Enzyme Activities and Properties of Lysosomes and Brush Borders in Jejunal Biopsies from Control Subjects and Patients with Coeliac Disease

Heath, James R.; Wansbrough‐Jones, Mark; Doe, William F.

doi:10.1042/cs0480259

Cited by 53 publications

(40 citation statements)

References 31 publications

(31 reference statements)

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“…Other portions were analysed by enzyme microassay and subcellular fractionation, as described before (Peters & Seymour, 1978a). Lysosomal integrity, indicated by latent P-Nacetyl-D-glucosaminidase activity, was determined as described before (Peters, Heath, Wansbrough-Jones & Doe, 1975).…”

Section: Biochemical Studiesmentioning

confidence: 99%

Subcellular Fractionation Studies on Hepatic Tissue from a Patient with Pompe's Disease (Type II Glycogen-Storage Disease)

1980

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1. An homogenate of liver biopsy taken from a patient with Pompe's disease (type II glycogen-storage disease) was analysed by enzyme microassay and subcellular fractionation by sucrose-density-gradient centrifugation. 2. A strikingly low activity of lysosomal acid alpha-D-glucosidase was noted but there was also increased activity of neutral alpha-D-glucosidase (found in endoplasmic reticulum). 3. Activities of the acid hydrolases, acid phosphatase and beta-N-acetyl-D-glucosaminidase, were elevated compared with those of controls. Measurement of latent beta-N-acetyl-D-glucosaminidase activity and results of subcellular fractionation experiments, indicated a marked fragility of certain populations of lysosomes. Lysosomes containing predominantly acid phosphatase and beta-D-glucuronidase activities appeared to have normal integrity. 4. Assessment of organelle pathology by enzyme microassay indicated low mitochondrial and peroxisomal enzyme activities. In addition there was evidence of mitochondrial damage as reflected by increased sucrose permeability and by ultrastructural studies.

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Section: Biochemical Studiesmentioning

confidence: 99%

Subcellular Fractionation Studies on Hepatic Tissue from a Patient with Pompe's Disease (Type II Glycogen-Storage Disease)

1980

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“…The latent activity of the lysosomal enzyme, N-acetyl-/3-glucosaminidase was assayed in the liver homogenate by the fluorometric measurement of N-acetyl-P-glucosaminidase after incubation of suitably diluted liver homogenates in the presence or absence of 0.1% Triton X-100 (Peters et al, 1975).…”

Section: Methodsmentioning

confidence: 99%

Biochemical and biophysical investigations of the ferrocene‐iron‐loaded rat

Ward

Florence

Baldwin

et al. 1991

European Journal of Biochemistry

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Male Wistar rats fed with ferrocene had high hepatic iron loading (7.24 1.97 mg Fe/g tissue) after 6 weeks, principally located in lysosomes, which was comparable to the levels and distribution determined in human haemochromatosis.The two iron-storage proteins, ferritin and haemosiderin were isolated from the livers of the ferrocene-loaded rats and their iron cores were investigated by Mossbauer spectroscopy and inductively coupled plasma-emission spectrometry. Ferrihydrite was the predominant form of iron present in both ferritin and haemosiderin, while haemosiderin contained higher amounts of phosphorus, magnesium, calcium and barium, then either normal or ferrocene-loaded ferritin.Free-radical-mediated damage in the iron-loaded livers was inferred by the significant depletion of a-tocopherol in both the livers and subcellular hepatic lysosomal fraction, which inversely correlated with the inereasink iron ' content (r = -0.61 ; P < 0.05) and was associated with increased fragility of the lysosomal membranes.

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“…We used a modification (Freiburghaus, Hauri, Green & Hadorn, 1978) of the method of Schmitz, Preiser, Maestracci, Ghosh, Corda & Crane (1973). Activities of aminopeptidase N (modified from Saifuku, Sekine, Namihisa, Takahashi & Kanaoka, 1978), dipeptidyl aminopeptidase IV (Kato, Nagatsu, Kimura & Sakakibara, 1978), alkaline phosphatase (Fernley & Walker, 1965), and a-glucosidase (Peters, Heath, Wansbrough-Jones & Doe, 1975) were assayed using published methods in brush-border and non-brush-border membrane fractions. Protein was assayed by the fluorescamine method (Udenfriend, Stein, Bohlen, Dairman, Leimgruber & Weigele, 1972).…”

Section: Cell Culturementioning

confidence: 99%

Acid‐base transport systems in a polarized human intestinal cell monolayer: Caco‐2

Osypiw¹,

Gleeson²,

Lobley³

et al. 1994

Experimental Physiology

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SUMMARYAcid-base transport systems have been incompletely characterized in intact intestinal epithelial cells. We therefore studied the human cell line Caco-2, cultured on Teflon membranes to form confluent monolayers with apical microvilli on transmission electron microscopy and progressive enrichment in microvillar hydrolases. Monolayers (16-to 25-day-old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyethyl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvette to allow selective superfusion of apical and basolateral surfaces with Hepes-or HCO3--buffered media. Intracellular pH (pH.) was measured by dual-excitation spectrofluorimetry; calibration was with standards containing nigericin and 110 mm K+, corresponding to measured intracellular [K+] in Caco-2 cell monolayers. In HCO3--free (Hepes-buffered) media, bilateral superfusion with 1 mm amiloride or with Na+-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4C1 pulse) by 86 and 98 % respectively. Selective readdition of Na+ to the apical or basolateral superfusate also induced a pHi recovery, which was inhibited by ipsilateral but not by contralateral amiloride (I mM). The pH. recovery induced by apical Na+ readdition had a Michaelis constant (K,R) for Na+ of 30 mm and a relatively high inhibitor constant (K,) for amiloride of 45 5 /lM. Initial pHi in HCO3--buffered media was lower than in the absence of HCO3-(7 35 vs. 7 80). pH, recovery from an acid load in HCO3-was Na+ dependent but was inhibited only 18 % by I mM amiloride. The amiloride-independent pH. recovery was inhibited 49 % by pre-incubation of cells in 5 mm DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). These data suggest that Caco-2 cells possess: (a) both apical and basolateral membrane Na+-H+ exchange mechanisms, the apical exchanger being relatively resistant to amiloride, similar to apical Na+-H+ exchangers in several normal epithelia; and (b) a Na+-dependent HCO3-transport system, either Na+-HCO3-cotransport or Na+-dependent Cl--HCO3-exchange.

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Enzyme Activities and Properties of Lysosomes and Brush Borders in Jejunal Biopsies from Control Subjects and Patients with Coeliac Disease

Cited by 53 publications

References 31 publications

Subcellular Fractionation Studies on Hepatic Tissue from a Patient with Pompe's Disease (Type II Glycogen-Storage Disease)

Subcellular Fractionation Studies on Hepatic Tissue from a Patient with Pompe's Disease (Type II Glycogen-Storage Disease)

Biochemical and biophysical investigations of the ferrocene‐iron‐loaded rat

Acid‐base transport systems in a polarized human intestinal cell monolayer: Caco‐2

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