Fractionation of pepsin-digested, denatured collagen. III. Characterization of the small fragments

The survey of the fragments obtained from pepsin-digested, denatured rat tail tendon collagen is completed. Three additional fragments could be renatured and two of them (490 A and 670 A) were located in tropocollagen by electron microscopy. Data are given on the amino acid compositions of the various fractions. Certain fragments probably originated from the associated non-collagenous material belonging to the "acidic structural proteins".     

Cardiolipin degradation by rat liver lysosomes.

The lysosomal subcellular fraction of rat liver contains acid hydrolases which can carry out the degradation of cardiolipin to yield water-soluble products and free fatty acids. The time course of appearance of the products indicates that the major catabolic route involves the sequential removal of three of the fatty acids, followed by hydrolysis to acylglycerophosphoryl glycerol (from which the fatty acid is subsequently removed) and d-glycerophosphate (which is hydrolysed to give free phosphate and glycerol). The phospholipase A activity responsible for removal of the first fatty acid is located in the lysosomal fraction.     

Neutral-sugar transport by rat liver lysosomes.

Transport of D-glucose was studied in Percoll-gradient-purified rat liver lysosomes. D-Glucose uptake had a Km of 22 mM and a t1/2 of approx. 30 s. D-Fucose, 2-deoxyglucose and methyl alpha-glucoside were the most effective competitors for uptake of D-glucose, although D-galactose, D-mannose, D-xylose and L-fucose also appeared to compete for uptake. L-Glucose was a poor competitor for uptake. No competition was observed with N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, D-glucuronic acid, N-acetylneuraminic acid, D-glucosamine or the amino acids L-glycine, L-lysine and L-proline. Uptake was unaffected by N-ethylmaleimide, dithiothreitol, KCl, NaCl, ATP/Mg or alteration of buffer pH. D-Glucose efflux from lysosomes was temperature-dependent, with a Q10 of 2.3, and was inhibited by cytochalasin B. Counter-transport could not be demonstrated. In contrast, L-fucose uptake had a Km of 65 mM and was largely unaffected by 5 M excess of neutral D-sugars. Both uptake and efflux of L-fucose were inhibited by cytochalasin B. It appears that lysosomes possess a facilitated transport system for D-glucose and perhaps other neutral D-sugars that is discrete from transport systems for acetylated and acidic sugars.     

Sulfate transport by rat liver lysosomes

Sulfate transport was examined using membrane vesicles (pH 7.0 inside) prepared from rat liver lysosomes. Sulfate uptake was dependent upon external pH with increased uptake at lower buffer pH. The Km for uptake was 160 microM at pH 5.0 while at pH 7.0, a lower affinity system with a Km of 1.4 mM was present. The protonophore carbonyl cyanide m-chlorophenylhydrazone increased uptake at pH 5.0 while valinomycin/KCl had no effect. In contrast, at pH 7.0, valinomycin-induced changes in membrane potential stimulated uptake. Countertransport of sulfate at pH 7.0 was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonic acid, and a variety of anions: SO4(2-) greater than MoO4(2-) greater than Cl- greater than HPO4- greater than HCO3-. Trans-stimulation of sulfate uptake at pH 7.0 was observed with MoO4(2-) and, to a lesser extent, with S2O3(2-) while Cl-, HPO4-, and HCO3- had little effect. However, chloride loading of vesicles resulted in marked stimulation of sulfate uptake at pH 5.0. It appears that sulfate and protons exit lysosomes in exchange for chloride by a specific, pH-regulated anion transport system.     

Insulin degradation by lysosomal extracts from rat liver; model for a role of lysosomes in hormon degradation.

The presence of both glutathione-insulin transhydrogenase and “insulin specific protease” in highly purified lysosomal extracts from rat liver is presented. Although both of these enzyme activities were known to exist in the cytosol, it was difficult to understand their participation in insulin degradation, for it is known that insulin binds to plasma membrane receptors. Thus, the presence of both enzymes in lysosomes is of much interest for it suggests an explanation and/or model for the degradation via pinocytosis of insulin and possibly of other hormones which bind to receptors in the plasma membrane.     

Transport of N-acetyl-D-glucosamine and N-acetyl-D-galactosamine by rat liver lysosomes

Transport of N-acetyl-D-glucosamine and N-acetyl-D-galactosamine, products of lysosomal glycoprotein and glycosaminoglycan degradation, was examined in Percoll gradient purified rat liver lysosomes. Uptake of these two sugars was competitive and quite specific remaining largely unaffected by the presence of L-fucose, D-glucosamine, D-glucose, D-glucuronic acid, D-mannose, or N-acetylneuraminic acid. Kinetic studies revealed a Km of 4.4 mM for both N-acetyl-D-glucosamine and N-acetyl-D-galactosamine uptake. Temperature dependence studies revealed a Q10 of 2.3. N-Acetyl-D-glucosamine uptake was not dependent upon NaCl, KCl, MgCl2, or ATP/MgCl2 and was unaffected by 5 mM dithiothreitol or variation of buffer pH between 6.0 and 8.0. Cytochalasin B at a concentration of 50 microM effectively inhibited uptake of N-acetyl-D-glucosamine by 90% and N-acetyl-D-galactosamine by 65%. Prior incubation of lysosomes in 20 mM N-acetyl-D-glucosamine stimulated uptake of both sugars 3-4-fold, although it had no effect on the uptake of D-glucose. Countertransport was unaffected by neutral and cationic amino acids demonstrating independence from these amino acid transport systems. We conclude that lysosomes possess a highly specific transport system for N-acetyl-D-glucosamine and N-acetyl-D-galactosamine.     

Hydrolysis of dolichyl esters by rat liver lysosomes

Dolichyl ester hydrolase activity is broadly distributed among the organs of the rat. The highest activity was found in spleen, brain, lung, and thyroid tissues, whereas this activity is very low in stomach and intestine. The esterase involved is localized to the lumen of lysosomes and, to some extent, in the plasma membranes. Hydrolysis occurs with both alpha-saturated and alpha-unsaturated polyisoprenes esterified with different fatty acids, but the rate of hydrolysis is strongly dependent on the nature of the substrate. The enzyme involved is inhibited by divalent cations, EDTA and EGTA and also by one of the products, dolichol. The esterase is activated by 3-[(3-cholamidopropyl) dimethylammonio]-1-propranesulfonic acid and taurodeoxycholate and inhibited by Triton X-100. Dolichyl esterase activity is completely inhibited by alpha- and beta-naphthyl acetate, phenylmethylsulfonyl fluoride, and beta-chloromethylmercurisulfate. These inhibitors, as well as the pH optimum for dolichyl ester hydrolysis, clearly differentiate the enzyme involved from cholesteryl esterase and triglyceride lipase. Microsomal phospholipase A hydrolyzes dolichyl esters at a slow rate only. In vivo labeling experiments with [3H]mevalonate demonstrated that newly synthesized dolichol is transported in esterified form to the lysosomes, where this lipid is slowly hydrolyzed by the esterase. The possibility is raised that the role of the fatty acyl moiety may be to target dolichol to its final location in the cell.     

Antibody binding by native and denatured myosin and paramyosin.

By the techniques of immunodiffusion and fluorescent immunohistochemistry we show that antibodies to both the native and the SDS-denatured forms of the proteins, paramyosin and myosin, react with the native, SDS-denatured and glutaraldehyde-fixed forms of their respective antigens. Anti-denatured myosin also binds to both native and denatured forms of the proteolytic subfragments of myosin: globular subfragment-1 and alpha-helical LMM. Anti-native myosin, on the other hand, while able to bind to both native and denatured LMM or rod and to native and glutaraldehyde-fixed S-1, does not bind to SDS-denatured S-1.     

Diadenosine triphosphate splitting by rat liver extracts.

A splitting activity on diadenosine triphosphate has been found in rat liver. One of the products of the cleavage is ADP. A Km of 10 μM has been found. This activity on diadenosine triphosphate seems to be specific as diadenosine tetraphosphate, a nucleotide previously described by others to occur in rat liver at very low concentration, is not a substrate of the reaction. The occurrence of diadenosine triphosphate in rat liver has not been so far reported, but a dinucleoside triphosphate structure has been described at the 5′ end of certain mRNAs. The possibility that this enzymatic activity may be involved in the hydrolysis of diadenosine triphosphate or in the processing of mRNAs is suggested.