Lysosomal enzyme

LTB4, PAF, IL-1, IL-8, reactive oxygen species, lysosomal enzymes... 

Previous studies indicate that osmotic gradients promote membrane fusion, while hyperosmotic conditions inhibit membrane fusion during exocytosis. Consistent with this idea is the observation that the release of lysosomal enzymes from rabbit neutrophils, induced by the chemotactic peptide J -formylmethionyl-leucyl-phenylalanine (FMLP), is inhibited almost 80% in a 700-mosmol/kg medium. Inhibition is immediate (within 10 s), increases with osmolality, and is independent of the osmoticant. Neutrophils loaded with the calcium indicator indo-1 exhibit an FMLP-induced calcium signal that is inhibited by hyperosmolality. Hyperosmolality (700 mosmol/kg) increases basal calcium levels and reduces the peak of the calcium signal elicited by FMLP at concentrations ranging from 10 ° to 10 M. 

Neutrophils represent an ideal system for studying osmotic effects on exocytosis. Stimulation of cytochalasin-B-treated neutrophils with the chemotactic peptide Jlf-formylmethionyl-leucyl-phenyl-alanine (FMLP) results in a rapid compound exocytosis up to 80% of lysosomal enzymes are released within 30 s (9-14). Secretion appears to be triggered by a rise in the level of cytosolic free calcium (15-18) promoted in part by entry of extracellular calcium through receptor-gated channels and in part by release of calcium that is sequestered or bound at some intracellular site (19-21). In this presentation, we augment our previously published data (22,23), which demonstrates that lysosomal enzyme release from neutrophils is inhibited under hyperosmotic conditions and that the rise in cytosolic calcium preceding secretion is inhibited as well. 

Figure 4. Effect of hyperosmolality on lysosomal enzyme release from rabbit neutrophils. Cells were preincubated 10 min at 37 C in either regular HEPES buffer at 320 mosmol/kg ( ) or in HEPES buffer with 0.3-M sucrose at 680 mosmol/kg ( ), 5 Mg/mL cyto-chalasin B was added, cells were stimulated with FMLP, and p-glucuronidase was released into the medium during a 6-min period measured.

Figure 5. Inhibition of lysosomal enzyme release from neutrophils by increased osmotic strength. Cells were preincubated for 10 min at 37°C in regular buffer containing no additions (o), or containing sodium sulfate ( ), sodium HEPES ( ), or sucrose ( ) to increase the osmotic strength. Cells were treated with cyto-chalasin B (5 arid FMLP (10" M) and p-glucuronidase was...

The only other examples of bromoconduritol inhibition reported so far are a cytosolic jff-D-glucosidase from calf liver and the lysosomal ff-D-glu-cosidase from calf spleen. In spite of the 6500-fold difference in their reactivity with conduritol B epoxide (see Table XI), both enzymes are rapidly inactivated by bromoconduritol F, with kj(max)/Kj 10 M min for the cytosolic enzyme and lq(max)/Ki 3.2 10 for the crude and 3.9 10 M min for the purified lysosomal enzyme. It should be noted that purification of the lysosomal jS-D-glucosidase had effects on the reactivity with bromoconduritol F similar to those it had on the reactivity with conduritol B epoxide (see Table XI). 

Interestingly, both the cytosolic and the lysosomal enzyme regained most of their activity on prolonged standing after they had been inactivated to the extent of 98% with bromoconduritol F. The rate of reactivation was larger at pH 6 than at pH 4.6. It was concluded that a labile ester-bond had been formed in the inactivation reaction. From the stereochemistry of the hydroxyl groups and the bromine substituent, it could have been with the carboxyl group presumed to act as acid catalyst in the activation of substrate or epoxide (see Scheme 6). 

I-Cell Disease Results From Faulty Targeting of Lysosomal Enzymes... 

As indicated above, Man 6-P serves as a chemical marker to target certain lysosomal enzymes to that organelle. Analysis of cultured fibroblasts derived from patients with I-cell (inclusion cell) disease played a large part in revealing the above role of Man 6-P. I-cell disease is an uncommon condition characterized by severe progressive psychomotor retardation and a variety of physical signs, with death often occurring in the first decade. Cultured cells from patients with I-cell disease were found to lack almost all of the normal lysosomal enzymes the lysosomes thus accumulate many different... 

Figure 48-12. Schematic illustration of some aspects of the role of the osteoclast in bone resorption. Lysosomal enzymes and hydrogen ions are released into the confined microenvironment created by the attachment between bone matrix and the peripheral clear zone of the osteoclast. The acidification of this confined space facilitates the dissolution of calcium phosphate from bone and is the optimal pH for the activity of lysosomal hydrolases. Bone matrix is thus removed, and the products of bone resorption are taken up into the cytoplasm of the osteoclast, probably digested further, and transferred into capillaries. The chemical equation shown in the figure refers to the action of carbonic anhydrase II, described in the text. (Reproduced, with permission, from Jun-queira LC, Carneiro J BasicHistology. Text Atlas, 10th ed. McGraw-Hill, 2003.)...

The major biochemical features of neutrophils are summarized in Table 52-8. Prominent feamres are active aerobic glycolysis, active pentose phosphate pathway, moderately active oxidative phosphorylation (because mitochondria are relatively sparse), and a high content of lysosomal enzymes. Many of the enzymes listed in Table 52-4 are also of importance in the oxidative metabolism of neutrophils (see below). Table 52-9 summarizes the functions of some proteins that are relatively unique to neutrophils. 

Lipoxygenases catalyse the regio-specific and stereoselective oxygenation of unsaturated fatty acids. The mammalian enzymes have been detected in human platelets, lung, kidney, testes and white blood cells. The leukotrienes, derived from the enzymatic action of the enzyme on arachidonic acid, have effects on neutrophil migration and aggregation, release of lysosomal enzymes, capillary permeability, induction of pain and smooth muscle contraction (Salmon, 1986). 

In inflammatory conditions, activated PMNs may pro-teolytically (by release of lysosomal enzymes) and oxidatively (by release of HOCl) inactivate ai-antitrypsin. Studies of synovial fluid samples from patients with RA showed that a i-antitrypsin was both cleaved and oxidized, resulting in inactivation (Chidwick et al., 1991, 1994). Free-radical attack on ai-antitrypsin and its subsequent inactivation may contribute to the destruction of joint tissues in arthritis due to the imbalance between elastase and its inhibitors. 

Cellular Effects of Complement Activation. C5a is chemotactic for neutrophils, activates their oxidative metabolism, and induces secretion of lysosomal enzymes from the granulocytes and macrophages. C5a may also induce the production of cytokines and prostaglandins (H19, S14). 

In some human studies where clinical chemistry measurements but no renal biopsies were performed, the only parameter of renal function shown to be affected was an increase in the levels of NAG in the urine. NAG is a lysosomal enzyme present in renal tubular cells that has been shown to be a sensitive indicator of early subclinical renal tubular disease. The mechanism by which lead affects the release of NAG from renal tubular cells is not known, but it is suggested that lead could attach to kidney cell membranes and alter membrane permeability (Chia et al. 1994). 

Srivastava L, Tandon SK. 1984. Effects of zinc on lead-induced changes in brain lysosomal enzymes in the chick embryo. Toxicol Lett 20 111-114. 

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