Histology and cytochemistry of human skin. XI. The distribution of beta-glucuronidase

1. Various amounts of beta-glucuronidase activity may be found in all of the cutaneous appendages. 2. In the epidermis, the basal layer and the Malpighian layer contain a moderate amount of it, but a band of cells, including the stratum granulosum and the cells immediately above it, is rich in beta-glucuronidase. 3. The cells of the duct of eccrine sweat glands have moderately strong enzyme activity, but those in the secretory coil are strongly reactive; small and large reactive granules are crowded in the reactive cytoplasm. 4. The cells of the secretory coil of the apocrine glands contain more beta-glucuronidase than any other cutaneous appendage. 5. In the sebaceous glands, a very strong concentration of enzyme activity is found in the undifferentiated peripheral cells, a smaller amount of it is found in the differentiating cells. 6. In active hair follicles, the largest amount of beta-glucuronidase is found in the outer root sheath and in the bulb. In the outer sheath, the strongest concentration is found around the level of the keratogenous zone of the cortex. The dermal papilla is strongly reactive. In quiescent hair follicles, the outer root sheath has a moderate amount of enzyme concentration, but the dermal papilla is unreactive. 7. In the dermis, the fibroblasts in the papillary layer, the smooth muscle cells of the arrectores pilorum and the tunica media of arteries, and the fat cells all exhibit enzyme activity. Mast cells show a great concentration of beta-glucuronidase.     

QUANTITATIVE ESTIMATION OF LYO- AND DESMOENZYMES IN TISSUE SECTIONS WITH AND WITHOUT FIXATION

1. Tissue sections eight microns thick were exposed to various experimental conditions used in histochemistry, and the effect upon the activities of esterase, the phosphatases, leucine aminopeptidase, β-glucuronidase, and arylsulfatase was determined colorimetrically. 2. Significant differences were found in the amounts of the lyo and desmo fractions of these enzymes. The desmo components were found to be for esterase, alkaline phosphatase, leucine aminopeptidase, acid phosphatase, β-glucuronidase, and arylsulfatase, ⅓, 2/3, 2/3, ½, ⅛, and ⅛ of the total enzymatic activity respectively. 3. Variations in the time and in the temperature at which diffusion was studied and of the pH and salt concentration of the solution into which the sections were placed, resulted in differences in the amount of enzymatic activity which remained in the tissue section. Some enzyme loss by diffusion was noted even after fixation of the tissue section. 4. The significance of the findings with respect to some of the concepts of localization of enzymes in tissue sections was discussed.     

HISTOLOGY AND CYTOCHEMISTRY OF HUMAN SKIN : IX. THE DISTRIBUTION OF NON-SPECIFIC ESTERASES

1. In the epidermis non-specific esterase activity outlines a strongly reactive band between the stratum granulosum and the stratum corneum. In the epidermis of the palm, there is no such esterase-rich band. 2. The outer sheath of active hair follicles has strong enzyme activity. The degenerating hair bulb in catagen follicles is very strongly reactive, and clusters of cells around the hair club in quiescent follicles are rich in enzyme activity. 3. Strong enzyme activity is found in young sebaceous cells, while decaying sebaceous cells and newly formed sebum are unreactive. Old sebum, however, is very intensely reactive. 4. Only the "dark" cells of eccrine sweat glands show a reaction; the "clear" cells are negative. 5. The cells of axillary apocrine glands abound in enzyme.     

Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-¹⁴C]glucosamine or l-[U-¹⁴C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.     

BETA GLUCURONIDASE-RICH CYTOPLASMIC PARTICLES IN ANDROGEN-STIMULATED MOUSE KIDNEY : A Cytobiochemical Study

Androgens produced by stimulating mouse testis with gonadotropic hormones cause a rise in renal β-glucuronidase but not an increase in acid or alkaline phosphatase. All subcellular components increase in β-glucuronidase activity, with a relatively greater increment in particulate enzyme as compared with that free in the cytoplasm (non-sedimentable). A small percentage of recovered β-glucuronidase, acid phosphatase, and alkaline phosphatase is found in material which rises to the surface during centrifugation in sucrose media (fraction I). The specific activity of β-glucuronidase and acid phosphatase in this fraction is normally quite high with respect to the homogenate, while that of alkaline phosphatase is not. On the other hand, the fraction I material from androgen-stimulated mice exhibits a further increase in specific activity with respect to β-glucuronidase and not acid phosphatase. It thus appears that there is an independence in the behavior of individual enzymes in response to physiologic stimuli in spite of obvious morphologic proximity.     

Histology and cytochemistry of human skin. IX. The distribution of non-specific esterases

1. In the epidermis non-specific esterase activity outlines a strongly reactive band between the stratum granulosum and the stratum corneum. In the epidermis of the palm, there is no such esterase-rich band. 2. The outer sheath of active hair follicles has strong enzyme activity. The degenerating hair bulb in catagen follicles is very strongly reactive, and clusters of cells around the hair club in quiescent follicles are rich in enzyme activity. 3. Strong enzyme activity is found in young sebaceous cells, while decaying sebaceous cells and newly formed sebum are unreactive. Old sebum, however, is very intensely reactive. 4. Only the "dark" cells of eccrine sweat glands show a reaction; the "clear" cells are negative. 5. The cells of axillary apocrine glands abound in enzyme.     

CCN2 modulates hair follicle cycling in mice

It is critical to understand how stem cell activity is regulated during regeneration. Hair follicles constitute an important model for organ regeneration because, throughout adult life, they undergo cyclical regeneration. Hair follicle stem cells—epithelial cells located in the follicle bulge—are activated by periodic β-catenin activity, which is regulated not only by epithelial-derived Wnt, but also, through as-yet-undefined mechanisms, the surrounding dermal microenvironment. The matricellular protein connective tissue growth factor (CCN2) is secreted into the microenvironment and acts as a multifunctional signaling modifier. In adult skin, CCN2 is largely absent but is unexpectedly restricted to the dermal papillae and outer root sheath. Deletion of CCN2 in dermal papillae and the outer root sheath results in a shortened telogen-phase length and elevated number of hair follicles. Recombinant CCN2 causes decreased β-catenin stability in keratinocytes. In vivo, loss of CCN2 results in elevated numbers of K15-positive epidermal stem cells that possess elevated β-catenin levels and β-catenin–dependent reporter gene expression. These results indicate that CCN2 expression by dermal papillae cells is a physiologically relevant suppressor of hair follicle formation by destabilization of β-catenin and suggest that CCN2 normally acts to maintain stem cell quiescence.     

Conditions Favorable for the Somatic Embryogenesis in Carrot Cell Culture Enhance Expression of the roIC Promoter-GUS Fusion Gene

We obtained carrot (Daucus carota) cells possessing the 5′-noncoding sequence of the ORF12 gene (roIC) of TL-DNA of the Ri plasmid and a structural gene of bacterial β-glucuronidase by Agrobacterium-mediated transformation. When such cells were cultured in medium containing 2,4-dichlorophenoxyacetic acid, substantial reduction in β-glucuronidase activity was observed. Upon transferring the cells from a 2,4-D-containing medium to one devoid of 2,4-dichlorophenoxyacetic acid, enhanced expression of β-glucuronidase in somatic embryo development was recorded. Activation by gibberillic acid and suppression by abscisic acid of β-glucuronidase activities, in concord with embryogenesis, were also noted.     

Apparent TRANS Control of Murine βglucuronidase Synthesis by a Temporal Genetic Element

A difference in the heat-inactivation kinetics between the β-glucuronidases of C3HeB/FeJ and C57Bl/6J mice was utilized to assess the mode of action of a temporal genetic element in controlling the expression of the β-glucuronidase structural gene Gus. The heat-inactivation kinetics of liver and kidney β-glucuronidase from F₁ C3HeB/FeJ x C57Bl/6J animals were intermediate with respect to the parental enzyme patterns, suggesting that equal concentrations of the two allelic products were present in β-glucuronidase tetramers of F₁ progeny. β-glucuronidase heteropolymers assembled in vivo under conditions where equal concentrations of the two structural alleles of the enzyme were known to be present also exhibited intermediate heat-inactivation kinetics. These observations are consistent with a trans mode of action of a genetic element that controls the rate of murine β-glucuronidase synthesis.     

Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.     

Tissue Distribution of beta-Cyanoalanine Synthase in Leaves

β-Cyanoalanine synthase, which catalyzes the reaction between cysteine and HCN to form β-cyanoalanine and H₂S, was assayed in leaf tissues from cyanogenic (Sorghum bicolor × Sorghum sudanense [sorghum]) and noncyanogenic (Pisum sativum [pea], Zea mays [maize], and Allium porrum [leek]) plants. The activity in whole leaf extracts ranged from 33 nanomoles per gram fresh weight per minute in leeks, to 1940 nanomoles per gram fresh weight per minute in sorghum. The specific activities of β-cyanoalanine synthase in epidermal protoplasts from maize and sorghum and in epidermal tissues from peas were in each case greater than the corresponding values for mesophyll protoplasts or tissues, or for strands of bundle sheath cells.

The tissue distributions for this enzyme were determined for pea, leek, and sorghum: the mesophyll protoplasts and tissues in these three plants contained 65% to 78% of the enzyme, while epidermal protoplasts and tissues contained 10% to 35% of the total leaf activity. In sorghum, the bundle sheath strands contained 13% of the leaf activity. The presence of β-cyanoalanine synthase in all tissues and species studied suggests a fundamental role for this enzyme in plant metabolism.

     

Metabolite Proofreading in Carnosine and Homocarnosine Synthesis: MOLECULAR IDENTIFICATION OF PM20D2 AS β-ALANYL-LYSINE DIPEPTIDASE*

Carnosine synthase is the ATP-dependent ligase responsible for carnosine (β-alanyl-histidine) and homocarnosine (γ-aminobutyryl-histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a “metabolite repair” enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic β-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with β-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine, and γ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on β-alanyl-arginine and γ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from β-alanine or γ-aminobutyrate, PM20D2 also acted at lower rates on some “classic dipeptides” like α-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was ∼100–200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (β-alanyl-lysine, β-alanyl-ornithine, γ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.     

Induction of transforming growth factor-beta 1 by androgen is mediated by reactive oxygen species in hair follicle dermal papilla cells

The progression of androgenetic alopecia is closely related to androgen-inducible transforming growth factor (TGF)-β1 secretion by hair follicle dermal papilla cells (DPCs) in bald scalp. Physiological levels of androgen exposure were reported to increase reactive oxygen species (ROS) generation. In this study, rat vibrissae dermal papilla cells (DP-6) transfected with androgen receptor showed increased ROS production following androgen treatment. We confirmed that TGF-β1 secretion is increased by androgen treatment in DP-6, whereas androgeninducible TGF-β1 was significantly suppressed by the ROSscavenger, N-acetyl cysteine. Therefore, we suggest that induction of TGF-β1 by androgen is mediated by ROS in hair follicle DPCs. [BMB Reports 2013; 46(9): 460-464]     

Host-Pathogen Interactions: II. Parameters Affecting Polysaccharide-degrading Enzyme Secretion by Colletotrichum lindemuthianum Grown in Culture

The effect of a number of physiological variables on the secretion of polysaccharide-degrading enzymes by culture-grown Colletotrichum lindemuthianum (Saccardo and Magnus) Scribner was determined. The number of spores used to inoculate cultures grown on isolated bean hypocotyl cell walls affects the time after inoculation at which enzyme secretion occurs, but has no significant effect on the maximal amount of enzyme ultimately secreted. Cell walls isolated from bean leaves, first internodes, or hypocotyls (susceptible to C. lindemuthianum infection), when used as carbon source for C. lindemuthianum growth, stimulate the fungus to secrete more α-galactosidase than do cell walls isolated from roots (resistant to infection). The concentration of carbon source used for fungal growth determines the final level of enzyme activity in the culture fluid. The level of enzyme secretion is not proportional to fungal growth; rather, enzyme secretion is induced. Maximal α-galactosidase activity in the culture medium is found when the concentration of cell walls used as carbon source is 1% or greater. A higher concentration of cell walls is necessary for maximal α-arabinosidase activity. Galactose, when used as the carbon source, stimulates α-galactosidase secretion but, at comparable concentrations, is less effective in doing so than are cell walls. Polysaccharide-degrading enzymes are secreted by C. lindemuthianum at different times during growth of the pathogen on isolated cell walls. Pectinase and α-arabinosidase are secreted first, followed by β-xylosidase and cellulase, then β-glucosidase, and, finally, α-galactosidase.     

The Primitive Endoderm Segregates from the Epiblast in β1 Integrin-Deficient Early Mouse Embryos

We analyzed the mechanism of developmental failure in implanted β1 integrin-null blastocysts and found that primitive endoderm cells are present but segregate away from, instead of forming an epithelial layer covering, the inner cell mass. This cell segregation phenotype was also reproduced in β1 integrin-null embryoid bodies, in which primitive endoderm cells segregated and appeared as miniature aggregates detached from the core spheroids, and a primitive endoderm layer failed to form on the surface. Restricted β1 integrin gene deletion in embryos using Ttr-Cre or Sox2-Cre indicated that the loss of integrin function in the cells of the inner core rather than the outer layer is responsible for the failure to form a primitive endoderm layer. We conclude that β1 integrin is essential for the attachment of the primitive endoderm layer to the epiblast during the formation of a basement membrane, a process concurrent with the transition from cadherin- to integrin-mediated cell adhesion.     

beta-Amylases in Cereals : A Study of the Maize beta-Amylase System

β-Amylase of maize (Zea mays L.) caryopses was studied during development and germination by means of enzymic, electrophoretic, and immunochemical techniques. β-Amylase activity increased during caryopsis development to a maximum value at the beginning of the water content plateau (at this stage the enzyme was located primarily within the pericarp) and then decreased. Almost no β-amylase (activity or antigen) was found in either free or bound forms in the mature maize caryopsis. The activity increased again during seedling growth and reached much higher values. Both the aleurone layer (to a major extent) and the scutellum produced and secreted β-amylase during germination, the secretion being stimulated by Ca²⁺. No posttranslational modification of the enzyme was detected during germination. The molecular specific activity of the enzyme remained unchanged during the observed periods, indicating that the regulation of the activity is based essentially on protein turnover. The enzyme from developing and germinating caryopses was found to be identical in terms of antigenicity, isoelectric point, and molecular mass to the β-amylases extracted from the roots and the leaves of the maize seedling. The maize β-amylase resembles in all respects the ubiquitous β-amylase described for rye and wheat, whereas the major β-amylase of those cereals appears to be lacking in the maize caryopsis.     

β-Glucan from Saccharomyces cerevisiae alleviates oxidative stress in LPS-stimulated RAW264.7 cells via Dectin-1/Nrf2/HO-1 signaling pathway

β-Glucan from Saccharomyces cerevisiae has been described to be effective antioxidants, but the specific antioxidation mechanism of β-glucan is unclear. The objectives of this research were to determine whether the β-glucan from Saccharomyces cerevisiae could regulate oxidative stress through the Dectin-1/Nrf2/HO-1 signaling pathway in lipopolysaccharides (LPS)-stimulated RAW264.7 cells. In this study, we examined the effects of β-glucan on the enzyme activity or production of oxidative stress indicators in LPS-stimulated RAW264.7 cells by biochemical analysis and the protein expression of key factors of Dectin-1/Nrf2/HO-1 signaling pathway by immunofluorescence and western blot. The biochemical analysis results showed that β-glucan increased the LPS-induced downregulation of enzyme activity of intracellular heme oxygenase (HO), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) while decreasing the production of reactive oxygen species (ROS) and malondialdehyde (MDA). Furthermore, immunofluorescence results showed that β-glucan can activate the nuclear factor erythroid 2-related factor 2 (Nrf2). The antioxidant mechanism study indicated that β-glucan activated dendritic-cell-associated C-type lectin 1 (Dectin-1) receptors mediated Nrf2/HO-1 signaling pathway, thereby downregulating the production of ROS and thus produced the antioxidant effects in LPS-stimulated RAW 264.7 cells. In conclusion, these results indicate that β-glucan potently alleviated oxidative stress via Dectin-1/Nrf2/HO-1 in LPS-stimulated RAW 264.7 cells.     

aguA, the Gene Encoding an Extracellular α-Glucuronidase from Aspergillus tubingensis, Is Specifically Induced on Xylose and Not on Glucuronic Acid

An extracellular α-glucuronidase was purified and characterized from a commercial Aspergillus preparation and from culture filtrate of Aspergillus tubingensis. The enzyme has a molecular mass of 107 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 112 kDa as determined by mass spectrometry, has a determined pI just below 5.2, and is stable at pH 6.0 for prolonged times. The pH optimum for the enzyme is between 4.5 and 6.0, and the temperature optimum is 70°C. The α-glucuronidase is active mainly on small substituted xylo-oligomers but is also able to release a small amount of 4-O-methylglucuronic acid from birchwood xylan. The enzyme acts synergistically with endoxylanases and β-xylosidase in the hydrolysis of xylan. The enzyme is N glycosylated and contains 14 putative N-glycosylation sites. The gene encoding this α-glucuronidase (aguA) was cloned from A. tubingensis. It consists of an open reading frame of 2,523 bp and contains no introns. The gene codes for a protein of 841 amino acids, containing a eukaryotic signal sequence of 20 amino acids. The mature protein has a predicted molecular mass of 91,790 Da and a calculated pI of 5.13. Multiple copies of the gene were introduced in A. tubingensis, and expression was studied in a highly overproducing transformant. The aguA gene was expressed on xylose, xylobiose, and xylan, similarly to genes encoding endoxylanases, suggesting a coordinate regulation of expression of xylanases and α-glucuronidase. Glucuronic acid did not induce the expression of aguA and also did not modulate the expression on xylose. Addition of glucose prevented expression of aguA on xylan but only reduced the expression on xylose.     

CHANGES IN "DROPLET" FRACTIONS FROM RAT KIDNEY CELLS AFTER INTRAPERITONEAL INJECTION OF EGG WHITE

1. Kidney homogenates from rats injected with egg white and from control rats were fractionated simultaneously into six fractions and the content of acid phosphatase, ribonuclease, desoxyribonuclease, cathepsin, and β-glucuronidase in corresponding fractions from treated and untreated animals was compared. These observations were correlated with the amount of dark brown bottom sediments in fractions NDrI, DrII, and DrIII, and with the number of droplets in fraction NDrI. 2. It was found that after injection of egg white the amount of small droplets decreased as indicated by the decrease of the dark brown bottom layer in the sediment of fraction DrIII and by the concomitant decrease of hydrolytic enzymes in the same fraction, and that the number of large droplets increased as indicated by the increase of brown sediment in fraction NDrI and the increase in the number of droplets counted in a bacterial counting chamber in the same fraction. It was concluded that the treatment with egg white induced the transformation of small droplets into large droplets. 3. The decrease of hydrolytic enzymes in the fractions containing the small droplets was accompanied by a marked increase of these enzymes in the supernatant fluid. The enzyme content of fraction NDrI was not increased after treatment, although it contained greatly increased numbers of large droplets. Counting of the droplets in this fraction showed decreased enzymatic activity of the average large droplet after treatment with egg white. It was suggested that during the transformation of small into large droplets, a portion of the hydrolytic enzymes was released into the surrounding cytoplasm, and that this was partly responsible for the increased enzyme content of the supernatant fluid after fractionation of the kidney homogenate. In contrast to the four other hydrolytic enzymes, β-glucuronidase was not increased in the supernatant fluid. 4. Eighteen hours after intraperitoneal injection of egg white, the specific enzymatic activities of kidney homogenates showed a 25 to 35 per cent increase for cathepsin, ribonuclease, and desoxyribonuclease, no change for acid phosphatase and β-glucuronidase, and approximately a 7 per cent decrease for cytochrome oxidase. The increase of cathepsin, ribonuclease, and desoxyribonuclease in the total homogenate was interpreted as an indication of the formation of new enzymes, and it was suggested that this partly accounted for the increase of these enzymes in the supernatant fluid. 5. The activation of the enzymes by osmotic effects was investigated in vitro by incubation of droplet fractions in the presence of different concentrations of sucrose.