Subcellular localization of diamine oxidase in rabbit kidney cortex

The intracellular localization of diamine oxidase (EC 1.4.3.6) in rabbit kidney cortex was studied. The distribution of diamine oxidase in the subcellular fractions, obtained by modifying the classical method of Wattiaux-De Coninck, S., Rutgeerts, M.T. and Wattiaux, R. (Biochim. Biophys. Acta (1965) 105, 446-459) demonstrated that this activity is concentrated (greater than 60%) in the microsomal fraction. Biochemical and morphological data indicate a 20-30% contamination of this fraction by plasma membrane and brush border fragments. Subfractionation of the microsomes, obtained by centrifuging in a continuous sucrose-Ficoll gradient (d 1.038-1.064) for 75 min, showed that diamine oxidase is concentrated in membrane deriving from the endoplasmic reticulum. In fact the bulk of diamine oxidase activity was recovered in a subfraction of the gradient which was shown both biochemically and morphologically to derive from the endoplasmic reticulum. The possible significance of this result is discussed.     

Association of diamine oxidase with microsomal membranes in rabbit liver

Diamine oxidase (diamine O₂ oxidoreductase, EC 1.4.3.6, DAO) of rabbit liver is localized in the microsomal fraction (1, 2). Microsomes, freed of adsorbed and intraluminal proteins by washing procedures, still retain all DAO activity. This indicates that DAO belongs to the microsomal membrane itself. While the release of DAO activity from washed microsomes occurs exclusively in the presence of high concentrations of deoxycholate, tight binding of this enzyme to the membrane structure is apparent.     

Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration

The effect of a single administration of ethanol (2 g/kg body weight) on hepatic and renal diamine oxidase activity was studied in fasted rats. Diamine oxidase activity significantly increased in liver and kidney 6 h after ethanol intubation. Pyrazole (an inhibitor of alcohol dehydrogenase), cycloheximide or actinomycin D (inhibitors of macromolecular syntheses), as well as prior adrenalectomy, prevented the ethanol-induced stimulation of diamine oxidase in the liver, but not in the kidney. The results demonstrated that the enhancement of diamine oxidase activity in the liver was due to an enzyme induction mediated by alcohol metabolism as well as by adrenals. In contrast, the stimulation of diamine oxidase activity in the kidney did not depend on synthesis of new enzyme molecules and was not mediated by ethanol metabolism or adrenal hormones.     

Polyamine levels and diamine oxidase activity in hypertrophic heart of spontaneously hypertensive rats and of rats treated with isoproterenol

Polyamine levels and diamine oxidase (EC 1.4.3.6) activity were studied in hypertrophic heart of spontaneously hypertensive rats as well as in the heart of Wistar rats during the development and regression of cardiac hypertrophy induced by isoproterenol administration. In spontaneously hypertensive rats, putrescine content and diamine oxidase activity were higher than those found in normotensive Kyoto-Wistar control rats. During the development of cardiac hypertrophy induced by isoproterenol, there was an increase in polyamine content and diamine oxidase activity. The administration of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity during the first 24 h after isoproterenol administration, demonstrating that the rise in diamine oxidase activity was due to synthesis of new enzyme. Following the cessation of isoproterenol treatment, cardiac hypertrophy regressed and polyamine levels and diamine oxidase activity diminished toward control values. The administration of aminoguanidine to isoproterenol-treated rats caused in the heart an inhibition of diamine oxidase activity that led to an increase in putrescine level beyond the values found in animals given isoproterenol alone. The results suggest that the enhancement of diamine oxidase activity plays a role in the regulation of putrescine level in hypertrophic heart.     

Location of glycerol phosphate acyltransferase in the transverse plane of mitochondrial outer membrane of guinea pig lung

Incubation of guinea pig lung mitochondrial suspension in an isotonic low ionic strength buffer containing various proteolytic enzymes caused significant stimulation of the glycerophosphate acyltransferase activity. The maximal stimulation range between 20 and 105%, and the order was as follows: bromelain greater than chymotrypsin greater than pronase greater than trypsin greater than papain greater than nagarse. Under hypotonic conditions, over 85% of GAT was destroyed by all the proteolytic enzymes. Microsomal enzyme activity was consistently inhibited (greater than 95%) by exposure to any of these proteases even under isotonic conditions. These results suggest that GAT is located on the inner aspect of the mitochondrial outer membrane. Also, it is likely that a portion of this enzyme or that of a modulator is present in the outer side of the outer membrane and proteolysis of this component causes stimulation.     

Effects of pronase on passive ion permeability of the human red blood cell

Summary The effects of pronase fromStreptomyces griseus on sulfate, potassium, sodium, and erythritol permeability of human red blood cells were studied. It was found that the proteolytic enzyme reduces anion permeability, increases cation permeability and has no effect on the nonfacilitated component of the flux of the nonelectrolyte. These findings can be explained on the basis of the fixed charge hypothesis by the assumption that the enzyme exerts its effects by altering the density of positive fixed charges in the membrane.The effects of pronase are qualitatively similar to those of the amino reactive agent, dinitrofluorobenzene (DNFB). Therefore, attempts were made to discover if this similarity is due to alterations of the same membrane sites by the enzyme and the chemical modifier. It was found that the effects of pronase and DNFB were not additive. Hence, the enzyme and the amino reactive agent do not seem to act on two independent and parallel channels. A more detailed analysis of the data suggests that DNFB and pronase affect functionally identical sites.Proteolytic enzymes frequently exhibit some esterase activity. However, the amino-N content of lipid extracts of red cell membranes remained virtually unaltered after exposure of the cells to pronase. This finding indicates that the positive charge of the bulk of the lipid amino groups is not involved in the control of passive ion permeability. The carbohydrate amino groups of the red cell membrane are N-acylated and hence cannot contribute to the positive membrane charge. It seems reasonable to conclude that the effects of pronase on ion permeability are primarily due to alterations of the density of charged protein amino groups in the red cell membrane.     

Induction of diamine oxidase activity in rat kidney during compensatory hypertrophy

Diamine oxidase (EC 1.4.3.6) activity, measured as [¹⁴C]Δ¹-pyrroline formation from [¹⁴C] putrescine, was studied in homogenates of rat kidney during compensatory hypertrophy after unilateral nephrectomy. Acetaldehyde and to a lesser degree phenobarbital, at concentrations which did not modify the activity of a preparation of hog kidney diamine oxidase, increased Δ¹-pyrroline formation in kidney homogenate, which suggests that aldehyde-metabolizing enzymes present in this tissue may interfere with yield of Δ¹-pyrroline and that the use of acetaldehyde may give better information on kidney diamine oxidase activity. Other inhibitors of aldehyde-metabolizing enzymes such as chloral hydrate, disulfiram, and pyrazole cannot be used for diamine oxidase determination since they stimulated or depressed this enzyme activity. In rat kidney undergoing compensatory hypertrophy the levels of putrescine, spermidine, and spermine increased rapidly and were followed by an increase in diamine oxidase activity that presented a first peak on day 2 and a second peak on day 6. The administration of cycloheximide or actinomycin D to nephrectomized rats prevented the increase in diamine oxidase activity. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 14 h in normal and hypertrophic kidney. These results suggest that the increase in diamine oxidase activity in renal hypertrophy was due to the synthesis of new enzyme rather than to a slowing of its degradation.     

Detection of xanthine oxidase and immunologically related proteins in fractions from bovine mammary tissue and milk after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate.

A solid-phase immunoassay was used to detect xanthine oxidase in fractions from bovine mammary glands after electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Under these conditions the major proportion of xanthine oxidase in either mammary tissue or mild could be recovered as a protein of mol.wt. 150 000. In mammary tissue approx. 80% of the enzyme was in a soluble form and the remainder was accounted for in either 'mitochondrial' or microsomal fractions after tissue homogenization and fractionation. Affinity chromatography of either detergent-solubilized microsomal membranes or postmicrosomal supernatants on immobilized antibody to xanthine oxidase yielded a single protein that cross-reacted with antibody to the enzyme. In milk presumptive degradation products of the enzyme were detected in minor quantities with mol.wts. of 43 000 in the whey fraction and 90 000 in fat-globule membrane. Only the undegraded enzyme was present in the skim-milk membrane fraction. Xanthine oxidase is therefore synthesized and secreted as a protein with a monomeric mol.wt. of 150 000 and is not subjected to extensive proteolytic degradation during the storage of milk in mammary alveoli. The significance of the results is discussed in relation to the overall protein composition of the membranes of milk-fat globules and skim milk.     

Enhanced binding of low and high density lipoproteins to human adipocyte plasma membranes: effects of temperature and proteases

The specific binding of 125I-labelled low density lipoprotein ([125I]LDL to human adipocyte plasma membranes was higher at 37 than at 0 degree C. Prior treatment of membranes with pronase had no effect on LDL binding measured at 0 degree C but consistently stimulated binding at 37 degrees C. Plasmin was similar to pronase in enhancing LDL-specific binding, but thrombin was not as effective. 125I-labelled high density lipoprotein ([125I]HDL2) specific binding to human adipocyte plasma membranes was similarly sensitive to temperature and pronase treatment. Addition of the protease inhibitor aprotinin in the adipocyte membrane binding assay significantly reduced [125I]LDL binding at 37 degrees C (p less than 0.05), suggesting the involvement of a protease activity intrinsic to the lipoproteins and (or) membranes. These data demonstrate that both LDL and HDL binding in human adipocyte plasma membranes can be "up-regulated" by specific proteolytic perturbations in a temperature-dependent manner.     

Spermidine oxidase in human pregnancy serum. Probable identity with diamine oxidase.

Diamine oxidase was previously measured in human pregnancy serum with putrescine or histamine as substrate. We have now documented the presence of spermidine oxidase activity in pregnancy serum by means of a specific radioactive assay with [14C]spermidine as substrate and Dowex 50 cation-exchange chromatography to separate products from substrate. The apparent Km of a partially purified preparation of this enzyme for spermidine was 10.9 microM and the Ki for aminoguanidine was 0.8 microM. The pH optimum (pH 9.0) and temperature optimum (55 degrees C) were identical with those for diamine oxidase. Spermidine oxidase activity and diamine oxidase activity eluted in a concerted fashion when pregnancy serum was subjected to cadaverine-Sepharose chromatography, gel filtration and ion-exchange chromatography. Spermidine oxidase became detectable in serum during pregnancy in the human approx. 8 weeks after the last menstrual period and increased with gestational age in concert with the increase in diamine oxidase activity, reaching a plateau at 20 weeks of gestation. Foetal-cord serum displayed virtually no activity of either enzyme. A 400-fold-purified preparation of diamine oxidase retained the same diamine oxidase/spermidine oxidase ratio as exhibited by crude pregnancy serum. These data suggest that in pregnancy serum, unlike foetal bovine serum, spermidine oxidase and diamine oxidase activity may be a single enzyme protein.     

Effects of chronic alcohol consumption on the steady-state kinetics properties of cytochrome oxidase in rat liver

The effect of chronic alcohol consumption on steady-state kinetic characteristics of cytochrome oxidase in rat liver was studied using submitochondrial particles prepared from ethanol-fed and control rats. Preparations from both control and alcoholic rats had equivalent apparent Km values for cytochrome c of 13 microM in the presence of phenazine methosulfate or 19 microM with N,N,N',N'-tetramethylphenylene diamine as oxidation-reduction mediators at physiological ionic strength. Both preparations showed comparable stimulation (approx. 3-fold) of oxidase activity following detergent solubilization of the membrane and similar temperature dependence for oxidase activity. Under all conditions, preparations from alcohol-fed rats displayed 30 to 50% lower rats of cytochrome oxidase activity per unit membrane protein than those from control rats. The diminution in specific activity per mg protein was accompanied by a similar decline in heme aa3 content, as has been noted in previous studies. When expressed on a turnover number basis, the molecular activity of cytochrome oxidase (natoms O/min per nmol heme a) was equivalent in both alcoholic and control preparations. The results indicate that the intrinsic kinetic characteristics of cytochrome oxidase are not changed by alcohol consumption. The data suggest that the characteristic decline in heme aa3 content and cytochrome oxidase specific activity seen in ethanol-fed rats does not arise from alterations in the accessibility of the oxidase towards cytochrome c, or from changes in bulk phase lipid composition or physical properties. The results support the conclusion that ethanol consumption decreases the membrane content of functionally active oxidase molecules, but does not change the catalytic properties of these oxidase molecules.     

Sidedness of Phosphatidylcholine-Synthesizing Enzymes in Rat Brain Microsomal Vesicles

The sidedness of CDP-choline:1,2-diradylglycerol choline phosphotransferase (EC 2.7.8.2) and of the choline base-exchange activity has been studied in rat brain microsomal vesicles. Proteases (trypsin and pronase) and mercury-dextran have been used as reagents for membrane surface components. All of them could inactivate both enzymes to a good extent, without affecting the morphology or the permeability to sucrose of the vesicles. It is therefore concluded that CDP-choline:1,2-diradylglycerol choline phosphotransferase and the choline base-exchange activity are localized on the outer surface of rat brain microsomal vesicles.     

Membrane proteins and urea and acetamide transport in the human erythrocyte.

Previous studies have shown that urea and acetamide traverse the erythrocyte membrane by way of facilitated diffusion. The nature of this selective pathway is unknown. The present studies investigate the effects of proteolytic enzymes and crosslinking agents on amide transport. Cleavage of the erythrocyte membrane surface by pronase or trypsin had no effect on urea and acetamide permeability or inhibition by phloretin. These findings suggest that the sialoglycopeptide segment of the sialoglycoproteins is not critical to urea and acetamide transport. In addition, extensive crosslinking of membrane proteins with glutaraldehyde had no effect on amide transport in the absence or presence of phloretin.     

Diamine oxidase activity induction in regenerating rat liver

The synthesis and turnover of diamine oxidase (EC 1.4.3.6) activity was studied in regenerating rat liver after partial hepatectomy using inhibitors of protein and RNA syntheses. The administration to animals of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity normally observed during the first hours after hepatectomy. The study of the turnover rate of diamine oxidase with cycloheximide demonstrated that the half-life of this enzyme was about 15 h in normal and regenerating liver. These results suggest that the rise in diamine oxidase activity in regenerating rat liver was due to the synthesis of new enzyme rather than to a lengthening of its turnover.     

Protease inhibitors but not proteases inhibit the glucose-6-phosphatase of native rat liver microsomes.

Controlled proteolytic digestion by trypsin or bacterial proteases limited to the cytosolic side of the native microsomal membrane is not efficient to inhibit glucose-6-phosphate hydrolysis. Modification of the microsomes with deoxycholate prior to protease treatment is prerequisite to allow accessibility of the integral protein and inhibition of enzyme activity. Glucose-6-phosphatase of native microsomes, however, is rapidly inactivated by micromolar concentrations of TPCK as well as TLCK. In deoxycholate-modified microsomes both reagents do not affect glucose-6-phosphate hydrolysis. These results indicate that in the native, intact microsomal membrane glucose-6-phosphatase is not accessible to proteolytic attack from the cytoplasmic surface. The putative inhibitory effect of some trypsin or bacterial protease preparations on glucose-6-phosphatase of native microsomes observed most possibly is a result of contaminating agents as TPCK or TLCK.     

Release of intestinal diamine oxidase by histamine in rats

Diamine oxidase activity was measured in the intestinal mucosa, lymph, and in the serum of rats, to determine whether histamine, a substrate of diamine oxidase, liberates this enzyme from its mucosal storage site(s). Histamine induced a sharp rise in intestinal lymph flow, lymph protein, and lymph diamine oxidase, lasting less than 1 h after the histamine injection. The rise in lymph diamine oxidase activity was dose dependent over a narrow concentration range (0.05-0.2 mmol/kg, i.v. and 0.15-0.6 mmol/kg i.d.). It did not correlate with the dose dependent increase in lymph flow or lymph protein. A single maximal intraduodenal dose of histamine caused a 41.6-fold increase in the lymph diamine oxidase activity and a 2.4-fold increase in the serum enzyme level temporarily. A second injection of histamine, 2 h after the first, resulted in a comparatively smaller increase in the lymph enzyme. The extent of the reduction was dependent on the magnitude of the first injection. The results suggest that histamine causes a limited liberation of diamine oxidase from the intestinal mucosa. The function of this enzyme release may be a protective response by the mucosa to reduce toxic levels of free histamine, either liberated by the mucosal tissue or absorbed from the intestinal lumen.     

Diamine Oxidase in Cotyledons of Pisum sativum Develops as a Result of the Supply of Oxygen through the Embryonic Axis during Germination

The activity of diamine oxidase (EC 1.4.3.6.) in pea, Pisum sativum cv Alaska, cotyledons was studied. The rapid hydration caused by soaking seeds in water, the excision of the embryonic axis, and the suppression of the elongation of the embryonic axis by indoleacetic acid generate anaerobic conditions in these cotyledons that suppress diamine oxidase activity. These results show that oxygen is essential for the induction of diamine oxidase activity in pea cotyledons. During germination cotyledonary diamine oxidase develops as a result of the supply of oxygen through the embryonic axis of the intact pea seedling.     

Response of tissue diamine oxidase activity to polyamine administration.

The administration to rats of putrescine (750 mumol/kg body wt.) caused in liver, kidney and heart an increase in putrescine at 1 h and in diamine oxidase (EC 1.4.3.6) activity within 3-6 h. An increase in spermidine was observed at 9 h in liver and at 6 h in kidney, whereas in heart there was no change. The increase in diamine oxidase activity by exogenous putrescine was prevented by the administration of actinomycin D and cycloheximide, suggesting that syntheses of mRNA and protein are involved. Equimolar doses of 1,3-diaminopropane, 1,5-diaminopentane and monoacetylputrescine stimulated, similarly to putrescine, hepatic, renal and cardiac diamine oxidase activity. After the injection of a non-toxic dose of spermidine (750 mumol/kg body wt.), the increase in diamine oxidase activity occurred at 9 h in all the tissues studied, when a substantial putrescine formation from spermidine occurred. sym-Norspermidine, which is unable to form putrescine, did not cause an increase in enzyme activity. The possibility that the tissue contents of putrescine might regulate diamine oxidase activity is discussed.     

Interaction of photosynthetic pigments with various organic solvents. Magnetic circular dichroism approach and application to chlorosomes

Reduction of extracellular ferricyanide by intact cells reflects the activity of an as yet unidentified trans-plasma membrane oxidoreductase. In human erythrocytes, this activity was found to be limited by the ability of the cells to recycle intracellular ascorbic acid, its primary trans-membrane electron donor. Ascorbate-dependent ferricyanide reduction by erythrocytes was partially inhibited by reaction of one or more cell-surface sulfhydryls with p-chloromercuribenzene sulfonic acid, an effect that persisted in resealed ghosts prepared from such treated cells. However, treatment of intact cells with the sulfhydryl reagent had no effect on NADH-dependent ferricyanide or ferricytochrome c reductase activities of open ghosts prepared from treated cells. When cytosol-free ghosts were resealed to contain trypsin or pronase, ascorbate-dependent reduction of extravesicular ferricyanide was doubled, whereas NADH-dependent ferricyanide and ferricytochrome c reduction were decreased by proteolytic digestion. The trans-membrane ascorbate-dependent activity was also found to be inhibited by reaction of sulfhydryls on its cytoplasmic face. These results show that the trans-membrane ferricyanide oxidoreductase is limited by the ability of erythrocytes to recycle intracellular ascorbate, that it does not involve the endofacial NADH-dependent cytochrome b(5) reductase system, and that it is a trans-membrane protein that contains sensitive sulfhydryl groups on both membrane faces.     

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.