Isolation of a Golgi apparatus-rich fraction from rat liver. II. Enzymatic characterization and comparison with other cell fractions.
TL;DR: The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Gol Gi apparatus-derived material.
Abstract: Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20-30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2-3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.
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TL;DR: Subcellular localization of Parkin protein in patients with AR‐JP or Parkinson's disease (PD) and in controls by immunoblotting and immunohistochemistry using antibodies raised against the Parkin molecule is reported.
Abstract: Autosomal recessive juvenile parkinsonism (AR-JP) is a distinct clinical entity characterized by a selective degeneration of nigral neurons. Recently, the parkin gene responsible for AR-JP has been identified. Now, we report the subcellular localization of Parkin protein in patients with AR-JP or Parkinson's disease (PD) and in controls by immunoblotting and immunohistochemistry using antibodies raised against the Parkin molecule. Parkin protein was absent in all regions of the brains of patients with AR-JP. Parkin protein was not decreased in the brains of sporadic PD patients. Immunoreactivity was detected in a few Lewy bodies. Parkin protein was located in both the Golgi complex and cytosol.
274 citations
TL;DR: The results obtained allowed us to assess the importance of knowing the carrier and removal status of canine coronavirus, as a source of infection for other animals, not necessarily belonging to the same breeds.
271 citations
TL;DR: The usefulness of cytochemical methods in monitoring the fractionation procedure has been shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements.
Abstract: Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF1, GF2, GF3) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF1 and GF2, and along the outside of the cisternal membranes in GF3. In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF1 and within many VLDL-filled vacuoles in GF1 and GF2, indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF3 and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF1 and GF2, and was not found in the cisternae in GF3. The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF1 and GF3, representing primarily trans-Golgi elements from the secretory Golgi face, and GF3 consisting largely of cis-Golgi components from the opposite face.
254 citations
TL;DR: Electron microscopic analysis of a Golgi-rich fraction isolated from rat liver revealed the presence of structures very similar to those of the Golgi apparatus in intact cells, namely stacked cisternae, secretory vesicles, and tubular elements.
218 citations
References
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TL;DR: Procedures are described for measuring protein in solution or after precipitation with acids or other agents, and for the determination of as little as 0.2 gamma of protein.
289,852 citations
"Isolation of a Golgi apparatus-rich..." refers methods in this paper
...... were determined according to the procedures referenced glucose-6-phosphatase, EC 3 .1.3.9(26);succinic dehydrogenase (succinate dehydrogenase), EC 1 as succinate-2-(p-indophenyl)-3-(,p-nitrophenyl)-5phenyltetrazolium (INT) reductase (22);5'-nucleotidase (5'-mononucleotidase), EC 3 .1.3.5(10);cytochrome oxidase, EC 1 .9.3 .1(25);uric acid oxidase (urate : 02-oxidoreductase), EC 1 .7.3.3(14).Protein was determined by the Lowry procedure ......
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18,029 citations
TL;DR: The results are shown to favour the ferryl ion structure, or an isomer of this structure, for the higher oxidation state, and theHigher oxidation state may provisionally be named ferrylmyoglobin.
Abstract: more acidic solutions, with pH < 7 0, a few seconds elapsed before equilibrium was attained. Taking into account the ionization of the haem-linked group on MetMb and the higher oxidation state, the variation of Kb,. with pH is shown to confirm the conclusion that 2 moles ofH+ are liberated/mole of acidic MetMb. Using 6-1 for the pK of the group in MetMb as established in other studies, the results give apK of75 for the group in the higher oxidation state at 200 and I= 0 04. 3. The variation of KRb, with temperature gives AHO = 10-0 ± 2*0 kcal./g.mol.: if the ionization of the haem-linked group is allowed for, the value 9*0 ± 1.0 kcal./g.mol. is obtained. 4. The dependence of Kob. on ionic strength is in accord with a change in charge from + 1 on MetMb to zero on the higher oxidation state. 5. The results are shown to favour the ferryl ion structure, or an isomer of this structure, for the higher oxidation state. The isomeric structures would, in general, require the presence of another ionizing group in myoglobin, but no evidence for such an ionization could be found. With other direct evidence favouring the ferryl ion structure this is to be preferred, and the higher oxidation state may provisionally be named ferrylmyoglobin.
3,654 citations
TL;DR: The discovery of a muscular dystrophy in a strain of mice has facilitated the search for biochemical alterations in myopathy and it seems probable that many, at least, of the secondary biochemical changes may be common to various types of muscle disease.
Abstract: Little is known about biochemical abnormalities which accompany or are responsible for the morphological changes which occur in the primary myopathies. A number of such diseases have been characterized in humans (Walton & Nattrass, 1954). The discovery (Michelson, Russell & Harman, 1955) of a muscular dystrophy in a strain of mice, inherited by an autosomal recessive gene, has facilitated the search for biochemical alterations in myopathy. Although this condition may not be identical with any of the types of human muscular dystrophy, its investigation may help to throw light on the biochemistry ofhuman muscular dystrophies. It seems probable that many, at least, of the secondary biochemical changes may be common to various types of muscle disease. A few workers (e.g. Weinstock, Epstein & Milhorat, 1958; Hazzard & Leonard, 1959; White, 1959) have reported altered concentrations of certain muscle enzymes in the mouse myopathy. In view of reported morphological abnormalities in mitochondria of dystrophic mouse muscle (Dr G. W. Pearce, unpublished work; Ross, Pappas & Har-
980 citations
TL;DR: The possibility that the plasma-membrane preparations were contaminated by microsomal elements is discussed, and, on account of the available evidence, the conclusion is reached that there is no reason to assume that this was the case.
700 citations
"Isolation of a Golgi apparatus-rich..." refers methods in this paper
...Golgi apparatus-rich fractions were prepared as described in the preceding report (17).The plasma membrane fraction was obtained by a modification ( 10 ) of Neville's (21) procedure . Microbodies were isolated by sucrose gradient centrifugation (7, 8) . Mitochondria were recovered from the 1.6-1.8 M interface of the sucrose gradients used to obtain Golgi apparatus (17) and were purified through two Downloaded from jcb.rupress.org on ......
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...conditions were those of Emmelot et al. ( 10 )....
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