Dual localization of β-glucuronidase and acid phosphatase in lysosomes and in microsomes

Summary The dual localization of certain hydrolases in lysosomes and in endoplasmic reticulum as studied in enzyme staining reactions is now supported by cytobiochemical studies on mouse liver and kidney -glucuronidase and acid phosphatase. Use was made of the renal -glucuronidase response to endogenous androgen for both studies. Accordingly, sucrose homogenates were prepared of liver and kidney of male BALB/C mice previously injected with gonadotrophin along with control animals receiving saline instead. The homogenates were subjected to differential ultracentrifugation yielding six fractions. These were characterized as to their organelle composition by measurements of marker enzymes and by observations with the electron microscope. In all subcellular fractions, -glucuronidase was uniformly increased 5 to 8 times over the corresponding control value and, in fractions rich in lysosomes, this enzyme was easily released by alternate freezing and thawing. On the other hand, the microsomal -glucuronidase and acid phosphatase enzymes were not liberated by freezing and thawing nor were they after treatment with 0.1 % Triton X-100 and by employing other reagents and conditions which are known to release lysosomal enzymes. In contrast to microsomal acid phosphatase, microsomal -glucuronidase activity could be liberated by treatment with hyaluronidase. This soluble -glucuronidase showed the same optimum pH, Michaelis Constant and heat inactivation behavior as the lysosomal -glucuronidase prepared by freezing and thawing treatment. These observations define two populations of microsomal vesicles each identifiable by an individual membrane-associated acid hydrolase. One of these -glucuronidase, increases in specific activity in the animal on androgens and is released by hyaluronidase and the other, acid phosphatase, does not respond to androgen and is not released by hyaluronidase. There would appear to be a variety of mechanisms by which hydrolases enter into association with the membranes of the endoplasmic reticulum and from there, a variety of routes to the lysosomes. A comment is made concerning the question of acid phosphatases and -glucuronidase as enzyme markers for lysosomes in mouse kidney.Aided in part by Research Grant, P-106, of the American Cancer Society, Inc., New York, and by U.S.P.H.S. Grant CA-07538 and by a Research Career Award, CA-K6-18453 to William H. Fishman.     

Multiple forms of β-glucuronidase in rat liver lysosomes and microsomes

Multiple forms of β-glucuronidase have been demonstrated using sucrose gradient and polyacrylamide gel isoelectric focusing techniques in 6 m urea. Microsomal β-glucuronidase, a membrane-bound enzyme, was solubilized from lysosome-free, Ca²⁺-precipitated microsomes by detergents and isolated by chromatography on columns of rabbit anti-rat preputial gland β-glucuronidase antibody bound to Sepharose. The enzyme has a pI of 6.7. Polyacrylamide gel isoelectric focusing resolves the microsomal enzyme into three components, each of which is protease sensitive. The protease-modified microsomal enzyme is very similar to several forms of β-glucuronidase in lysosomes. The lysosomal β-glucuronidase, isolated from osmotically shocked lysosomes, is very heterogeneous after isoelectric focusing over the range pI 5.4–6.0. The lysosomal enzyme can be resolved into 10–12 bands by polyacrylamide gel isoelectric focusing. The more acid forms of the lysosomal enzyme are neuraminidase sensitive, suggesting they may be sialoglycoproteins.     

Immunohistochemical localization of 14 kDa β-galactoside-binding lectin in various organs of rat

Summary Immunohistochemical localization of 14 kDa -galactoside-binding lectin in various organs of adult rat was achieved using a monospecific antibody raised against lectin purified from rat lung. The antibody-stained cells were formed into small aggregates, thin fascicles, or thick bundles in the walls of blood vessels, gastrointestinal tracts and urogenital organs. From the patterns of distribution, as well as their organization, these immunoreactive cells were regarded as smooth muscle cells. This was confirmed by a double immunofluorescence study using a mixture of anti 14 kDa lectin and anti -smooth muscle-specific actin antibodies. Strong 14 kDa lectin immunoreactivity was seen in the pericellular matrix of smooth muscle cells in intact organs as well as in detergent-treated organs from which all cellular components were extracted. From these findings, it is suggested that the 14 kDa lectin may be externalized by smooth muscle cells into their pericellular matrix and participate in the crosslinking of the complementary glycoconjugate(s) localized at that site. The macromolecular complex of glycoconjugates thus formed around smooth muscle cells may play a role in anchoring smooth muscle cells to the pericellular connective tissue thereby permitting the force of muscle contraction to be efficiently transmitted to the surrounding connective tissue proper.     

The localization of the β-subtype of protein kinase C (PKC-β) in rat sympathetic neurons

Summary The localization of PKC- was studied in rat sympathetic neurons using a polyclonal antibody specific for the ₁- and ₂-subspecies. The tissues studied included the superior cervical (SCG) and hypogastric (HGG) ganglia and the target tissues of the SCG and HGG neurons: the submandibular gland, iris, prostate and vas deferens. PKC--LI was found in nerve fibers in both ganglia. A proportion of the fibers in the SCG disappeared after decentralization, suggesting that the fibers were of both pre- and postganglionic origin. The somata of the HGG and SCG neurons expressed varying amounts of PKC--LI, the majority of SCG neurons being labelled only after colchicine treatment. In all target tissues there were PKC--immunoreactive nerve fibers in bundles, but the most peripheral branches of the fibers were negatively labelled. The results show that PKC--LI is widely present in sympathetic postganglionic neurons with mainly quantitative differences. The lack of PKC- in the most peripheral branches of nerve fibers might be a general feature of sympathetic postganglionic neurons, suggesting that the participation of PKC- in neurotransmitter release and in other functions in nerve terminals in sympathetic adrenergic neurons is unlikely.     

The dual location of β-oxidation enzymes in germinating pea cotyledons

-Oxidation enzymes were detected both in the mitochondria and microbodies of pea cotyledons. Intact mitochondria did not show -oxidation enzyme activity but in ruptured mitochondria this activity was high. It is apparent that the mitochondrial membrane barrier prevents rapid access of acyl-CoA substrates to matrix -oxidation sites. Removal of the membrane barrier permits rapid access of acyl-CoAs and these enzyme activities may then be measured.     

The expression of β-glucuronidase during preimplantation development of mouse embryos

β-Glucuronidase activity was measured in mouse embryos during the preimplantation period of development by using a microfluorometric assay. A 100-fold increase in activity was observed between 57 (8-cell stage) and 84 hr (morulae) of development. Activity changes between 30 and 60 hr were also significant. Genetic variants of β-glucuronidase occur between the strains of mice $\text{C57BL}\text{6J}$ and $\text{C3H}\text{HeJ}$ which differ in levels of activity and heat denaturation kinetics. Activity changes and heat denaturation kinetics of β-glucuronidase in $\text{C57BL}\text{6}\text{,}\text{C3H}\text{HeJ}$ and F₁ hybrid embryos were compared, and it was demonstrated that paternal genes were expressed during the 100-fold increase in activity and that embryonic genes may be functioning between 30 and 60 hr of development.     

Cloning of a cDNA complementary to rat preputial gland β-glucuronidase mRNA

Messenger RNA was isolated from rat preputial glands by guanidine HCl extraction, ethanol and salt precipitation, followed by chromatography on oligo(dT) cellulose. Double-stranded cDNA was synthesized from the mRNA and inserted into the Pst 1 site of the plasmid pBR322 by the poly(dG)·poly(dC) tailing and annealing procedure. The hybrid plasmids were used to transform $\text{E. coli}$ HB101. Recombinant clones were screened for those containing cDNA inserts complementary to β-glucuronidase mRNA by a hybridization-selection procedure. One clone, containing an insert of about 1.2 kilobases, hybridized to preputial gland mRNA which, when translated $\text{in vitro}$, gave a product that migrated with the β-glucuronidase subunit on polyacrylamide gels.     

Sudan-β-d-glucuronides and their use for the histochemical localization of β-glucuronidase activity in transgenic plants

 Synthesis of five different Sudan-β-d-glucuronides (I, II, III, IV, and RedB) was performed by condensation of a set of red Sudan diazo dyes with methyl (1-deoxy-2,3,4-tri-O-acetyl-1-trichloroacetimidoyl-α-d-glucopyran)uronate. After the acid and alcohol groups had been deprotected, the resulting compounds were used for histochemical localization of β-glucuronidase (GUS) activity in transgenic plants (Petunia hybrida, Arabidopsis thaliana, and Nicotiana tabacum) that contained the GUS reporter system. Because the cleavage of the β-glucuronide results in the liberation of an insoluble Sudan dye, Sudan substrates gave no diffusion artifacts as described for the commonly used 5-bromo-4-chloro-3-indolyl-β-d-glucuronide (X-gluc). A comparison of assays with different Sudan glucuronides and X-gluc demonstrated that the SudanIV variant is a valuable glucuronide substrate for the precise histochemical localization of GUS activity in transgenic plants. Received: 9 December 1999 / Revision received: 25 January 2000 / Accepted: 26 January 2000     

Ultrastructural localization of amyloid β/A4 protein precursor in the normal rat brain

The ultrastructural localization of amyloidβ/ A4 protein precursor (APP) was studied immunohistochemically in normal rat brains using antibodies against different portions of APP. In cerebral cortical neurons and Purkinje cells, APP reaction products were located in the cytoplasm and on cell surface membranes. Some Golgi apparatuses and rough endoplasmic reticulum also showed APP immunoreactivity on their membranes and some vesicles near the trans face of the Golgi apparatuses were stained. In the neuropil of the cerebral cortex and the cerebellar molecular layer, many cell processes, which surrounded synapses and were considered to be astrocytic, were APP-positive. Foot processes around capillaries and subpial astrocytic processes were also immuno-positive. At the ultrastructural level, APP-positive astrocytic processes were identified.     

Detection of endogenous β-glucuronidase activity in Aspergillus niger

 An endogenous β-glucuronidase that hydrolyses the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc) in Aspergillus niger is reported. The activity was induced when the fungus was grown in media containing xylan, but was either very low, or absent, when grown on glucose. Endogenous β-glucuronidase was primarily located in newly formed hyphae, and was apparent at pH values between 3 and 6. Hydrolysis of X-gluc was sensitive to the inhibitor D-saccharic acid 1,4-lactone and was irreversibly inactivated by heating. The bacterial uidAβ-glucuronidase reporter gene was strongly expressed in the hyphae of transformed A. niger but, in contrast to the endogenous activity, the enzyme was also active at pH 7–8.5. Histochemical localization of uidA expression in A. niger, without interference from the endogenous β-glucuronidase activity, was achieved by staining at this pH. Received : 22 March 1995/Received last revision : 17 August 1995/Accepted : 22 August 1995     

Immunohistochemical localization of β1-adrenergic receptors in the liver of male and female F344 rat

The distribution of beta(1)-adrenergic receptors in the liver of Fischer 344 (F344) rat has been examined by an immunohistochemical method. The study was carried out on formalin-fixed and paraffin-embedded livers from young adult, middle-aged, and old female and male F344 rats. An antibody specific for the beta(1)-adrenoreceptor subtype was used. A positive reaction was found in the liver parenchyma of female and male rats from all age groups. Within the liver lobule, a clear zonation is observed, with the beta(1)-adrenoreceptor positivity most evident in pericentral zone hepatocytes and a gradual fading of the immunostaining from pericentral to periportal zone hepatocytes, which may be completely negative. Immunoreactivity is localized on the cell membrane and on the membrane of peripheral cytoplasmic vesicles, and is mostly confined to the cell side facing vascular space. The intensity of immunostaining seems to be slightly higher in the 6- and 10-month-old female rats as compared to the matched male rats and to the senescent female rats. No age-related changes in the intensity of immunostaining are appreciable in male rats. However, no definite conclusion could be drawn about the existence of gender-related differences or age-related changes in the density of beta(1)-adrenoreceptors. A low density of beta1-adrenoreceptor was observed in the spontaneous preneoplastic lesions of the livers from senescent rats.     

The locations of cathepsin activity and β-glucuronidase in the Guerin T8 tumour

Tumour homogenate fractions, isolated by differential centrifugation, were subfractionated by density-gradient centrifugation. Biochemical and electron microscopic analyses revealed that beta-glucuronidase and cathepsin activity were associated with a class (possibly two) of lysosomal particles of density greater than those of mitochondria and the endoplasmic reticulum. Lysosomes sedimented by low g forces were vacuolar, electron-dense, delineated by a unit membrane and about 0.2mum in diameter. beta-Glucuronidase was also apparently associated with ribosomes whereas cathepsin was bound in part to the endoplasmic reticulum. Catalase and glucose 6-phosphatase possessed slightly different density-gradient sedimentation profiles.     

The synthesis and processing of β-glucuronidase in normal and egasyn deficient mouse kidney

The presence of a precursor form of β-glucuronidase, with a subunit molecular weight of 75,000 was demonstrated in mouse kidney. This was later processed to the mature form, with subunit molecular weight of 71,500. Tissue fractionation revealed that the precursor was associated with the microsomes whereas the mature form was associated with the lysosomes. In mice lacking egasyn both forms of β-glucuronidase were present, but the rate of processing was elevated compared to normal.     

Progressive induction of β-glucuronidase in individual kidney epithelial cells

The magnitude and kinetics of β-glucuronidase induction in mouse kidney are determined by a cis-acting regulatory gene, Gus-r, that is closely linked to the enzyme structural gene. The accumulation of β-glucuronidase mRNA during induction is much slower than the turnover time of the mRNA, suggesting progressive acquisition of mRNA synthesizing capacity during induction. Counts of the numbers of induced cells present at various times of induction in strains carrying three different alleles of Gus-r show that all potentially responsive cells respond immediately. The level of induction is progressive in individual cells and does not involve continued recruitment of new cells into the induced population. It appears that during induction each chromosome becomes progressively more active in directing the synthesis of β-glucuronidase.     

Roles of β-catenin in somitogenesis in rat embryos

Summary We studied the roles of β-catenin in somitogenesis using immunostaining and antisense experiments in rat embryos. High levels of β-catenin appeared transiently in the developing rat somites. Initially, β-catenin accumulation was observed in the core cells of presomitic cell aggregates and then in the lumen of epithelial vesicles. Subsequently, it was confined to the dermomyotomes and their lumen and then the myotomes. High levels of cyclin D1 were observed in the core cells, in the lumen of epithelial vesicles, in myotomes, and in mesenchymal sclerotomes. When embryos were cultured in medium supplemented with β-catenin antisense oligodeoxynucleotide (ODN), the accumulation of β-catenin, but not of cyclin D1, in the nascent somites and dermomyotomes was suppressed, while the number of somites was the same as that observed in control embryos. The number of myosin-positive somites and the amount of myosin per somite in embryos treated with the antisense ODN were lower than those in controls. These results suggested that β-catenin promotes development of myotomal cells during somitogenesis. The function of β-catenin in the development of myotomes may not be correlated to cyclin D1.     

Histochemical localization of β-glycosidases in roots ofZea mays

Summary A simultaneous coupling azo dye technique using 6-bromo-2-naphthyl-D-glucoside as substrate has been developed for the localization of-glucosidase in hand sections of plant root tissue. This technique is also applicable to-galactosidase localization. Using corn root hairs and epidermal cells as an experimental system, the localization obtained in simultaneous coupling has been compared with that obtained in the older post-coupling method, and in various control sections. It is concluded that the localization obtained in root hairs in the post-coupling method is an artifact produced by diffusion of the product before coupling to substantive non-enzymatic sites in the cytoplasm.     

Immunohistochemical localization of thymosin β9 in bovine tissues

Summary Using an indirect fluorescent antibody technique with frozen sections, the localization of thymosin ₉ was investigated for the first time in bovine thymus, spleen, lung, muscle and liver. The antibodies used have been raised against the N-terminal fragment 1–14 of thymosin ₉ in order to minimize the cross-reactivity with thymosin ₄ which was found to be also present in bovine tissues. The specific antibodies against thymosin ₉ raised in our laboratory allowed us to localize this peptide in presence of the highly homologous and always accompanying thymosin ₄ in different tissues. Although thymosin ₉ was first isolated from calf thymus, it could be also detected in other bovine organs. The highest density of positive immunoreaction was found to be in spleen sections. In the muscle tissue a pronounced fluorescence intensity was present in the region of the sarcolemn.     

Histochemical localization of β-glycosidases in roots ofZea mays

Summary With 6-bromo-2-naphthyl--D-glucoside as substrate, -glucosidase activity has been histochemically localized in the lateral root meristems ofZea mays. Enzyme activity is highest in the epidermis and root cap of the young primordium. In contrast, enzyme activity is low in the parent root cortex, including the damaged areas around the lateral root. The results indicate that -glucosidase is involved in the development of the lateral root rather than its penetration through the cortex.