1
Protein degradation in isolated rat hepatocytes was measured as the release of [14C]valine from pre-labelled protein. To reduce background radioactivity, the intracellular [14C]valine pool was depleted by serial extraction at 37 °C, effecting equilibration between the intracellular pool and the valine-free extracellular medium. After extraction, a small, non-equilibrating intracellular [14C]valine pool remained; this pool could only be labelled in the presence of ongoing protein synthesis, and might represent valine and valine-containing oligopeptides derived from protein degradation.

2
The [14C]valine released from degraded protein was not significantly re-utilized for protein synthesis intracellularly (no effect of cycloheximide or high concentrations of unlabelled valine), reflecting the low rate of protein synthesis and the rapid transport of valine into the extracellular medium, both characteristic of isolated hepatocytes.

3
From cells pre-labelled for 24 h in vivo, [14C]valine was released linearly at a rate of 5%/h, probably representing the true over-all protein degradation rate. The lysosomotropic inhibitor ammonia (10 mM NH4Cl) inhibited 70% of the degradation, presumably the contribution by the lysosomal pathway.

From 1-h pre-labelled cells, [14C]valine was released at a declining rate, and ammonia inhibited degradation only by 45%, consistent with the view that the majority of short-lived proteins are degraded by the non-lysosomal pathway(s).

4
Chloroquine and methylamine, which accumulate in lysosomes by virtue of their weak base properties, inhibited hepatocytic protein degradation to the same extent as ammonia, with no additivity. These compounds therefore seem to block the lysosomal pathway of protein degradation selectively and completely. Leupeptin, which binds to and inhibits the activity of certain lysosomal proteases, also inhibited protein degradation almost to the same extent as ammonia, but with a small part of the effect (< 20%) being additive to the NH3 effect and thus probably reflecting a slight inhibition of non-lysosomal protein degradation as well.

5
Of the four inhibitors tested, only the effect of ammonia was rapidly reversible within the experimental period (2 h). Leupeptin, on the other hand, was the only degradation inhibitor which did not also affect protein synthesis. Chloroquine caused significant cell death at concentrations above 0.2 mmol/l in this protein-free medium, i.e. in the concentration range needed for maximal inhibition of protein degradation.

6
Incubation of hepatocytes under anoxic conditions resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of ammonia, i.e. most of the degradation by the lysosomal pathway and more than one-half of the degradation by the non-lysosomal pathways appears to be energy-dependent.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1979.tb12956.x

Inhibition of the Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes by Ammonia, Methylamine, Chloroquine and Leupeptin

The keto analog of leucine, alpha-ketoisocaproate (KIC), is formed intracellularly from leucine and is released, in part, into the systemic circulation. Therefore. KIC can be used to estimate intracellular leucine tracer enrichment in man during labeled-leucine tracer experiments without requiring tissue biopsy samples. This approach was studied in young, healthy, male adults maintained on different dietary protein intakes from generous (1.5 g kg-1d-1) to deficient (0.0 g kg-1d-1) for 5-7 day periods. At the end of each dietary period, the volunteers were given a primed, continuous infusion of L-[1-13C]leucine either after an overnight fast (postabsorptive state) or while being fed hourly aliquots of the same diet. The plasma concentrations of all 3 branched-chain amino and keto acid pairs were measured from early morning blood samples taken from 4 subjects at 4 different levels of protein intake. Leucine concentration showed a weak correlation, and valine concentration showed a strong correlation with protein intake; isoleucine and the 3 keto acids did not. However, each branched-chain amino acid concentration was strongly correlated with its corresponding keto acid concentration. In plasma samples obtained during the L-[1-13C]leucine infusions, the ratio of [1-13C]KIC to [1-13C]leucine enrichment ratio remained relatively constant (77 +/- 1% over the wide range of dietary protein intakes and for both the fed and postabsorptive states. For the tissues from which the plasma KIC originates, the rate of plasma leucine into cells will account for approximately 77% of the intracellular leucine flux with the remaining 23% coming primarily from leucine release via protein breakdown. The constant nature of the plasma KIC to leucine 13C enrichment ratio implies that relative changes in leucine kinetics will appear the same under many dietary circumstances regardless of whether plasma leucine or KIC enrichments are used for the calculations.

Relationship of plasma leucine and α-ketoisocaproate during a L-[1-13C]leucine infusion in man: A method for measuring human intracellular leucine tracer enrichment

Measurement of the rate of protein synthesis and compartmentation of heart phenylalanine.

I. General Considerations and Background.- I.1. Perspectives.- I.2. Definitions.- I.2.1. General Functional Categories.- I.2.2. Outline of Lysosome Functioning in Phagocytes.- I.2.3. Additional Terms: Heterophagy and Autophagy.- I.3. Characterization of Lysosomes.- I.3.1. Basic Biochemical Characteristics of Lysosomes.- I.3.1.1. Key Features of Lysosomes.- I.3.1.2. The Lysosomal Enzymes.- I.3.1.3. Digestion in Lysosomes.- I.3.1.4. Lysosomal "Permeability".- I.3.2. Some Cytochemical and Morphological Characteristics.- I.3.2.1. Cytochemical Methods.- I.3.2.2. Cytochemical Studies of Exogenous Tracers.- I.3.2.3. Additional Cytochemical Features of Lysosomes: "Matrix" Materials and Some Other Non-Enzymatic Components.- I.4. Morphological Categories of Lysosomes.- II. Lysosome Formation, Functioning and Fate.- II. 1. Heterophagy.- II.1.1. Background.- II.1.2. Specificity.- II.1.3. The Transport of Acid Hydrolases.- II.1.3.1. The Formation of Primary Lysosomes in PMN Leukocytes.- II.1.3.2. Primary Lysosomes in Macrophages.- II.1.4. Fusion Phenomena, Lysosome "Reuse" and Lysosome Membranes.- II.1.4.1. Lysosome "Recycling".- II.1.4.2. Energetics and Control of Movements in Heterophagy.- II.1.4.3. Lysosome Membranes Fusion.- II.1.4.4. Other Features of the Lysosome Surface: Enzymes, Changes and Stability.- II.1.4.5. Acidification.- II.2. Hydrolase Transport in Cells Other than Phagocytes.- II.2.1. GERL.- II.2.2. Endoplasmic Reticulum and Lysosomes Some Biochemical Findings.- II.3. Autophagy.- II.3.1. Basic Morphology.- II.3.2. Mode of Formation of Autophagie Vacuoles.- II.3.2.1. Source of the Delimiting Membranes.- II.3.2.2. Source of the Hydrolases.- II.3.3. Crinophagy.- II.3.4. Some General Aspects of the Control and Specificity of Autophagy.- II.4. Multivesicular Bodies (MVB's).- II.4.1. Heterophagic Roles.- II.4.2. Participation in Autophagy.- II.4.2.1. Incorporation of Secretory Material and of Intact Vesicles.- II.4.2.2. Degradation of Membranes Participating in Endocytosis.- II.4.2.3. Possible Roles in Degrading Other Types of Membrane.- II.4.2.4. Microautophagy.- II.5. The Fate of Lysosomes.- II.5.1. Release vs. Retention.- II.5.2. "Telolysosomes" and Lipofuscin.- II.6. Lysosome Heterogeneity.- III. Lysosomes in Turnover and Modulation.- III.1. Turnover of Cells and Tissues of Higher Animals and Features of Developmental Remodelling.- III.1.1. Background and an Example: The Red Blood Cell Life History.- III.1.1.1. Lysosomes in the Maturation of Red Blood Cells.- III.1.1.2. The Destruction of Red Blood Cells.- III.1.1.3. Iron Storage and Release.- III.1.2. Developmental "Remodelling".- III.1.2.1. Case Histories: Cell Destruction in Insect Metamorphosis.- III.2. Turnover of Extracellular Materials.- III.2.1. Connective Tissue Components.- III.2.1.1. Hydrolase Release in Cartilage.- III.2.1.2. Osteoclasts.- III.2.1.3. Collagenases.- III.3. Turnover of Circulating Macromolecules.- III.3.1. Selectivity.- III.4. Intracellular Turnover.- III.4.1. Some Methodological Perspectives and Problems.- III.4.2. Turnover in Bacteria.- III.4.3. Turnover of Organelles and Intracellular Macromolecules in Eucaryotes.- III.4.3.1. Some General Points and Some Experiments Paralleling those Done with Procaryotes.- III.4.3.2. Turnover of Macromolecules that are not Components of Membrane-Delimited Organelles.- III.4.3.3. Aspects of the Turnover of Membrane-Delimited Cytoplasmic Organdies.- III.5. Turnover of Photoreceptor Membranes.- IV. Pathology.- IV.1. Lysosomal Storage Diseases.- IV.1.1. Lipidoses and Polysaccharidoses.- IV.1.2. Etiological Aspects.- IV.1.3. Some Interesting Disorders of Uncertain Status.- IV.2. Lysosomes and Infection.- IV.2.1. The Entry of Structures with Macromolecular Dimensions into Cells: Viruses and Toxic Proteins.- IV.2.2. Potentially Instructive Failures of Defenses.- IV.2.3. Malaria.- IV.3. The "Pathological" Release of Enzymes to Extracellular Spaces Arthritis, Inflammation, and Related Phenomena.- IV.4. The Intracellular Release of Hydrolases: Lysosome Fragility, Labilizers, and Stabilizers.- IV.4.1. Methodological Problems.- IV.4.2. Silica and Uric Acid.- IV.4.3. Labilizers, Stabilizers, Drugs, and Inhibitors.- IV.5. Lysosomes in Immune Responses.- IV.5.1. Macrophages and Some Other Cells in the "Processing" or "Presentation" of Antigens.- IV.5.2. Lysosomes in Lymphocyte Activation.- IV.5.3. The Transfer of Maternal Antibodies.- V. Some Special Topics and Some Loose Ends.- V.1. Lysosomes in Plant Cells.- V.1.1. Senescence and Cell Death.- V.1.2. Autophagy.- V.1.3. Extracellular Hydrolases.- V.2. Hydrolases in Secretory Cells.- V.2.1. Lysosomes and Secretory Processes the Thyroid Gland.- V.2.2. Hydrolases in the Golgi Apparatus and Secretion Granules.- V.2.3. Melanin.- V.3. Lysosomes in Animal Gametes.- V.3.1. Sperm.- V.3.2. Eggs.- V.4. Closing Comments.- Acknowledgements.

Lysosomes: A Survey

Compartmentation of Free Valine and Its Relation to Protein Turnover in Perfused Rat Liver

Protein Catabolism in Rat Liver Homogenates A RE-EVALUATION OF THE ENERGY REQUIREMENT FOR PROTEIN CATABOLISM

The kinetics of transfer of plasma amino acids to tissues, and the turnover rates of liver and muscle proteins: The use of continuous infusion technique

Free lysine compartments in rat liver cells

Ferrets that were given 5-5 diphenylhydantoin developed various degrees of gingival hyperplasia. Analysis for 5-5 diphenylhydantoin did not show a correlation between the degree of hyperplasia, concentrations of the drug in the various tissues, or turnover of protein-3H proline. Highest concentratrations of the drug were found in oral mucosa, salivary glands, and adrenal glands, and gingiva concentrations were low.

Henry Jeffay

Papers

Protein Catabolism in Rat Liver Homogenates A RE-EVALUATION OF THE ENERGY REQUIREMENT FOR PROTEIN CATABOLISM

The kinetics of transfer of plasma amino acids to tissues, and the turnover rates of liver and muscle proteins: The use of continuous infusion technique

Free lysine compartments in rat liver cells

Lack of Relationship Between the Degree of Induced Gingival Hyperplasia and the Concentration of Diphenylhydantoin in Various Tissues of Ferrets

Protein catabolism in rat liver nuclei.