In vitro fusion of Acanthamoeba phagolysosomes. I. Demonstration and quantitation of vacuole fusion in Acanthamoeba homogenates.

Fusion of phagolysosomes (PLs) has been demonstrated to occur in vitro. Two separate cell homogenates of the ameba Acanthamoeba sp. (Neff) were prepared, each rich in PLs labeled with distinctive particulate markers. Portions of each were incubated together in vitro and fusion occurred as evidenced by the appearance of PLs containing both types of markers. Fusion was confirmed by electron microscopy, including serial sectioning. The membranes of fused vacuoles excluded the dye eosin Y. Surviving cells in the homogenates were not responsible for the observed fusion. Fusion was obtained using either synthetic markers (polystyrene and polyvinyltoluene latex) or biological markers (autoclaved yeast cells and glutaraldehyde-fixed goat red blood cells), or a combination of both. The specificity of PL fusion in vivo appeared to be maintained in vitro. As determined by light and electron microscopy, the fusion reaction was dependent on time and temperature, and on the initial presence of membrane around both marker particles. A minimum of 10% of the vacuoles fused by 10 min of incubation at 30 degrees C, and no rupture of the vacuoles was detected during this time. After 10 min of incubation, vacuole rupture began and fusion ceased. At a constant initial vacuole concentration, the extent of PL fusion in vitro was quantitatively reproducible. This appears to be a promising system for further investigation of membrane fusion in the lysosomal system.

In vitro fusion of Acanthamoeba phagolysosomes. III. Evidence that cyclic nucleotides and vacuole subpopulations respectively control the rate and the extent of vacuole fusion in Acanthamoeba homogenates.

Fusion of phagolysosomes has been previously demonstrated to occur during the incubation of phagolysosome-containing homogenates of Acanthamoeba (Oates and Touster, 1978, J. Cell Biol. 79:217-234). Further studies on this system have shown that methylxanthines (0.2 mM) and/or cAMP (0.5-1 mM) markedly accelerate the average rate, but not the extent, of the in vitro phagolysosome fusion process. Adenosine, 5'-AMP, and ADP (0.5-1 mM) were without effect. ATP (0.5-1 mM) caused variable stimulation, whereas beta, gamma-methylene-ATP (1 mM) caused pronounced inhibition, as did GTP (1 mM) and cGMP (1 mM). Stimulation by 3-isobutyl-1-methylxanthine was blocked by GTP, but not by ATP or cAMP. These results indicate that the rate of phagolysosome fusion in Acanthamoeba homogenates may be regulated by cyclic nucleotides, with enhancement of the fusion rate by cAMP and inhibition of the rate by cGMP. The extent of the reaction increased spontaneously and markedly during the first few hours after preparation of the homogenates. This activation appears to be because of a slow conversion of a significant fraction of the vacuole population from a fusion-incompetent to a fusion-competent, cyclic nucleotide-sensitive state.

In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques.

To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial characterization of the fusion reaction by this method was followed by the development of a more rapid light microscope assay. Results obtained by the two methods were found to be in close agreement. By use of these new techniques, the in vitro PL fusion reaction was demonstrated to occur in a quantitatively reproducible manner. Under the present conditions employed, PL breakdown was not detected at any time during the in vitro incubation, while PL fusion was observed to proceed linearly for approximately 10 min, at which time the reaction ceased. Incubation of mixtures of two distinct PL types resulted in increases in hybrid PL types that were paralleled by decreases in nonhybrid PL types. The relative changes in PL concentrations observed were quantitatively consistent with PL fusion occurring randomly with respect to PL type. PL fusion was strongly inhibited by low concentrations of KF (50% inhibition at 2.7 mM), and by approximately tenfold higher concentrations of KCl, while KCN and 2,4-dinitrophenol (2,4-DNP) had little effect. In addition to further defining the nature of the PL fusion reaction in this system, these results demonstrate that, by use of the techniques described, quantitative study of the biochemical properties of this reaction is now possible.

The isolation of lysosomes from Ehrlich ascites tumor cells following pretreatment of mice with Triton WR-1339.

A method is described for obtaining highly purified lysosomes from Ehrlich ascites tumo cells grown in mice injected with Triton WR-1339. The isolated particles show a high specific activity for aryl sulfatase, representing an 80-90-fold purification over the homogenate, and a 15-18% yield of the total enzyme activity. Mitochondrial and microsomal marker enzymes are present in negligible amounts (0.2% of the activity of the homogenate). The biochemical evidence for a rather high degree of homogeneity of the fraction is supported by the electron microscopic examination of the purified lysosomes. The intracellular localizations of N-acetyl-beta-glucosaminidase, NADH-cytochrome c reductase and NADPH-cytochrome c reductase in Ehrlich ascites cells are also reported, the first two being present in highest concentration in the combined mitochondrial-lysosomal fraction and the third in the microsomal fraction.

Isolation of rat liver plasma membranes. Use of nucleotide pyrophosphatase and phosphodiesterase I as marker enzymes.

Nucleotide pyrophosphatase and phosphodiesterase I of rat liver have been found to be localized primarily in cell particulates highly enriched with respect to the most commonly accepted plasma membrane marker, 5'-nucleotidase, and therefore should themselves be assigned a plasma membrane localization. The observation that plasma membranes sediment in isotonic sucrose with both nuclear and microsomal fractions was exploited to obtain plasma membrane preparations from each fraction. Both preparations are similar in chemical and enzymic composition. Moreover, the preparative method developed in this study appears to give the best combination of yield, purity, and reproducibility available. The question of the possible identity of nucleotide pyrophosphatase and phosphodiesterase I is considered, and evidence is presented suggesting that these activities may be manifestations of the same enzyme.

Effects of swainsonine on rat liver and kidney: biochemical and morphological studies.

Among the reported effects of the plant toxin swainsonine in animals are a decreased level of Golgi mannosidase II activity, an increase in lysosomal alpha-D-mannosidase activity, oligosaccharide accumulation, vacuolization of cells, and neurological changes. We now find that, in the rat, the alkaloid rapidly induces vacuolization of both liver and kidney cells, but oligosaccharides accumulate only in the latter. We demonstrate by enzyme- and immunocytochemistry that the induced pleomorphic vacuoles are lysosomal in nature. The vacuoles do not appear to be derived from the Golgi apparatus, which retains its typical ultrastructural appearance, but are formed by autophagy. In swainsonine-fed rats, the lysosomal system is highly developed in hepatocytes, Kupffer cells, and cells of the proximal convoluted tubules. The relation of this hypertrophy of the lysosomal system to the known effects of swainsonine on glycoprotein biosynthesis and on Golgi and lysosomal alpha-mannosidases is not clear. In addition, in liver there occurs a marked increase in mitotic figures in the hepatocytes. This occurred in the absence of both cell death and increased liver size as estimated by gross morphology.

Cell type-dependent variations in the subcellular distribution of alpha-mannosidase I and II.

alpha-mannosidases I and II (Man I and II) are resident enzymes of the Golgi complex involved in oligosaccharide processing during N-linked glycoprotein biosynthesis that are widely considered to be markers of the cis- and medial-Golgi compartments, respectively. We have investigated the distribution of these enzymes in several cell types by immunofluorescence and immunoelectron microscopy. Man II was most commonly found in medial- and/or trans- cisternae but showed cell type-dependent variations in intra-Golgi distribution. It was variously localized to either medial (NRK and CHO cells), both medial and trans (pancreatic acinar cells, enterocytes), or trans- (goblet cells) cisternae, or distributed across the entire Golgi stack (hepatocytes and some enterocytes). The distribution of Man I largely coincided with that of Man II in that it was detected primarily in medial- and trans-cisternae. It also showed cell type dependent variations in its intra-Golgi distribution. Man I and Man II were also detected within secretory granules and at the cell surface of some cell types (enterocytes, pancreatic acinar cells, goblet cells). In the case of Man II, cell surface staining was shown not to be due to antibody cross-reactivity with oligosaccharide epitopes. These results indicate that the distribution of Man I and Man II within the Golgi stack of a given cell type overlaps considerably, and their distribution from one cell type to another is more variable and less compartmentalized than previously assumed.

Isolation and partial characterization of the oligosaccharide moieties of rat preputial beta-glucuronidase.

The oligosaccharide alditols of rat preputial gland beta-glucuronidase have been isolated by reductive alkaline hydrolysis of the enzyme followed by gel filtration. Since the product was about decasaccharide in size, and as beta-glucuronidase contains about 20 sugar residues per subunit, the enzyme must contain two oligosaccharides per subunit. Two major subfractions were obtained. Chemical and enzymatic studies on the initial oligosaccharide alditol fraction and on the separated subfractions indicated the presence of a mannosyl-chitobiosyl core and permitted formulation of the following structures for the two different oligosaccharide alditols: Man3 leads to alpha [(alpha Man, GlcNAc) leads to Man] leads to beta GlcNAc leads to beta GlcNAc-ol Man 3 leads to 4 leads to alpha[Man2 leads to alpha Man] leads to beta GlcNAc leads to beta GLcNAc-ol.

Studies on dehydrogenases of the glucuronate-xylulose cycle in the livers of diabetic mice and rats.

In view of reports that accessory pathways of glucose oxidation are enhanced in the diabetic state, we have determined the levels of key enzymes of the glucuronate-xylulose cycle in the livers of diabetic mice and rats. Genetically diabetic mice (db/db) were found to have increased levels of two NADP-linked enzymes of this cycle [NADP-xylitol dehydrogenase and NADP-L-hexonate dehydrogenase (aldehyde reductase II)], whereas other NAD- and NADP-linked dehydrogenase activities of the pathway were not changed. On the other hand, the livers of streptozotocin-diabetic mice and rats showed normal levels of all these enzymes. In the course of this study, evidence was obtained for the presence in db/db mouse liver of low molecular weight material inhibitory for glucose 6-phosphate dehydrogenase. The use of these animal models in diabetes research is briefly discussed.

On the utility of 13C-n.m.r. spectroscopy in the identification of the primary structures of manno-oligosaccharides and glycopeptides.

The utility of 13C-n.m.r. spectroscopy in the identification of the primary structures of mannose-containing glycans is investigated. Unlike 1H resonances where the chemical shifts reflect multiple short- and long-range effects, the chemical shifts of 13C resonances are dependent largely upon short-range effects classified as glycosylation (linkage) and substitution effects. These effects are parametized for glycans composed of mannose and encoded in a FORTRAN algorithm. Applications of this program to "unknown" sets of experimental chemical shifts for the resonances of anomeric carbons gave the following conclusions. (1) This program can be used to produce a sub-set of possible structures inclusive of the "known" structure. (2) For other than simple oligosaccharides, it is unlikely that a single structure is consistent with the data for anomeric carbons alone, even when the linkage composition of the glycan has been assessed from other spectral data. (3) When used in conjunction with other chemical techniques, this program can provide a powerful tool for primary analysis of the structure of mannose-containing glycans.