Lysosomes intact

LDL (apo B-lOO, E) receptors occur on the cell surface in pits that are coated on the cytosolic side of the cell membrane with a protein called clathrin. The glycoprotein receptor spans the membrane, the B-lOO binding region being at the exposed amino terminal end. After binding, LDL is taken up intact by endocytosis. The apoprotein and cholesteryl ester are then hydrolyzed in the lysosomes, and cholesterol is translocated into the cell. The receptors are recycled to the cell surface. This influx of cholesterol inhibits in a coordinated manner HMG-CoA synthase, HMG-CoA reductase, and, therefore, cholesterol synthesis stimulates ACAT activ-... 

E). Basally internalized conjugate (F) may also be directed to a lysosomal compartment (B) but, it may also cross the cell via a non-lysosomal proteolytic compartment (G) where the PLL moiety can be selectively cleaved. Free and intact HRP (H) can then be released into the apical medium. 

After initial filtration many proteins are actively reabsorbed (endocytosed) by the proximal tubules and subjected to lysosomal degradation, with subsequent amino acid reabsorption. Thus, very little intact protein actually enters the urine. 

Schneider, D.L. (1981). ATP-dependent acidification of intact and disrupted lysosomes Evidence for an ATP-driven proton pump. J. Biol. Chem. 256, 3858-3864. 

For studies of membrane composition, the first task is to isolate a selected membrane. When eukaryotic cells are subjected to mechanical shear, their plasma membranes are torn and fragmented, releasing cytoplasmic components and membrane-bounded organelles such as mitochondria, chloroplasts, lysosomes, and nuclei. Plasma membrane fragments and intact organelles can be isolated by centrifugal techniques described in Chapter 1 (see Fig. 1-8). 

Many biological cells contain degradative enzymes (proteases) that catalyze the hydrolysis of peptide linkages. In the intact cell, functional proteins are protected from these destructive enzymes because the enzymes are stored in cell organelles (lysosomes, etc.) and released only when needed. The proteases are freed upon cell disruption and immediately begin to catalyze the degradation of protein material. This detrimental action can be slowed by the addition of specific protease inhibitors such as phenylmethyl-sulfonyl fluoride or certain bioactive peptides. These inhibitors are to be used with extreme caution because they are potentially toxic. 

This increase has two-fold implications. First, it implies that the fractured dendrimer is more flexible than the intact dendrimer and second, that the fractured dendrimer may expand due to an increase in positive charge at lower pH, a quantity that was described as essential for PEI mediated transfection. Indeed, when branched PEI was subjected to the same set of experiments, the same three-fold increase in viscosity was observed, lending support to the idea that PAMAM dendrimers act as proton sponges. Evidence of lysosomal buffering capability of PAMAM dendrimers was shown by Kukowska-Latallo et al. (1996) when they observed that the efficiency of G5-EDA dendrimers was enhanced by the addition of chloroquine, while the same molecule could not enhance transfection of G10-EDA dendrimers, which contain 40-fold more surface amine groups for proton absorption. 

Proteases degrade the unbound antibody molecules. (5) The degraded products reside in the lysosome part. (6) The intact IgG molecules bound to Fc-Rn are transported to the cell membrane where they can be returned to the extracellular space. 

The resultant decrease in pH value leads to an increase in binding affinity to the protective Fc-Rn, and to degradation by proteases of unbound antibody molecules. The degradation products will reside in the lysosome, while the intact IgG molecules bound to Fc-Rn will be transported to the cell membrane and can be returned intact to the extracellular space. The Fc-Rn can transport the bound antibody bidirectionally to the apical and basolateral membrane, delivering it to the interstitial fluid or the systemic circulation. As a result, the residence time of the intact antibody in the body is prolonged. 

Fig. 3.2. General mechanisms for the uptake and transport of macromolecules by an enterocyte. Intracellular uptake-, after absorption and endocytosis by the microvillous membrane, macromolecules are transported in small vesicles and larger phagosomes. Intracellular digestion occurs when lysosomes combine to form phagolysosomes. Intact molecules that remain after digestion are deposited in the intercellular space by exocytosis. Intercellular uptake-, alternatively, macromolecules may cross the tight junction barrier between cells and diffuse into the intercellular space. (After Walker, W. A. Isselbacher, K. J. Uptake and transport of macromolecules by the intestine possible role in clinical disorders. Gastroenterology, 6T. 531-50, by Williams Wilkins (1974).)...

---