Publisher Summary Catalase exerts a dual function: (1) decomposition of H 2 O 2 to give H 2 O and O 2 (catalytic activity) and (2) oxidation of H donors, for example, methanol, ethanol, formic acid, phenols, with the consumption of 1 mol of peroxide (peroxide activity) The kinetics of catalase does not obey the normal pattern Measurements of enzyme activity at substrate saturation or determination of the K s is therefore impossible In contrast to reactions proceeding at substrate saturation, the enzymic decomposition of H 2 O 2 is a first-order reaction, the rate of which is always proportional to the peroxide concentration present Consequently, to avoid a rapid decrease in the initial rate of the reaction, the assay must be carried out with relatively low concentrations of H 2 O 2 (about 001 M) This chapter discusses the catalytic activity of catalase The method of choice for biological material, however, is ultraviolet (UV) spectrophotometry Titrimetric methods are suitable for comparative studies For large series of measurements, there are either simple screening tests, which give a quick indication of the approximative catalase activity, or automated methods

Catalase in vitro

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Endocytic mechanisms control the lipid and protein composition of the plasma membrane, thereby regulating how cells interact with their environments. Here, we review what is known about mammalian endocytic mechanisms, with focus on the cellular proteins that control these events. We discuss the well-studied clathrin-mediated endocytic mechanisms and dissect endocytic pathways that proceed independently of clathrin. These clathrin-independent pathways include the CLIC/GEEC endocytic pathway, arf6-dependent endocytosis, flotillin-dependent endocytosis, macropinocytosis, circular doral ruffles, phagocytosis, and trans-endocytosis. We also critically review the role of caveolae and caveolin1 in endocytosis. We highlight the roles of lipids, membrane curvature-modulating proteins, small G proteins, actin, and dynamin in endocytic pathways. We discuss the functional relevance of distinct endocytic pathways and emphasize the importance of studying these pathways to understand human disease processes.

Mechanisms of Endocytosis

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms Recent reviews have described the range of assays that have been used for this purpose(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi) Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-4sx3t3he7d.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Peroxisomes (microbodies and related particles).

Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L-α-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage.

/pdf/the-large-scale-separation-of-peroxisomes-mitochondria-and-2qo1u10n1t.pdf

The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions.

/pdf/tissue-fractionation-studies-17-intracellular-distribution-110scd191g.pdf

Tissue fractionation studies. 17. Intracellular distribution of monoamine oxidase, aspartate aminotransferase, alanine aminotransferase, D-amino acid oxidase and catalase in rat-liver tissue.

Numerous observations made over the last five years have established the widespread occurrence of cellular autophagy as well as its importance in the processes whereby living cells achieve the degradation of their own constituents (For a review, see reference 5). In all the cases that have been investigated in this respect, autophagic vacuoles have been found to stain positively for acid phosphatase, suggesting strongly that lysosomal enzymes are the main agents of the digestive processes taking place within them. However, the mechanism of the segregation phenomenon and the manner in which hydrolases become associated with the sequestered area are still far from being understood. One theory, proposed by Novikoff et al. (9), considers areas of endoplasmic reticulum, assumed to be involved in the intracellular transport of newly synthesized hydrolases, as providing both the surrounding membrane of autophagic vacuoles and their enzymic complement. According to this theory, cellular autophagy represents a mechanism, though not necessarily the only one, whereby new lysosomes may arise in a living cell. Other workers have expressed similar ideas concerning the origin of lysosomes (1, 8). An alternative hypothesis, put forward by de Duve and Wattiaux (5), considers preexisting lysosomes as the source of the enzymes that are found in autophagic vacuoles, on the assumption, either that the particles actually participate in the sequestration phenomenon, or that they fuse with initially hydrolase-less \"autophagosomes\" as they are known to do with the \"heterophagosomes\" formed by endocytosis. In a recent investigation, Deter and de Duve (7) have found that the injection of a large dose of glucagon, a powerful inducer of autophagy in liver, causes a transient, but manifest increase in the mechanical and osmotic fragility and in the median sedimentation coefficient of rat-liver lysosomes. These changes were tentatively attributed to an enlargement of the lysosomes, possibly reflecting extensive participation of these particles in the autophagic process. But in the absence of morphological data, no firm conclusion could be reached. The present communication reports the first results of a quantitative morphological study correlating the appearance of autophagic vacuoles with a marked decrease in the number of recognizable dense bodies present in particulate fractions separated from hepatic homogenates 45 min after glucagon injection.

https://rupress.org/jcb/article-pdf/35/2/C11/1384274/c11.pdf

Participation of lysosomes in cellular autophagy induced in rat liver by glucagon.

Tissue fractionation studies. 18. Resolution of mitochondrial fractions from rat liver into three distinct populations of cytoplasmic particles by means of density equilibration in various gradients.

Pierre Baudhuin

Papers

Peroxisomes (microbodies and related particles).

The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions.

Tissue fractionation studies. 17. Intracellular distribution of monoamine oxidase, aspartate aminotransferase, alanine aminotransferase, D-amino acid oxidase and catalase in rat-liver tissue.

Participation of lysosomes in cellular autophagy induced in rat liver by glucagon.

Tissue fractionation studies. 18. Resolution of mitochondrial fractions from rat liver into three distinct populations of cytoplasmic particles by means of density equilibration in various gradients.