisbar. ATPase reagiert an den ersten Lebenstagen in den Zellen der Drüsenanlage apikal kräftig positiv; von der 2. Woche an findet sie sich vorwiegend basal. Alkalische Phosphatase ist in den Drüsenzellen während der gesamten Beobachtungszeit nur schwach aktiv, dagegen läßt sie sich in den Myoepithelzellen vom 8. Tage an in zunehmender Intensität nachweisen.Die B-Zellen zeigen zwischen dem 10. und 14. Lebenstage eine höhere LAP-Aktivität als die A-Zellen und heben sich vom 21. Tage an durch kräftig positive Dehydrogenasen- und Diaphorasen-Reaktionen von ihnen ab.Die fermenthistochemische Entwicklung der Harderschen Drüse ist gegen Ende der 4. Lebenswoche abgeschlossen. Anhaltspunkte für geschlechtsspezifische Unterschiede fanden sich nicht.

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The postnatal development of the rat harderian glandII. Enzyme-histochemical studies

Summary Following a previous lightmicroscopic-morphological investigation (1st communication), the Harderian glands of rats, age ranging from newly born to 13 weeks, are studied by means of enzyme-histochemical methods (ATPase, alkaline and acid Pase, LAP, LDH, G-6-PDH, -GPDH, -HBDH, SDH, MDH, IDH, Cyt-Ox, NADH-T-Red, NADPH-T-Red). The following results are obtained:All the enzymes examined react weakly during the first days after birth. The beginning of synthesis activity of the glandular cells in 6 days old rats corresponds to an increase of dehydrogenase- and diaphorase-activity. This increase continues until the age of 3 weeks.Acid phosphatase can only be observed in the 1st postnatal week, leucine-amino-peptidase during the 1st to 3rd week. ATPase is localized in the apical part of the gland cells at the first days post partum, and predominandly in the basal part from the first week forth. The alkaline phosphatase activity is low in the glandular cells during all the period observed, whereas in myoepithelial cells its intensity keeps increasing from the 8th day.B-cells show between the 10th and 14th day of age a stronger leucine-amino-peptidase-reaction than A-cells, and after the 21st day differenciate from these by having a more intensive dehydrogenase and diaphorase activity.The Harderian gland enzyme-histochemical developing is finished at the end of the 4th week. Histochemically it was not possible to find sex-dependent differences.

     

ATPase and acid phosphatase activities associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Phosphatase activities were measured in preparations of vacuoles isolated from storage roots of red beet (Beta vulgaris L.). The vacuoles possessed both acid phosphatase and ATPase activities which could be distinguished by their susceptibility to inhibition by low concentrations of ammonium molybdate [(NH₄)₆Mo₇O₂₄·4H₂O]. The acid phosphatase was completely inhibited by 100 M ammonium molybdate but the ATPase was unaffected. The acid phosphatase was a soluble enzyme which hydrolysed a large number of phosphate esters and had a pH optimum of 5.5. In contrast, the ATPase was partially membrane-bound, had a pH optimum of 8.0 and hydrolysed ATP preferentially, although it was also active agianst PP_i, GTP and GDP. At pH 8.0 both the ATPase and PPase activities were Mg²⁺-dependent and were further stimulated by KCl. The ATPase and PPase activities at pH 8.0 may be different enzymes. The recovery and purification of the ATPase during vacuole isolation were determined. The results indicate that the Mg²⁺-dependent, KCl-stimulated ATPase activity is not exclusively associated with vacuoles.Abbreviations BSA bovine serum albumen - MES 2-(N-Morpholino)ethanesulphonic acid - MOPS 3-(N-Morpholino)propanesulphonic acid - Na₂EDTA ethylenediaminetetra-acetic acid, disodium salt - P_i inorganic phosphate - PP_i inorganic pyrophosphate - PPase inorganic pyrophosphatase - TCA trichloroacetic acid - TES N-tris(hydroxymethyl)methyl-2-amino-ethanesulphonic acid - Tris tris(hydroxymethyl)methylamine     

Histochemical changes in the fowl small intestine associated with enhanced absorption after feed restriction

Summary The morphology and histochemical enzyme pattern of the small intestine were investigated in chicks undergoing feed restriction. Corresponding intestinal sites were compared in both restricted birds and in control birds under normal feeding. Intestines from the restricted birds showed some atrophy, the villi being slightly shorter and thinner than normal after eight days restriction, and there was an increase in the activity of alkaline phosphatase, leucine naphthylamidase, acid phosphatase, -glucuronidase, non-specific esterase and succinic dehydrogenase in the absorptive cells.The significance of these findings has been discussed in relation to the enhanced absorptive capacity of the intestine during feed restriction and its similarity to other dietary stress factors that produce enhanced absorption. Possible mechanisms for the production of such mucosal changes have been considered. It was concluded that the enhanced absorption of nutrients in semi-starved animals is correlated with increased mucosal enzyme activities.     

Comparison of rhizobitoxine-induced inhibition of β-cystathionase from different bradyrhizobia and soybean genotypes

The enzyme -cystathionase catalyzes the conversion of cystathionine to homocysteine in both plants and bacteria. Preparations of this enzyme taken from both Salmonella and spinach (Spinacia oleracea L.) have been shown to be irreversibly inhibited by low concentrations of rhizobitoxine (RT), a chlorosis-inducing phytotoxin produced by some strains of soybean bradyrhizobia. The sensitivities of -cystathionase from bradyrhizobia and soybean are not well characterized. Therefore, we purified -cystathionase from selected bradyrhizobia and soybean genotypes that have been shown to exhibit differences in RT production and apparent RT sensitivities, respectively. Enzyme purified from E. coli strain DH52 was used for comparison. The enzymes differed in their physiological properties and RT sensitivities. Overall, the -cystathionase enzymes purified from bradyrhizobia were more sensitive to RT than were those from the soybean cultivars. Kinetic studies showed that the nature of the RT-induced inhibition also differed between the two sources. The enzymes from bradyrhizobia exhibited inhibition that was [RT]-dependent, whereas the enzymes from soybean showed a time-dependent inhibition. These contrasting characteristics may in part reflect differences in active site accessibility, amino acid components, and associated RT diffusion rates. However, in all cases the inhibition caused by RT showed a typical substrate-competitive inhibition pattern.Abbreviations RT rhizobitoxine - PLP pyridoxal phosphate - DTT dithioerythritol - PMSF phenylmethylsulfonyl fluoride - HTP hydroxyapatite Published as Paper No. 1571 in the Journal Series of the Delaware Agricultural Experiment Station.     

Isozymes in nutrient medium of suspension-cultured apple cells (Malus sylvestris Mill.)

The time course of the activities of esterase, -galactosidase, and -glucosidase in cell sap and nutrient medium in in vitro cultured apple cells (Malus sylvestris Mill.) was studied. The corresponding isozyme patterns and the intracellular and extracellular isozyme patterns of acid phosphatase and polyphenol oxidase were compared using isozyme visualization methods adapted to ultra-thin-layer isoelectric focusing. Neither quantitative (total activity) nor qualitative (isozyme pattern) data were congruent for cell saps and nutrient media. Malate dehydrogenase, malic enzyme, and glutamate dehydrogenase occurred in cell sap only. The extracellular activities probably originate to a great part from a programmed release by intact cells. Nutrient media of plant cell cultures constitute a rich source of active plant isozymes.