Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6

The acidification of various ligands was measured on a cell by cell basis for cell suspensions by correlated dual fluorescence flow cytometry. Mouse 3T3 cells were incubated with a mixture of fluorescein- and rhodamine-conjugated ligands, and the ratio of fluorescein and rhodamine fluorescence was used as a measure of endosome pH. The calibration of this ratio by both fluorometry and flow cytometry is described. Dual parameter histograms of average endosome pH per cell versus amount of internalization were calculated from this data, for samples in the absence and presence of chloroquine added to neutralize acidic cellular vesicles. The kinetics of acidification of insulin were measured and compared with previous results obtained with the chloroquine ratio technique. Rapid acidification of internalized ligand was observed both for insulin, which was mostly internalized via nonspecific pathways, and for alpha 2-macroglobulin, which was mainly internalized by specific receptor-mediated endocytosis. The average pH observed for internalized insulin was less than pH 6 within 10 min after addition of insulin. At 30 min, the average pH began to decrease to approximately pH 5, presumably because of fusion of endosomes with lysosomes.     

Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants

During endocytosis in Chinese hamster ovary (CHO) cells, Semliki Forest virus (SFV) passes through two distinct subpopulations of endosomes before reaching lysosomes. One subpopulation, defined by cell fractionation using free flow electrophoresis as "early endosomes," constitutes the major site of membrane and receptor recycling; while "late endosomes," an electrophoretically distinct endosome subpopulation, are involved in the delivery of endosomal content to lysosomes. In this paper, the pH-sensitive conformational changes of the SFV E1 spike glycoprotein were used to study the acidification of these defined endosome subpopulations in intact wild-type and acidification-defective CHO cells. Different virus strains were used to measure the kinetics at which internalized SFV was delivered to endosomes of pH less than or equal to 6.2 (the pH at which wild-type E1 becomes resistant to trypsin digestion) vs. endosomes of pH less than or equal to 5.3 (the threshold pH for E1 of the SFV mutant fus-1). By correlating the kinetics of acquisition of E1 trypsin resistance with the transfer of SFV among distinct endosome subpopulations defined by cell fractionation, we found that after a brief residence in vesicles of relatively neutral pH, internalized virus encountered pH less than or equal to 6.2 in early endosomes with a t1/2 of 5 min. Although a fraction of the virus reached a pH of less than or equal to 5.3 in early endosomes, most fus-1 SFV did not exhibit the acid-induced conformational change until arrival in late endosomes (t1/2 = 8-10 min). Thus, acidification of both endosome subpopulations was heterogeneous. However, passage of SFV through a less acidic early endosome subpopulation always preceded arrival in the more acidic late endosome subpopulation. In mutant CHO cells with temperature-sensitive defects in endosome acidification in vitro, acidification of both early and late endosomes was found to be impaired at the restrictive temperature (41 degrees C). The acidification defect was also found to be partially penetrant at the permissive temperature, resulting in the inability of any early endosomes in these cells to attain pH less than or equal to 5.3. In vitro studies of endosomes isolated from mutant cells suggested that the acidification defect is most likely in the proton pump itself. In one mutant, this defect resulted in increased sensitivity of the electrogenic H+ pump to fluctuations in the endosomal membrane potential.     

Internalization and degradation of heparin binding growth factor-I by endothelial cells

The fate of 125I-labeled heparin binding growth factor I (125I-HBGF-I) after binding to its cell surface receptor has been studied using murine lung capillary endothelial cells (LEII). Binding of 125I-HBGF-I to its receptor at 4 degrees C shows pH dependence with optimal binding at pH 6.5-7.5. The majority (approximately 80%) of 125I-HBGF-I bound to cells at 4 degrees C can be removed by washing with low pH medium, but rapidly becomes acid resistant upon shifting cells to 37 degrees C, with 50% of the 125I-HBGF-I becoming acid resistant after 20 minutes. Electrophoretic analysis of internalized 125I-HBGF-I shows that degradation begins approximately 2 hours after internalization with the appearance of two major labeled fragments of Mr 15,000 and Mr 10,000. Degradation of internalized 125I-HBGF-I is inhibited by the lysosomotropic agent chloroquine. These data suggest that cell-associated 125I-HBGF-I is rapidly internalized and directed to a lysosomal cellular compartment where it is slowly degraded.     

Fission of tubular endosomes triggers endosomal acidification and movement

The early endosome acts as a sorting station for internalized molecules destined for recycling or degradation. While recycled molecules are sorted and delivered to tubular endosomes, residual compartments containing molecules to be degraded undergo "maturation" before final degradation in the lysosome. This maturation involves acidification, microtubule-dependent motility, and perinuclear localization. It is currently unknown how sorting and the processes of maturation cooperate with each other. Here, we show that fission of a tubular endosome triggers the maturation of the residual endosome, leading to degradation. Use of the dynamin inhibitor dynasore to block tubular endosome fission inhibited acidification, endosomal motility along microtubules, perinuclear localization, and degradation. However, tubular endosome fission was not affected by inhibiting endosomal acidification or by depolymerizing the microtubules. These results demonstrate that the fission of recycling tubules is the first important step in endosomal maturation and degradation in the lysosome. We believe this to be the first evidence of a cascade from sorting to degradation.     

Cloning, purification, and properties of a phosphotyrosine protein phosphatase from Streptomyces coelicolor A3(2).

We describe the isolation and characterization of a gene (ptpA) from Streptomyces coelicolor A3(2) that codes for a protein with a deduced M(r) of 17,690 containing significant amino acid sequence identity with mammalian and prokaryotic small, acidic phosphotyrosine protein phosphatases (PTPases). After expression of S. coelicolor ptpA in Escherichia coli with a pT7-7-based vector system, PtpA was purified to homogeneity as a fusion protein containing five extra amino acids. The purified fusion enzyme catalyzed the removal of phosphate from p-nitrophenylphosphate (PNPP), phosphotyrosine (PY), and a commercial phosphopeptide containing a single phosphotyrosine residue but did not cleave phosphoserine or phosphothreonine. The pH optima for PNPP and PY hydrolysis by PtpA were 6.0 and 6.5, respectively. The Km values for hydrolysis of PNPP and PY by PtpA were 0.75 mM (pH 6.0, 37 degrees C) and 2.7 mM (pH 6.5, 37 degrees C), respectively. Hydrolysis of PNPP by S. coelicolor PtpA were 0.75 mM (pH 6.0, 37 degrees C) and 2.7 mM (pH 6.5, 37 degrees C), respectively. Hydrolysis of PNPP by S. coelicolor PtpA was competitively inhibited by dephostatin with a Ki of 1.64 microM; the known PTPase inhibitors phenylarsine oxide, sodium vanadate, and iodoacetate also inhibited enzyme activity. Apparent homologs of ptpA were detected in other streptomycetes by Southern hybridization; the biological functions of PtpA and its putative homologs in streptomycetes are not yet known.     

Absence of Na+,K(+)-ATPase regulation of endosomal acidification in K562 erythroleukemia cells. Analysis via inhibition of transferrin recycling by low temperatures.

Transferrin (Tf) acidification has been shown to be limited to pH 6 in murine Balb/c 3T3 fibroblasts, human A549 epidermoid carcinoma cells, and Chinese hamster ovary cells and is followed by alkalinization during recycling. In contrast, Tf acidification in the human erythroleukemic cell line K562 proceeds to below pH 5.5, and alkalinization of internal Tf during recycling is not observed. To explore the regulation of endosomal pH in K562 cells, we determined whether the existence of an early endosome of pH 6 could be demonstrated in K562 cells. The kinetics of Tf internalization, acidification, and recycling were determined at temperatures which block recycling of Tf in 3T3 cells. As in 3T3, Tf recycling in K562 was inhibited at 24 degrees C and below. At these temperatures, Tf internalization and acidification were delayed relative to 37 degrees C, yet the minimum pH achieved was below 5.5. Temperatures at or below 19 degrees C resulted in a complete block in recycling (at least over 40 min), which was rapidly reversible by incubation at 37 degrees C. Ouabain (a specific inhibitor of the Na+,K(+)-ATPase) had no effect on K562 Tf acidification, indicating that K562 endosomal pH is probably not regulated by the Na+,K(+)-ATPase. The results suggest that differentiation of the early endocytic pathway in erythroid cells involves changes such that the pH of Tf-containing compartments is not limited to 6 by the Na+,K(+)-ATPase.     

Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts.

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

Digestive proteinase activity of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae)

The khapra beetle, Trogoderma granarium, is one of the most important stored product pests worldwide. A study of digestive proteinases in T. granarium was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. The pH of guts was determined by addition of pH indicator solutions to broken open gut regions. The last instar larvae were dissected in cold distilled water and the whole guts were cleaned from adhering unwanted tissues. The pooled gut homogenates were centrifuged and the supernatants were used in the subsequent enzyme assay. Total proteinases activity of the gut homogenates was determined using the protein substrate azocasein. Optimal azocasein hydrolysis by luminal proteinases of the larvae of T. granarium was highly alkaline in pH 10-10.5, although the pH of luminal contents was slightly acidic (pH 6.5). The extract showed the highest activity at 55 degrees C (pH 6.5), 45 degrees C (pH 8) and 30 degrees C (pH 10). The proteolytic activity was strongly inhibited in the presence of phenylmethylsulphonyl fluoride (82.33+/-4.37% inhibition). This inhibition was decreased with increasing of the pH of assay incubating medium. N-p-tosyl-L-lysine chloromethyl ketone (51.6+/-3.3% inhibition) and N-tosyl-L-phenylalanine chloromethyl ketone (27.23+/-4.37 % inhibition) showed inhibitory effect on proteolysis. Addition of thiol activators dithiothreitol and L-cysteine had not enhanced azocaseinolytic activity. The data suggest that protein digestion in the larvae of T. granarium is primarily dependent on serine proteinases; trypsin- and chymotrypsin-like proteinases.     

Inhibition of the acidification of endosomes and lysosomes by the antibiotic concanamycin B in macrophage J774.

The antibiotic concanamycin B was found to inhibit oxidized-low-density-lipoprotein(LDL)-induced accumulation of lipid droplets in the macrophage J774 at a concentration of 5-10 nM. Concanamycin B inhibited cholesteryl-ester synthesis from [14C]oleate by 50% at 14 nM without affecting the synthesis of triacylglycerol and polar lipids. Degradation of internalized oxidized 125I-LDL was inhibited by about 80% in cells treated with 25 nM concanamycin B, while cell-surface binding of oxidized 125I-LDL at 4 degrees C, uptake of surface-bound oxidized 125I-LDL and microsomal acyl-CoA:cholesterol acyltransferase activity were not significantly affected by the antibiotic at 25 nM. When J774 cells were treated with 25 nM concanamycin B at 37 degrees C for 60 min, there was a reduction of about 50% in the activity of cell-surface receptors. This reduction appeared to be due to partial trapping of the receptors within the cells. Concanamycin B significantly inhibited ATP-dependent acidification of endosomes and lysosomes of the J774 cells at a concentration of 4 nM. Since acidic condition of these organelles is required for receptor recycling and hydrolysis of lipoproteins, the results demonstrate that concanamycin-B inhibition of oxidized-LDL-induced accumulation of lipid droplets and cholesteryl esters in macrophages J774 is associated with reduced ATP-dependent acidification of these organelles.     

Kinetics of alpha 2-macroglobulin endocytosis and degradation in mutant and wild-type Chinese hamster ovary cells

The production of Chinese hamster ovary (CHO) cell mutants which are defective in endocytosis has led to a greater understanding of the process by which cells sort ligands and their receptors. Robbins and coworkers have obtained CHO mutants which are resistant to diphtheria toxin, defective in the delivery of endocytosed lysosomal enzymes to lysosomes, and have a decreased uptake of iron from transferrin (Robbins et al.: J. Cell Biol. 96:1064-1071, 1983). We have previously shown that these CHO mutants are markedly deficient in the acidification of early endocytic compartments (Yamashiro and Maxfield: J. Cell Biol. 105:2713-2721, 1987). In this study we examined the endocytosis of alpha 2-macroglobulin (alpha 2M) to determine whether the defects in early endosome acidification would alter the processing of this ligand. We found that the CHO mutants DTG 1-5-4 and DTF 1-5-1 bind, internalize, and degrade 125I-alpha 2M in a manner similar to the wild-type cells. We also found that the CHO mutants retain the ability to recycle the receptors for alpha 2M. Since the binding of alpha 2M is greatly reduced at mildly acidic pH (approximately 6.8), only slight acidification of the endosomal compartment should be sufficient to achieve sorting of alpha 2M from its receptor. In contrast, lysosomal enzymes require more acidic conditions (pH less than 6.0) for dissociation. The different behavior of the two ligands provides biochemical evidence for a partial (but not complete) defect in early endosome acidification in the mutants. The data also indicate that pH regulation in a relatively narrow range can achieve differential sorting of various ligands.     

Acidification of phagosomes is initiated before lysosomal enzyme activity is detected

We have measured changes of pH in a protein's microenvironment consequent on its binding to the cell surface and incorporation into pinosomes. Changes of pH were measured from single, living cells and selected regions of cells by the fluorescence ratio technique using a photon-counting microspectrofluorimeter. The chemotactic agent and pinocytosis inducer, ribonuclease, labeled with fluorescein (FTC- RNase), adsorbed to the surface of Amoeba proteus, and was pinocytosed by cells in culture media at pH 7.0. The FTC-RNase entered an apparently acidic microenvironment, pH approximately 6.1, upon binding to the surface of amoebae. Once enclosed within pinosomes, this protein's microenvironment became steadily more acidic, reaching a minimum of pH approximately 5.6 in less than 10 min. FTC-RNase pinocytosed by the giant amoeba, Chaos carolinensis, entered pinosomes whose pH was correlated with their cytoplasmic location during the initial 30-40 min after pinocytosis. The majority of pinosomes containing FTC-RNase clustered in the tail ectoplasm of C. carolinensis during this interval and had a pH of approximately 6.5; those released into endoplasm and carried into the tip of cells had a pH below 5.0. As pinosomes became distributed at random in C. carolinensis (1-2 h after initial pinocytosis), differences in pH between tip and tail pinosomes vanished. We have also measured the pH within single phagosomes of A. proteus. Phagosomal pH dropped steadily to approximately 5.4 within 5 min after particle ingestion in 70% of the cells measured, and reached this level of acidity within 10 min in 90% of the cells measured. By contrast, stain for the lysosomal enzyme, acid phosphatase, was evident within only 20% of 5-min-old phagosomes visualized by light microscopy, and within only 40% of 10-min-old phagosomes. A microfluorimetric assay was used to simultaneously record changes in pH, and the initial deposition of lysosomal esterases, within phagosomes of single, living Amoeba proteus. Near complete acidification of the phagosome was recorded from some cells before phagosomal fusion was evident by this microfluorimetric assay. From other cells, however, continued acidification of phagosomes was recorded after lysosomal fusion was initiated. We conclude that acidification of phagosomes by A. proteus is initiated but not necessarily completed prior to phagosome-lysosome formation, and that the two events are closely linked in time. Initial acidification of endosomes is a property intrinsic to the plasma membrane which envelops particles at the cell surface, rather than the result of lysosomal fusion with phagosomes.     

Involvement of ATP-dependent Pseudomonas Exotoxin Translocation from a Late Recycling Compartment in Lymphocyte Intoxication Procedure

Pseudomonas exotoxin (PE) is a cytotoxin which, after endocytosis, is delivered to the cytosol where it inactivates protein synthesis. Using diaminobenzidine cytochemistry, we found over 94% of internalized PE in transferrin (Tf) -positive endosomes of lymphocytes. When PE translocation was examined in a cell-free assay using purified endocytic vesicles, more than 40% of endosomal ¹²⁵I-labeled PE was transported after 2 h at 37°C, whereas a toxin inactivated by point mutation in its translocation domain was not translocated. Sorting of endosomes did not allow cell-free PE translocation, whereas active PE transmembrane transport was observed after > 10 min of endocytosis when PE and fluorescent-Tf were localized by confocal immunofluorescence microscopy within a rab5-positive and rab4- and rab7-negative recycling compartment in the pericentriolar region of the cell. Accordingly, when PE delivery to this structure was inhibited using a 20°C endocytosis temperature, subsequent translocation from purified endosomes was impaired. Translocation was also inhibited when endosomes were obtained from cells labeled with PE in the presence of brefeldin A, which caused fusion of translocation-competent recycling endosomes with translocation-incompetent sorting elements. No PE processing was observed in lymphocyte endosomes, the full-sized toxin was translocated and recovered in an enzymatically active form. ATP hydrolysis was found to directly provide the energy required for PE translocation. Inhibitors of endosome acidification (weak bases, protonophores, or bafilomycin A1) when added to the assay did not significantly affect ¹²⁵I-labeled PE translocation, demonstrating that this transport is independent of the endosome-cytosol pH gradient. Nevertheless, when ¹²⁵I-labeled PE endocytosis was performed in the presence of one of these molecules, translocation from endosomes was strongly inhibited, indicating that exposure to acidic pH is a prerequisite for PE membrane traversal. When applied during endocytosis, treatments that protect cells against PE intoxication (low temperatures, inhibitors of endosome acidification, and brefeldin A) impaired ¹²⁵I-labeled PE translocation from purified endosomes. We conclude that PE translocation from a late receptor recycling compartment is implicated in the lymphocyte intoxication procedure.     

SNAPIN is critical for lysosomal acidification and autophagosome maturation in macrophages

We previously observed that SNAPIN, which is an adaptor protein in the SNARE core complex, was highly expressed in rheumatoid arthritis synovial tissue macrophages, but its role in macrophages and autoimmunity is unknown. To identify SNAPIN's role in these cells, we employed siRNA to silence the expression of SNAPIN in primary human macrophages. Silencing SNAPIN resulted in swollen lysosomes with impaired CTSD (cathepsin D) activation, although total CTSD was not reduced. Neither endosome cargo delivery nor lysosomal fusion with endosomes or autophagosomes was inhibited following the forced silencing of SNAPIN. The acidification of lysosomes and accumulation of autolysosomes in SNAPIN-silenced cells was inhibited, resulting in incomplete lysosomal hydrolysis and impaired macroautophagy/autophagy flux. Mechanistic studies employing ratiometric color fluorescence on living cells demonstrated that the reduction of SNAPIN resulted in a modest reduction of H⁺ pump activity; however, the more critical mechanism was a lysosomal proton leak. Overall, our results demonstrate that SNAPIN is critical in the maintenance of healthy lysosomes and autophagy through its role in lysosome acidification and autophagosome maturation in macrophages largely through preventing proton leak. These observations suggest an important role for SNAPIN and autophagy in the homeostasis of macrophages, particularly long-lived tissue resident macrophages.     

大鼠肾近球小管细胞胞饮体膜氢泵活性和水的渗透性转运的特征

本实验采用异硫氰酸-葡聚糖荧光素(fluorescein isothiocyanate-dextran,FITC-dextran)体内标记法,研究大鼠肾近球小管细胞胞饮体(endosome)膜上 H~+-ATP 酶的活性及水的渗透性转运。通过观察在胞饮体外加入一定量 ATP 后,胞饮体内 pH 值的时间反应曲线,从而测定 ATP-依赖的 H~+在胞饮体膜上的转运情况。胞饮体内的酸化速度及 pH 的最低值与加入的 ATP 浓度有关。在加入 ATP 前,胞饮体内的 pH 值为7.4,加入不同浓度的 ATP 后,即[ATP]为0.005,0.05,0.5,5和10mmol/L,胞饮体内 pH 最低值分别为7.30,6.99,6.68,6.38和6.39。此种由 ATP 引起的酸化反应,被0.5mmol/L N-ethylmaleimide(NEM)抑制97%,但不被钒酸盐和 oligomycin 所抑制。实验还同时观察了此种胞饮体水的渗透性转运机制。通过在胞饮体膜内外建立一个蔗糖浓度梯度。观察 FITC-dextran 荧光信号的快速动力学变化过程,从而测定由于渗透压梯度引起的水在胞饮体膜上转运的特征。在230℃时,水的渗透性通透系数(osmotic water permeability coefficient,P_f)为0.03cm/s;加入0.5mmol/L HgCl_2后,水的转运被抑制70%。此抑制反应可被5mmol/L 巯基乙醇(β-Mcrcaptoethanol)完全逆转。上述结果提示:大鼠肾近球小管胞饮体膜含有H~+-ATP 酶和水的转运通道。胞饮     

Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin.

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.     

Internalization and degradation of peptides of the bombesin family in Swiss 3T3 cells occurs without ligand-induced receptor down-regulation.

The binding of [125I]gastrin releasing peptide ([125I]GRP) to Swiss 3T3 cells at 37 degrees C increases rapidly, reaching a maximum after 30 min and decreasing afterwards. The decrease in cell-associated radioactivity at this temperature is accompanied by extensive degradation of the labelled peptide. At 4 degrees C equilibrium binding is achieved after 6 h and [125I]GRP degradation is markedly inhibited. Extraction of surface-bound ligand at low pH demonstrates that the iodinated peptide is internalized within minutes after addition to 3T3 cells at 37 degrees C. The rate of internalization is strikingly temperature-dependent and is virtually abolished at 4 degrees C. In addition, lysomotropic agents including chloroquine increase the cell-associated radioactivity in cells incubated with [125I]GRP. The binding of [125I]GRP to Swiss 3T3 cells was not affected by pretreatment for up to 24 h with either GRP or bombesin at mitogenic concentrations. Furthermore, pretreatment with GRP did not reduce the affinity labelling of a Mr 75,000-85,000 surface protein recently identified as a putative receptor for bombesin-like peptides. These results demonstrate that while peptides of the bombesin family are rapidly internalized and degraded by Swiss 3T3 cells, the cell surface receptors for these molecules are not down-regulated.     

Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.     

Autolysis of glycoproteins in rat kidney lysosomes in vitro. Effects on the isoelectric focusing behaviour of glycoproteins, arylsulphatase and β-glucuronidase

1. Rat kidney lysosomal glycoproteins, prelabelled in the N-acetylneuraminic acid and polypeptide portions with N-acetyl[(3)H]mannosamine and [(14)C]lysine, or with N-acetyl-[(14)C]glucosamine, were incubated under various conditions. Autolytic cleavage of labelled N-acetylneuraminic acid and peptide was maximum at pH5.0. 2. N-Acetylneuraminic acid was released more rapidly than peptide during incubation at 37 degrees or 4 degrees C at pH5. p-Nitrophenyloxamic acid, an inhibitor of bacterial neuraminidase (Edmond et al., 1966), inhibited the cleavage of N-acetylneuraminic acid and peptide, and also inhibited cathepsin D activity. 3. Galactono-, mannono-, and glucono-lactone, inhibitors of the corresponding glycosidases, blocked the autolytic cleavage of N-acetyl[(14)C]glucosamine and protein without inhibiting beta-N-acetylhexosaminidase or cathepsin D activity. These findings suggest that the carbohydrate side chains protect the polypeptide portion of the lysosomal glycoproteins against proteolytic attack by lysosomal cathepsins. 4. In electrofocusing experiments, autolysis was minimized by adding 0.1% p-nitrophenyloxamic acid to the media used for extraction and electrofocusing, and by maintaining an alkaline pH (pH8.8-9) during extraction and dialysis. Arylsulphatase occurred in two forms with pI values of 4.4 and 6.4-6.7, and beta-glucuronidase in two forms with pI values of 4.4 and 6.1. When [(14)C]lysine and N-acetyl[(3)H]mannosamine were given to rats 1.5 and 1 h before killing, (14)C and (3)H were largely restricted to highly acidic glycoprotein species with pI values of 2.1-5.1. 5. When a lysosomal extract was adjusted to pH5 and incubated at 20 degrees C for 16h and then at 37 degrees C for 1 h before electrofocusing, 32 and 58% of the labelled peptide and N-acetylneuraminic acid was cleaved and the pI values of the labelled glycoproteins were markedly increased. About 80% of the acidic form of arylsulphatase and beta-glucuronidase was recovered with the basic form, and the pI of the basic form of both enzymes rose to 7.0. Similar, though less marked changes, were observed when a lysosomal extract was kept at pH5 for 2h at 4 degrees C before electrofocusing. 6. When an acidic lysosomal fraction (pI4.2-4.6) was incubated at pH5 for 2.5h and refocused, 80% of the arylsulphatase now occurred in two forms with pI values of 5 and 6.4. When a basic lysosomal fraction (pI5.8-6.4) was similarly incubated, the pI of arylsulphatase increased from 6.4 to 7.2. The relative increase in pI of arylsulphatases was accompanied by a proportional loss of N-acetylneuraminic acid from the glycoprotein associated with these forms. 7. These experiments show that lysosomal glycoproteins and two representative hydrolases, when exposed to a mildly acidic pH, readily undergo autolytic degradation and their pI values increase. These observations may have a bearing on the origin of the molecular heterogeneity of the lysosomal enzymes.     

Thermochemical pretreatment of lignocellulose to enhance methane fermentation: II. Evaluation and application of pretreatment model

A model was developed and evaluated as a tool for predicting the formation of soluble products from staged thermochemical treatment of lignocellulosic materials under acidic conditions typical of autohydrolysis. The model was used to predict the general trend of hemi-cellulose and cellulose hydrolysis between pH 2 and 4 and temperatures of 170-230 degrees C, and results were compared with experimental data. When the model was evaluated for this range of temperatures and pH values, results indicated: (1) a relatively low temperature (175 degrees C) during the first stage allows hydrolysis of the hemi-cellulose polysaccharides without significant mono-saccharide decomposition, (2) subsequent stages at higher temperatures (equal or greater than 200 degrees C) are needed for significant cellulose hydrolysis, but glucose decomposition will also occur, and, (3) a pH in the range of 2-2.5 will enhance polysaccharide hydrolysis while limiting monosaccharide decomposition. The model's predictions, indicating that the formation of biodegradable products could be optimized using Pretreatments at pH 2-2.5 for the pH range evaluated, were confirmed in experiments with white fir as a representative lig nocellulose.