Enzyme Excretion Research Articles

FAECAL EXCRETION OF BRUSH BORDER MEMBRANE ENZYMES IN PATIENTS WITH CLOSTRIDIUM DIFFICILE DIARRHOEA

Purpose: To look for the presence of intestinal brush border membrane (BBM) enzymes in the faecal samples of patients with Clostridium difficile association. Methods: One hundred faecal samples were investigated for C.difficile toxin (CDT). Simultaneous assays for faecal excretion of intestinal BBM enzymes viz., disaccharidases, alkaline phosphatase (AP) and leucine aminopeptidase (LAP) were also done. Results:C.difficile toxin was detected in 25 (25%) of the samples with a titre ranging from 10 to 160. No significant difference (p>0.05) was seen between the CDT positive and negative groups with any of the disaccharidases studied. However, significant increase (p<0.05) in the AP excretion was observed in the CDT positive patients compared to the CDT negative group. In contrast to this, a significant decrease (p<0.001) in the LAP enzyme excretion was observed in the latter group. Conclusions: The results of this study suggest that there is a significant disturbance in the intestinal BBM enzymes in patients with C.difficile diarrhoea.

Synthesis of a palladium(II)-dithiocarbamate complex: biological assay and nephrotoxicity in rats.

A new palladium(II)-dithiocarbamate complex, [Pd(ESDT)Cl](n), was synthesised and its chemical characteristics are discussed. This complex was examined for its cytotoxic properties in human tumour cell lines; for comparison, the cytotoxicity of cisplatin was evaluated under the same experimental conditions. In particular, Pd(II)-complex cytotoxicity on ovarian carcinoma C13 cells, resistant to cisplatin, showed that there seemed to be no cross-resistance between [Pd(ESDT)Cl](n) and cisplatin. The effects on the kidney were also studied. Biochemical investigation on urinary parameters showed that the effects after a single injection are similar to those of cisplatin, with an increase of urinary proteins and enzyme excretion in urine, and a significant decrease of glutamine synthetase activity in the renal tissue. In addition, the Pd(II)-complex caused a significant decrease of p-aminohippuric acid uptake in renal cortical slices relative to cisplatin. On the other hand, histopathological findings showed that the effects of the Pd(II)-complex are more severe and diffuse than the damage caused by cisplatin. Biochemical and histopathological findings show that the Pd(II)-complex affects the pars recta and pars convoluta, in contrast to cisplatin, which only affects the pars recta.

Chronic cyanide toxicity and urinary enzyme excretion in rabbits

The time-course effect of sub-acute cyanide toxicity on urinary excretion of some marker enzymes for kidney damage was studied in two groups of three-month-old New Zealand White rabbits fed for eight weeks on either pure growers mash or mash plus 640ppm potassium cyanide. Weekly assays of lactate dehydrogenase (LDH), acid phosphatase (AP) and alkaline phosphatase (ALP) were carried out in urine for both groups, and in kidney tissues on termination of feeding. Results show that LDL and ALP were detected in the urine of cyanide-treated rabbits in the second and fifth weeks respectively and up to the eighth week. AP was not detected in urine, and its activity in the kidney was unaffected by cyanide. In contrast, cyanide treatment caused significant increases in kidney LDH while kidney ALP was significantly decreased relative to controls (p < 0.05). The pattern of enzyme changes in kidney and urine suggest cyanide-induced damage to kidney tubules most probably via tissue hypoxia. Key Words: Cyanide toxicity, enzyme excretion, urine [Jnl Medicine & Biomedical Res. Vol.1(2) 2002: 65-69]

Attenuation of oxalate-induced nephrotoxicity by eicosapentaenoate–lipoate (EPA–LA) derivative in experimental rat model

Hyperoxaluria is one of the major risk factors for the formation of urinary calcium oxalate stones. Calcium oxalate crystals and their deposition have been implicated in inducing renal tubular damage. Lipoic acid (LA) and eicosapentaenoic acid (EPA) have been shown to ameliorate the changes associated with hyperoxaluria. This prompted us to investigate the nephropr-otectant role of EPA-LA, a new derivative, in vivo in hyperoxaluric rats.Elevation in the levels of calcium, oxalate and phosphorus, the stone-forming constituents, were observed in calculogenic rats as a manifestation of crystal deposition. Tubular damage to the renal tissue was assessed by assaying the excretion of marker enzymes in the urine. Damage to the tubules was indicated by increased excretion of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), γ -glutamyl transferase (γ -GT), β -Glucuronidase (β -GLU) and N-Acetyl β -D glucosaminidase (NAG). Fibrinolytic activity was found to be reduced. Administration of EPA, LA and EPA-LA reduced the tubular damage and decreased the markers of crystal deposition markedly, which was substantiated by the reduction in weight of bladder stone formed. Our results highlight that EPA-LA is the most effective drug in inhibiting stone formation and mitigating renal damage caused by oxalate toxicity, thus confirming it as a nephroprotectant. Further work in this direction is warranted to establish the therapeutic effectiveness of this new derivative.

Decreased sensitivity of distal nephron and collecting duct to parathyroid hormone in pseudohypoparathyroidism type I.

Parathyroid hormone (PTH) transiently increases urinary excretion of the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase, which is distributed mainly in proximal tubules. The response is reduced in pseudohypoparathyroidism (PHP) type I, which is characterized by target-organ resistance to PTH. Evidenced by normal calcium resorption, distal tubule sensitivity to PTH has been believed to be normal in this disorder. This hypothesis was tested through a search for another marker of distal nephron sensitivity to PTH. In the human kidney, cathepsin D was expressed predominantly in distal segments of the nephron, cortical and medullary thick ascending limbs of Henle's loop, distal convoluted tubules, and connecting tubules and in cortical collecting ducts and medullary collecting ducts. PTH infusion transiently increased cathepsin D excretion in normal subjects. The cathepsin D response to PTH was reduced in the patients with PHP type I. The decrease in cathepsin D response in PHP type I indicates a resistance to PTH in the distal nephron (cortical thick ascending limbs of Henle's loop, distal convoluted tubules, and connecting tubules) and cortical collecting ducts. These observations suggest that the preservation of renal tubular sensitivity to PTH in this disorder may be confined to PTH-dependent calcium resorption in distal tubules.

Effects of renal sorbitol accumulation on urinary excretion of enzymes in hyperglycaemic rats.

We studied the effects of epalrestat, a specific inhibitor of aldose reductase, on renal sorbitol accumulation and the resulting urinary enzyme excretion in hyperglycaemic rats. The activities of proximal tubule-derived enzymes such as N-acetyl-beta-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), gamma-glutamyltranspeptidase (GGT) and dipeptidyl aminopeptidase IV (DAPIV) in urine were determined in five groups of male Wistar rats (each n = 7): (a) 0.9% saline-loaded, (b) 10% glucose-loaded, (c) 10% glucose-loaded with epalrestat pretreatment, (d) 10% mannitol-loaded and (e) 10% mannitol-loaded with epalrestat pretreatment. Epalrestat was given mixed in chow at a dose of 50 mg/kg body weight. Urinary NAG, AAP, GGT and DAPIV activities were significantly increased (P<0.005, P<0.05, P<0.01, P<0.01, respectively) by the induction of hyperglycaemia. In contrast, enzyme excretion was not increased in the mannitol- or saline-loaded groups. Pre-treatment with epalrestat completely prevented the increased urinary excretion of NAG, AAP and GGT. At the end of the infusion study, renal cortical glucose concentrations of the glucose-loaded groups with and without epalrestat pretreatment were approximately fivefold higher than those of the mannitol- or saline-loaded groups (P<0.005 each). Renal cortical sorbitol concentrations of the glucose-loaded group was also approximately twofold higher than those of the mannitol- or saline-loaded groups (P<0.01 each). However, in the group that received both glucose and epalrestat, renal cortical sorbitol concentration was not increased. These results suggest that accumulation of intracellular sorbitol leads to proximal tubular cell dysfunction and abnormal enzymuria.

Calculation of urinary enzyme excretion, with renal structure and function in dogs with pyometra

The urinary enzyme markers of renal damage, alkaline phosphatase (AP), γ-glutamyl transferase (GGT) and N-acetyl-β-glucosaminidase (NAG), glomerular filtration rate (GFR) and renal biopsies were studied to evaluate renal status in dogs with pyometra. After ovariohysterectomy, urinary enzymes were measured daily for 12 days in 55 dogs, and again at a later follow-up visit. Thirteen dogs had high levels of at least one enzyme at initial presentation. Seventeen dogs had a transient increase in urinary enzyme values between one and five days after surgery. Enzyme values usually declined to low activities within 12 post-operative days. Renal biopsies demonstrated tubular abnormalities in many dogs. Mean GFR was 2·4 and 2·0 ml min–1 kg–1, respectively on day 1 post-operatively and at the follow-up visit 1–4 months later. High urinary enzyme values often reflected extensive lesions in renal proximal tubular cells and sometimes reduced GFR.

INFLUENCE OF DIURESIS ON ENZYMURIA

Urinary excretion of renal brush border enzymes may serve as an early marker of renal injury. However, the distinction between physiological and pathological levels remains controversial, since enzymuria is affected by physiological parameters. To clarify the influence of diuresis, we investigated the urinary excretion of alanine-aminopeptidase (AAP; EC 3.4.11.2) as function of diuretic state. 17 healthy volunteers of both sexes were subjected to protocols with sudden or prolonged water load preceded and followed by a thirst period. Urinary excretion of AAP was measured using an enzyme kinetic assay. As expected AAP excretion increased with urine flow, the increments diminished yielding an overall excretion pattern that resembled saturation kinetics. This function is described by a mathematical model. This model assumes, that AAP is released in proximal tubules at a constant rate and reabsorbed or inactivated in the distal tubule and collecting duct. Non-linear fits of the model equation to our data allowed two parameters, χ and μ, to be defined. χ describes the rate of AAP release independent of urinary flow, and μ the ratio of distal tubular reabsorption or inactivation. If a substrate is not reabsorbed at all, μ approximates zero. Since μ fitted for AAP differed significantly from zero, this indicates reabsorption or inactivation of AAP in the distal nephron. Therefore, our study supports the theory of flow-dependent reabsorption or inactivation of AAP in the distal nephron.

Increased urinary excretion of tubular enzymes and proteins in children with epilepsy

Cross-sectional analysis of children undergoing treatment with anti-epileptic drugs has shown an increased urinary excretion of tubular enzymes and proteins. This has usually been interpreted as a consequence of subclinical renal–tubular damage or enzyme induction. We measured excretion of tubular enzymes and proteins in 29 children who suffered from epileptic seizures and in 27 control children. Investigations were undertaken at diagnosis before the start of treatment and 3–4 months later. At diagnosis we found a slightly, but statistically significant increased excretion of N -acetyl-β-glucosaminidase and α1-microglobulin. There was no significant difference between patients with an idiopathic and symptomatic aetiology of seizures or between patients with different seizure types. At the second investigation, in children treated with carbamazepine or valproate, no further increase occurred. We conclude that the increased excretion of tubular enzymes and proteins in children with epilepsy is most probably not due to a side-effect of the anti-epileptic drugs, but to a physiological alteration associated with the epilepsy itself. While the cause is unknown, the influence of serotonin metabolism is discussed.

Urinary protein excretion in Type 2 diabetes with complications

This study examined the association between urinary markers of early diabetic nephropathy and non-renal diabetic complications in 946 patients with type 2 diabetes mellitus. The association with hypertension was also studied. Data on macrovascular complications (ischaemic heart disease, stroke, peripheral vascular disease) and microvascular complications (retinopathy, peripheral neuropathy) were obtained from case records and clinical examination. Urine samples collected were analysed for albumin, β 2-microglobulin, retinol-binding protein (RBP), and N-acetyl-β- d-glucosaminidase (NAG). Results showed that urinary albumin, RBP and β 2-microglobulin levels were higher in patients with macro- and/or microvascular complications, compared to those without. NAG levels were higher only in patients with both types of complications. A higher proportion of patients with complications had abnormally raised urinary protein and enzyme levels, compared to those without. Patients with associated hypertension had higher urinary levels of albumin and β 2-microglobulin, regardless of whether complications were present or not. RBP excretion was, however, markedly higher only in patients with microvascular complications, whereas hypertension did not influence NAG excretion. Urine albumin and RBP excretion were predictive of microvascular, as well as both macrovascular and microvascular complications, whereas NAG excretion was predictive of macro- and microvascular complications. These findings could mean that increased urinary protein and enzyme excretion were associated with more severe disease in these patients.

Human neutral brush border endopeptidase EC 3.4.24.11 in urine, its isolation, characterisation and activity in renal diseases

Human neutral brush border endopeptidase (NEP) was purified from the urine of patients suffering from acute toxic tubulointerstitial nephropathy. An enzyme preparation with specific activity of 102 U g −1 protein was obtained. The urinary activities of neutral endopeptidase and alanine aminopeptidase were measured in patients with renal disease and in 30 control patients, resulting in a reference range from 0.1 to 0.7 U g −1 creatinine and 1.4–14.1 U g −1 creatinine, respectively. Urine enzyme activities were highest in patients with acute tubulotoxic renal diseases. Neutral endopeptidase and alanine aminopeptidase activities were found to be 6.5- and 10-fold higher than the upper value of the reference range, respectively. Smaller increases in the rate of excretion of these enzymes (2.5- and 3.5-fold), respectively, were observed in patients suffering from acute tubular insufficiency and even lower increases, 2- and 1.5-fold, respectively, were observed in patients with chronic renal diseases. In diabetics and kidney transplant patients the enzyme excretion rates were within the reference range. Assay of both transmembrane metalloproteinases in urine may prove valuable in serving as markers for renal toxicity. Together with β-NAG these enzymes could be employed as differentiation markers between acute and chronic tubular insufficiency.

Identification and characterization of an extracellular acid trehalase from the ectomycorrhizal fungus Amanita muscaria

The non‐reducing disaccharide trehalose is a major storage compound in most fungi. For a better understanding of carbon metabolism in the ectomycorrhizal‐forming basidiomycete Amanita muscaria, trehalase activity was analysed from mycelium growing in liquid culture or on agar plates, and from mycorrhizas. Trehalase activity was found in both the culture medium and in mycelial extracts. The excreted trehalase was purified to apparent homogeneity by anion‐exchange chromatography, gel filtration and preparative gel electrophoresis. The identified enzyme belongs to the group of acid trehalases. The apparent molecular mass of the native enzyme was estimated to be 165 kDa by gel filtration and 135 kDa by SDS–PAGE. Isoelectrofocusing indicated an isoelectric point (pI) of approx. 3.7, which is typical for acid trehalases. The enzyme is highly specific for its substrate trehalose, with an apparent Km of 0.38 mM. Validamycin and sucrose act as competitive inhibitors with Ki values of 45 nM and 15 mM, respectively. The activity of acid trehalase excreted into the growth medium is independent of the carbon source (glucose or trehalose), revealing that the enzyme is not regulated by its substrate trehalose. Nevertheless, fungal hyphae grown in the absence of an external carbon source showed enhanced enzyme excretion. The biochemical characteristics of the trehalase activity measured in the mycelium are in the same range as those determined for the excreted enzyme. The enzyme is localized in the cell wall and its activity is strongly decreased in ectomycorrhizas.

La dégradation des proies chez les plantes carnivores

Thanks to various digestive glands, carnivorous plants are able to exploit trapped preys. Those are hydrolysed to absorbable nutrients by enzymes secreted by the plant itself or, in some cases, by micro-organisms. For a few species, the secretion of digestive juice occurs in response to various, clearly defined stimuli. However, the exact mechanisms of transfer and excretion of digestive enzymes, as well as their origin, need further research to become thoroughly understood.

THE COMBINED EFFECT OF CYCLOSPORINE A AND GENTAMICIN ON ENZYMURIA IN THE SPRAGUE-DAWLEY RAT

Male Sprague-Dawley rats (8 per group) were administered a single oral dose of cyclosporine A (10, 30 and 50 mg/day) for 5 days or vehicle (corn oil, 1.5 mL/kg) and urinary enzymes excretion was monitored. Only minor changes in enzymuria were observed in the 10 and 30 mg/kg group. However, in the 50 mg/kg group, nephrotoxicity was evident by significant increase in the excretion of N-acetyl-β-D-glucosaminidase (NAG), glutamate dehydrogenase (GDH), and lactate dehydrogenase (LDH on day 2 of treatment. As chemotherapeutic drug interaction with cyclosporine A (CyA) is thought to aggravate its nephrotoxicity, the effect of combined CyA (30 mg/kg) and the antibiotic gentamicin (50 mg/kg) for 5 days was investigated. Gentamicin alone caused a significant enzymuria, whilst co-treatment of rats with CyA gave rise to increased changes in enzymuria on days 1 and 2, between the groups receiving gentamicin + vehicle and those receiving CyA + gentamicin. This was particularly marked by significant changes in LDH excretion. In contrast these observed differences were not paralleled by changes in serum creatinine and other functional parameters. Treatment with gentamicin, appears to enhance CyA nephrotoxicity, but only in the first 2 days, after this there was no significant differences between the two groups. Our data suggest that urinary enzyme measurements could serve as a valuable non-invasive means of monitoring renal performance in animals or humans who may be exposed to combination of drugs. CyA is found not to potentiate the nephrotoxic effect of gentamicin in the animal model used in this study. It therefore appears safe to use the combined therapy particularly in the treatment of transplant patients.

Relationship between Renal Dysfunction and Bone Metabolism Disorder in Male Rats after Long-Term Oral Quantitative Cadmium Administration.

To investigate the importance of the cadmium (Cd) exposure condition in the evaluation of toxic effect on renal function and bone metabolism, six groups of Male Wistar rats were given Cd at respective daily doses of 2, 5, 10, 20, 30 and 60 mgCd/kg (as CdCl2) via a gastric tube for 6 consecutive days a week for 60 weeks. In the groups given a low Cd dose (2, 5 and 10 mgCd/kg), relatively more Cd accumulated in the kidney without liver damage than in the liver. In the high Cd dose groups (20, 30 and 60 mgCd/kg), on the other hand, more Cd accumulated in the liver than in the kidney. The daily intake of Cd dose from the intestinal tract in each experimental group was deduced to be about 0.36%-0.54% of the cumulative dose of oral Cd administration. The daily intake of Cd into the body was estimated as 7, 22, 40, 100, 120, 260 microgCd/kg/day in the experimental groups of 2, 5, 10, 20, 30 and 60 mgCd/kg/day, respectively. Increase of plasma enzyme activity (GOT, GPT) and of urinary enzyme excretion (NAG, AAP, GST), reflecting hepatic damage and renal dysfunction, was found in the high Cd dose groups (30 and 60 mgCd/kg) from the 5th week. Non-CdMT concentration in the kidney was also significantly high in the high Cd dose groups. In the low Cd dose groups (2 and 5 mgCd/kg), although the renal Cd concentration was higher than that of the high Cd dose groups, prominent renal dysfunction and hepatic damage were not observed. Regeneration, vacuolization, and eosinophilic bodies in proximal tubular tissue were mainly observed in the groups subjected to 20, 30 and 60 mgCd/kg administration. Very slight regeneration was also observed in the renal proximal tubular tissue at the 30th week for the 5 mgCd/kg and 10 mgCd/kg groups, and at the 60th week for the 2 mgCd/kg group. Remarkable decrease of bone mineral density at the midpoint of the femur was found in the high Cd dose groups. Also, the decrease in bone mineral density was observed before or after the manifestation of the renal dysfunction, depending on the dose and the duration of Cd administration. Urinary excretion of Pyr, DPyr, and Ca increased and plasma BGP decreased in the higher Cd dose groups. Osteoid volume in the femur tissue was not increased significantly by Cd exposure. Based on these results, it was suggested that Cd exposure caused osteoporotic change. The results of the present study suggested that the toxic effect of Cd on renal function and that on bone metabolism were caused at different times and that renal Cd concentration after long-term oral Cd administration depended on the dose and the duration of Cd exposure.

Nitrogen excretion and expression of urea cycle enzymes in the atlantic cod (Gadus morhua l.): a comparison of early life stages with adults

For many years, the urea cycle was considered to be relatively unimportant in the life history of most teleost fishes. In previous studies, we were surprised to find that newly hatched freshwater rainbow trout embryos had relatively high activities of the key urea cycle enzyme, carbamoyl phosphate synthetase III (CPSase III), and other enzymes in the pathway, whereas adult trout had much lower or non-detectable activities. The present study tested the hypothesis that urea cycle enzyme expression is unique to early stages of rainbow trout. In marine Atlantic cod (Gadus morhua) embryos, CPSase III, ornithine transcarbamoylase (OTCase), glutamine synthetase (GSase) and arginase activities were all expressed prior to hatching. Urea excretion was detected shortly after fertilization and rates were high relative to those of ammonia excretion (50-100 % of total nitrogen excreted as urea nitrogen; total=ammonia+urea). Urea concentration was relatively constant in embryos, but ammonia concentration increased by about fourfold during embryogenesis. Two populations of cod embryos were studied (from Newfoundland and New Brunswick), and significant differences in enzyme activities and excretion rates were detected between the two populations. In adult cod, CPSase III was not detectable in liver, white muscle, intestine and kidney tissues, but OTCase, GSase and arginase were present. Adult cod excreted about 17 % of nitrogenous waste as urea. Taken together, these data indicate that early urea cycle enzyme expression is not unique to rainbow trout but is also a feature of Atlantic cod development, and possibly other teleosts. The relatively high urea excretion rates underline the importance of urea as the primary nitrogen excretory product in Atlantic cod during early embryogenesis.

Human parathyroid hormone (1–34) increases urinary excretion of lysosomal enzymes in rats

The kidney is the major target of parathyroid hormone (PTH), and PTH influences the urinary excretion of calcium, phosphate and hydrogen ions. It was previously reported that the urinary excretion of N-acetyl-β-D-glucosaminidase (NAG), a lysosomal enzyme, transiently increases after human PTH (hPTH) (1–34) infusion in normal subjects and idiopathic hypoparathyroidism patients, but not in pseudohypoparathyroidism type I patients. Here we report that intravenous infusion of hPTH(1–34) to rats transiently increased the urinary excretion of various lysosomal enzymes, such as β-glucuronidase and acid phosphatase as well as NAG. However, it did not affect the urinary excretion of tubular brush border membrane enzymes, i.e. alkaline phosphatase, leucine aminopeptidase and λ-glutamyl transpeptidase. Human PTH(1–34) dose-dependently increased the urinary excretion of NAG in rats with a peak at 30 min, which returned to a baseline within 60 min. The increase in the urinary NAG excretion caused by hPTH(1–34) positively correlated with the increase in the urinary cAMP excretion (r = 0.844, p < 0.01), and infusion of dibutyryl cAMP at a dose of 20 mg/kg similarly increased the urinary excretion of NAG. These results suggested that the increase in the urinary excretion of lysosomal enzymes caused by hPTH(1–34) may be a functional response to hPTH(1–34) occurring in the renal tubules via PTH signaling pathway.

Human parathyroid hormone (1-34) transiently increases the excretion of lysosomal enzymes into urine and the size of renal lysosomes.

It has been reported that the urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG), a lysosomal enzyme, transiently increases in human after treatment with human parathyroid hormone (hPTH)(1-34). We report here that hPTH(1-34) caused transient changes in the size and density of rat renal lysosomes following urinary excretion of NAG and other lysosomal enzymes tested. Percoll density gradient centrifugation revealed that hPTH(1-34) slightly but significantly increased the fraction of high density lysosomes (around 1.12 g/ml) 5-10 min after the treatment with hPTH(1-34), with a concomitant decrease in the fraction of intermediate density lysosomes (1.07-1.08 g/ml). On electron micrographs, some lysosomes in proximal tubules but not in distal tubules showed a change in morphology from circular to oval, and became enlarged and electron-dense 5-10 min after the treatment with hPTH(1-34). These responses to hPTH(1-34) were also reversible and transient. NAG excreted in urine after treatment with hPTH(1-34) had the molecular mass of a mature form in lysosomes and/or endosomes and was not a prepro-and/or pro-form of the enzyme. Thus, the changes in the density and size of renal lysosomes appear to be associated with the exocytosis of lysosomal enzymes by hPTH(1-34).

Calcitonin stimulates lysosomal enzyme release and uptake in LLC-PK1 cells.

Renal tubular targeted hormones increase urinary excretion of a lysosomal enzyme, N-acetyl-beta-D-glucosaminidase (NAG). To elucidate the mechanism of this event, the calcitonin effect on NAG handling by LLC-PK1 cells was examined. Calcitonin (1 nM to 1 microM), phorbol myristate (10 nM to 1 microM), and ionomycin (1 to 10 microM) promoted NAG release without any increase in lactate dehydrogenase release or any reduction of mitochondrial dehydrogenase activity. Treatment with 100 nM calphostin C or 50 microM KN-93 partially reversed the calcitonin effect on NAG release. Calcitonin promoted secretion of fluorescence ceramide, a reporter of protein transport from Golgi apparatus to cell surface. Calcitonin-stimulated NAG release was partially inhibited by 10 microg/ml brefeldin A, a blocker of protein transport through the Golgi apparatus. Calcitonin accelerated cellular uptake of exogenous NAG, which was inhibited by low temperature, 0.1 mM monodansyl cadaverine (receptor-mediated endocytosis inhibitor), and 10 mM mannose-6-phosphate. Furthermore, calcitonin promoted progression of intracellular membranes stained by a fluorescence membrane marker, styryl pyridinium dye, from cell periphery to perinuclear regions (commonly referred to as recycling vesicles) and increased dye release from preloaded cells. Fluorescence release from the cells pre-loaded with FITC-labeled NAG or albumin was also stimulated by calcitonin. These calcitonin effects on endocytotic and re-exocytotic pathways were inhibited by 100 nM cytochalasin D, 100 nM nocodazole, 0.1 to 1 microM bafilomycin A1, or 0.1 mM monodansyl cadaverine. Increased urinary NAG excretion has been considered to reflect renal tubular damage. However, it was demonstrated here that stimulation of secretory and recycling pathways may be an alternative mechanism for calcitonin-induced enzymuria, which will become a new indicator of renal tubular response to this hormone.

Oxidoreductase enzymes liberated by plant roots and their effects on soil humic material

Enzymes exuded by roots of 12 terrestrial plant species into sterile and unsterile soils were identified by spectrophotometry and gel electrophoretic methods. Aqueous extracts of planted unsterile soils contained up to 10 % of the high activities of peroxidases (EC 1.11.1.7), laccases (EC 1.10.3.2), monophenol monooxygenases (EC 1.14.18.1), and fluorescein diacetate hydrolyzing proteinase, esterase, and lipase enzymes which were found in planted sterile soils. Enzyme excretion was increased by minerally fertilized, dying, and water-stressed plants. The plant peroxidase/H 2O 2 system decreased the absorbance of humic macromolecules, and possibly, too, the reactivity of the humus phenolic groups to Fast Blue B salt. It is concluded that roots of certain members of Fabaceae, Gramineae, and Solanaceae release enough oxidoreductases to take part in the oxidative degradation of certain soil constituents.