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The effect of pH on cathepsin activities in mouse liver heterolysosomes
Abstract:
1. Mouse liver heterolysosomes containing (125)I-labelled albumin were incubated at 35 degrees C in 0.25m-sucrose-0.05m-mercaptoethanol, and various concentrations of buffers at pH4, 5, 7 or 8, and the degradation of intraparticulate protein was measured. 2. Buffers at pH4, 7 or 8 inhibited proteolytic activity and the inhibitions were greater with increasing concentrations of buffers. Tris-acetate buffer, pH8, was a more effective inhibitor. Tris-acetate buffer, pH5, neither inhibited nor stimulated proteolytic activity at concentrations up to 0.1m. 3. Inhibition by pH8 buffers could be accounted for by increased breakage rates of heterolysosomes. 4. Preincubation of heterolysosomes in 0.2m-sodium bicarbonate inhibited proteolytic activity when the particles were washed free of bicarbonate, but the inhibition was reversed if these particles were incubated in media containing pH5 buffer. The inhibition was shown not to be due to an increased breakage of heterolysosomes. 5. It was concluded that the pH within heterolysosomes is about 5, and it is possible to alter reversibly the pH within these particles. The possibility for the existence of an intralysosomal buffering system which maintains the pH at about 5 in heterolysosomes is discussed. This buffering system may be related to the presence of large quantities of acidic lipoproteins in lysosomes observed by other investigators.
Insights
Mouse liver heterolysosomes maintain an internal pH of approximately 5, crucial for protein degradation. This pH can be reversibly altered, suggesting an intralysosomal buffering system.
Area of Science:
- Cell Biology
- Biochemistry
- Lysosome Function
Background:
- Heterolysosomes are key organelles for intracellular degradation.
- Understanding lysosomal pH is critical for comprehending protein processing and turnover.
Purpose of the Study:
- To investigate the intralysosomal pH of mouse liver heterolysosomes.
- To determine the effect of external buffers on proteolytic activity within heterolysosomes.
- To explore the potential for reversible pH modulation within these organelles.
Main Methods:
- Incubation of (125)I-labelled albumin within mouse liver heterolysosomes at 35°C.
- Exposure to various buffer concentrations (pH 4, 5, 7, 8) and sodium bicarbonate.
- Measurement of intraparticulate protein degradation and heterolysosome breakage rates.
Main Results:
- Proteolytic activity was inhibited by pH 4, 7, and 8 buffers, with inhibition increasing at higher buffer concentrations.
- Tris-acetate buffer at pH 8 showed significant inhibition, potentially due to heterolysosome breakage.
- Sodium bicarbonate preincubation inhibited activity, but this was reversible upon incubation in pH 5 buffer, without increased heterolysosome breakage.
Conclusions:
- The internal pH of mouse liver heterolysosomes is approximately 5.
- The intralysosomal pH can be reversibly altered.
- A buffering system likely exists within heterolysosomes to maintain this acidic pH, possibly involving acidic lipoproteins.