[46] Enzymic diagnosis of the genetic mucopolysaccharide storage disorders

Hall, Clara W.; Liebaers, Ingeborg; Natale, Paola Di; Neufeld, Elizabeth F.

doi:10.1016/0076-6879(78)50048-7

Cited by 187 publications

(89 citation statements)

References 29 publications

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“…The details of the methods used in the assays for five different acid hydrolases (14)(15)(16) are given in Table 1. Cytochrome b S61 was estimated from the reduced minus oxidized difference spectra (17).…”

Section: Methodsmentioning

confidence: 99%

Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and percoll gradients

Gratzl

1984

Analytical Biochemistry

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emde chromaffin secretory vesicles, obtained by differential centrifugation, were further purified on isotonic (PereolI) gradients. The chromaffin vesicIe fractions recovered from the gradients contain acetylcholinesterase as well as lysosomal enzymes. With the aid of a subsequent sucrose gradient lysosomal enzymes could be removed from chromaffin vesicIe fractions. but not acetylcholinesterase. This suggests that lysosomal enzymes do not pass through the chromaffin vesicIes during the biogenesis of lysosomes but acetylcholinesterase does.

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Section: Methodsmentioning

confidence: 99%

Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and percoll gradients

Gratzl

1984

Analytical Biochemistry

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“…The homogenates were spun for 10 rain at 1,000 g. The supernatant was spun for 10 min at 3,000 g, and the resulting supernatant was layered on top of a step gradient consisting of 1 ml each of 30, 25, 20, 15, 12.5, 10, 7.5, 5 and 2.5% (vol/vol) iodixanol in homogenization buffer. After centrifugation at 126,000 g~v for 25 rain (SW 40.1 rotor; Beckman Instruments, Palo Alto, CA), I0 fractions were collected from the top of the gradient and analyzed for density, protein (Bradford, 1976), as well as the enzymatic activities of UDP-galactosyltransferase (Verdon and Berger, 1983), rotenone-insensitive NADH-cytochrome C reductase (Sottocasa et al,I967), and [3-hexosaminidase (Hall et al, 1978). To separate the particulate fractions from soluble proteins, 0.5 ml of each fraction was diluted with PBS and spun for 2 h at 100,000 gayThe resulting sediments were dissolved in Laemmli mix (Laemmli, 1970) under nonreducing or reducing (50 mM 2-mercaptoethanol plus 50 mM DTr) conditions and analyzed by SDS-PAGE (16% gels) using a mulfiphasic buffer system according to the method of Wiltfang et aL (1991) to detect free CTX-A2.…”

Section: Subeeuular Fractionationmentioning

confidence: 99%

Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.

Majoul¹,

Bastiaens²,

Söling³

1996

The Journal of Cell Biology

151

113

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Abstract. The A2 chain of cholera toxin (CTX) contains a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. We have, therefore, analyzed by immunofluorescence and by subcellular fractionation in Vero cells whether CTX can be used to demonstrate a retrograde transport of KDEL proteins from the Golgi to the ER. Immunofluorescence studies reveal that after a pulse treatment with CTX, the CTX-A and B subunits (CTX-A and CTX-B) reach Golgi-like structures after 15-20 rain (maximum after 30 min). Between 30 and 90 min, CTX-A (but not CTX-B) appear in the intermediate compartment and in the ER, whereas the CTX-B are translocated to the lysosomes. Subcellular fractionation studies confirm these results: after CTX uptake for 15 min, CTX-A is associated only with endosomal and Golgi compartments. After 30 min, a small amount of CTX-A appears in the ER in a trypsin-resistant form, and after 60 min, a significant amount appears. CTX-A seems to be transported mainly in its oxidized form (CTX-A1-S-S-CTX-A2) from the Golgi to the ER, where it becomes slowly reduced to form free CTX A1 and CTX-A2, as indicated by experiments in which cells were homogenized 30 and 90 rain after the onset of CTX uptake in the presence of N-ethylmaleimide. Nocodazol applied after accumulation of CTX in the Golgi inhibits the appearance of CTX-A in the ER and delays the increase of 3', 5'cAMP, indicating the participation of microtubules in the retrograde Golgi-ER transport.S OON after the detection of the COOH-terminal KDEL sequence in resident soluble proteins of the ER as a retention signal (Munro and Pelham, 1986), it became evident that the KDEL sequence is a retrieval rather than a retention signal (Munro and Pelham, 1987;Pelham, 1989). This view was mainly based on the observation that expression of the lysosomal enzyme cathepsin D with a COOH-terminal KDEL sequence led to the retention of most of the chimeric protein in the ER, but the glycan structure of the protein indicated that it had traversed the cis-and medium-Golgi cisternae before returning to the ER. Immunofluorescence (Lewis and Pelham, 1992;Hsu et al., 1992;Tang et al., 1993;SOnnichsen et al., 1994) and immunoelectronmicroscopical studies (Tang et al., 1993;Griffiths et al., 1994) showed that under steadystate conditions, the KDEL receptor ERD2 exists mainly in Golgi-like structures, particularly in the cis-Golgi network. The affinity of the KDEL receptor for the KDEL motif is increased under the slightly acidic conditions prevailing in the Golgi cisternae and is decreased under the neutral pH conditions in the ER . From these observations, it was deduced that: KDEL proteins can escape from the ER, they are "picked up" by the KDEL receptor, they are transported back to the ER together with the receptor, and are finally released in the ER. Up to now, however, the manner in which the occupied KDEL receptor is recycled to the ER remained unclear. Brefeldin A (Lippincott-Schwartz et al., 1990) as well as overexpression of the KDEL receptor (Hsu et al., 1992;Townsley et al., 1993) led to...

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“…Worthington Biochemical Corp ., Freehold, NJ) . Acid phosphatase activity was measured in 0.1% Triton X-100, 0.1 M acetate buffer (pH 5 .0) using p-nitrophenyl phosphate as a substrate (6). ß-hexosaminidase activity was measured in 0.1% Triton X-100, 0.1 M acetate buffer (pH 5.0) using p-aitrophenyl-N-acetyl-,6-Dglucosaminide as a substrate (6).…”

Section: Enzyme Assaysmentioning

confidence: 99%

Exocytosis of pinocytic contents by Chinese hamster ovary cells.

Adams¹,

Maurey²,

Storrie³

1982

The Journal of Cell Biology

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The extent of exocytosis of pinocytic vesicle contents was studied in suspensioncultured Chinese hamster ovary (CHO) cells using horseradish peroxidase (HRP) as a pinocytic content marker. HRP was shown to be internalized via fluid-phase pinocytosis in CHO cells.After an HRP pulse of 2 .5-10 min a rapid decrease of 30-50% in cell-associated HRP activity was observed within 10-20 min at 37°C. During this time the loss of cell-associated HRP was accompanied by an equivalent increase in extracellular HRP. After this rapid exocytosis of HRP, the remaining peroxidase activity decreased with a t1/2 of 6-8 h, the known lysosomal half-life of HRP. In pulse-chase experiments HRP was chased into a nonexocytic compartment. Based on cell fractionation and electron microscopic experiments, this nonexocytic compartment was identified as a lysosome and the compartment from which exocytosis occurs as a pinosome . The occurrence of pinocytic content exocytosis in cultured fibroblasts suggests that exocytosis of pinocytic vesicle contents is a general phenomenon .

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[46] Enzymic diagnosis of the genetic mucopolysaccharide storage disorders

Cited by 187 publications

References 29 publications

Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and percoll gradients

Distribution of chromaffin secretory vesicles, acetylcholinesterase, and lysosomal enzymes in sucrose and percoll gradients

Transport of an external Lys-Asp-Glu-Leu (KDEL) protein from the plasma membrane to the endoplasmic reticulum: studies with cholera toxin in Vero cells.

Exocytosis of pinocytic contents by Chinese hamster ovary cells.

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