Esterase. XXI. Es-9, a possibly new polymorphic esterase in Mus musculus genetically linked to Es-2

A so far undescribed gene controlling zone III esterases has been detected by means of disc gel electrophoresis of kidney homogenates from the two inbred mice strains NMRI and SK/Cam. The gene is tentatively designated Es-9, and the two codominant alleles are designated Es-9a and Es-9b. Es-9 esterases are present in many tissues, but, unlike the other zone III esterase (controlled by Es-5), are not found in the serum. Close linkage with the Es-2 gene leads us to map the Es-9 gene on chromosome 8.     

Esterase XXVII. Purification and characterization of esterase-9A of mouse kidney.

Esterase-9A, which appears electrophoretically as a triplet of the bands III-50, III-40 and III-30, was isolated from the kidneys of male NMRI-mice by isoelectrofocusing and refocusing followed by repeated molecular sieve chromography. The overall purification was approx. 250 fold and each of the three bands was isolated separately. The band of the triplet nearest to the cathode, III-50, changed in vitro into the satellite bands III-40 and III-30 and, further, into the band III-22 not observed before in the homogenate. It is assumed that the band III-50 represents the original gene product. The molecular weight (45 000) of the band III-50 is identical with those of III-40 and III-30, as measured by analytical electrophoresis, whereas the molecular weight obtained by thin-layer chromatography was 51 000. There were no obvious signs that esterase-9 was composed of subunits. The Km constant for 4-nitrophenyl proprionate was identical for each of three bands. The esterase-9A is the first testosterone-dependent isozyme of the mouse carboxylesterase (carboxylicester hydrolase, EC 3.1.1.1) system which has been isolated.     

Esterase XIX. Biochemical and ultrahistochemical investigations of the non specific esterase in nuclei from mouse liver.

The present investigations reveal that the non specific esterase from mouse liver nuclei is exclusively located in the perinuclear cistern and that the nuclear chromatin does not contain any esterase as a rule. However, esterase is associated with lipid droplets which are seldomly found within nuclei. Two different esterases are demonstrated with the employed substrates in the nuclear envelope. It is exemplified that the esterase, found in isolated nuclei, can neither be understood as qualitative nor as quantitative equivalent of the esterase demonstrated ultrahistochemically at nuclear membranes.     

Serum esterase genetics: Identification and hormone induction of the Es-1b esterase in inbred rats

A previously unrecognized esterase from the sera of the appropriate strains of the rat Rattus norvegicus was revealed by a discontinuous polyacrylamide gel electrophoretic technique. This esterase migrated in the albumin region, whereas a previously known major albumin esterase controlled by the Es-2 locus migrated in the postalbumin region when the method was used. The new albumin esterase component which separated from the Es-2 esterase was identified as the product of the Es-1b gene. The new albumin esterase was not detectable in the sera of sexually mature males of the appropriate genotype, because the activity level of this esterase was influenced by sex hormones, especially androgen.     

Es-6, a further polymorphic esterase in the rat

A further polymorphic rat esterase with broad tissue expression and restricted substrate specificity is described and tentatively called Es-6. Inbred rat strains have either fixed allele Es-6^F or fixed allele Es-6^S. Es-6 is not linked to the established esterase cluster consisting of the eight esterase loci Es-1, Es-2, Es-3M, Es-4M, Es-4W, Es-5 (=Es-3W), Es-7, and Es-8 in LG V of the rat or to RT1, Gc, c, a, and h. Esterases with apparently identical biochemical and genetical characteristics are Es-17 of the mouse and Es-A4 of humans.Supported by the Deutsche Forschungsgemeinschaft (Be 352/13 and Gu 105).     

The subcellular distribution of poly-A-degrading activity in mouse kidney.

Most (95%) of the poly-A-degrading activity of the mouse kidney was found in the cytoplasmic fraction and only 5% was found in the nuclear fraction; 43% of the poly-A-degrading activity of the cytoplasm was found in the mitochondria, 22% in the microsomes, and 30% in the soluble fraction. Differences in activity and specificity indicate that poly A is degraded in the nucleus by enzymes that are separate and distinct from the enzymes in the cytoplasm that degrade poly A. The nuclear poly-A-degrading activity can be separated into an endonuclease with a general specificity and exonuclease, similar to one found in Ehrlich ascites tumor cells, which shows some specificity for poly A.     

Esterase 13, a new mouse esterase locus with recessive expression and its genetic location on chromosome 9

A new esterase locus (Es-13) has been identified in Mus musculus. Strains AEJ/GnRk, LG/J, SJL/J, and SWR/J carry a recessive allele, Es-13 ^b, for a locus possibly involved in the posttranslational modification of a kidney esterase. All other strains observed carried the dominant Es-13 ^a allele. Es-13 was mapped on Chr 9 by recombinant inbred lines and by conventional backcrossing experiments. Backcross data produced the following gene order and map distances: Lap-1 (31.6±7.5 cM) Es-13 (2.6±2.6 cM) Mod-1.     

Esterase-16 (Es-16): Characterization,polymorphism, and linkage to chromosome 3 of a kidney esterase locus of the house mouse

A polymorphism for an isozyme of a presumed arylesterase, esterase-16 (EC 3.1.1.2), has been detected in kidney, heart, and spleen of the house mouse, Mus musculus, by means of isoelectric focusing and by disc electrophoresis. Three phenotypes can be distinguished: the ES-16A phenotype (IEP 5.9) was found in C57BL/10Sn and many other laboratory inbred strains; the ES-16B phenotype (IEP 6.1) was found in M. m. molossinus; and the ES-16C phenotype (IEP 5.9; very weak activity) was found in Peru-Coppock. Esterase-16 is strongly inhibited by 10^?3 m p-chloromercuribenzoate, but not by 2·10^?4 m bis-p-nitrophenyl phosphate or by 10^?3 m Diamox. It stains well with indoxyl acetate and other indigogenic substrates but only weakly with α-naphthyl acetate. Esterase-16 is completely insoluble in water. It is apparently governed by a structural gene locus, Es-16, with three alleles, Es-16 ^a , Es-16^b, and Es-16 ^c, respectively. Es-16 is closely linked to Car-1 and Car-2 on chromosome 3. Typing of 94 animals of the backcross (C57BL/10Sn × M. m. mol.) F₁ × M. m. mol. revealed a recombination frequency of 8.51±2.9%.     

Cellular and subcellular localization of uncoupling protein 2 in the human kidney

The uncoupling protein-2 (UCP2) is an anion transporter that plays a key role in the control of intracellular oxidative stress. In animal models UCP2 downregulation has several pathological sequelae, particularly affecting the vasculature and the kidney. Specifically, in these models kidney damage is highly favored in the absence of UCP2 in the context of experimental hypertension. Confirmations of these data in humans awaits further information, as no data are yet available concerning the cell-type and subcellular expression in the human kidney. In the present study, we aimed to characterize the UCP2 protein distribution in human kidney biopsies. In humans UCP2 is mainly localized in proximal convoluted tubule cells, with an intracytoplasmic punctate staining. UCP2 positive puncta are often localized at the interface between the endoplasmic reticulum and the mitochondria. Glomerular structures do not express UCP2 at detectable levels. The expression of UCP2 in proximal tubular cells may explain their relative propensity to damage in pathological conditions including the hypertensive disease.     

Fibre-type distribution and subcellular localisation of alpha and beta enolase in mouse striated muscle

Enolase is a dimeric glycolytic enzyme exhibiting tissue specific isoforms. During ontogenesis, a transition occurs from the embryonic alphaalpha towards the specific alphabeta, and betabeta isoforms in striated muscle. Immunocytochemical analyses on transverse sections of adult mouse gastrocnemius muscle, allowed us to compare the expression of alpha and beta subunits to that of myosin heavy chain (MHC) isoforms. Levels of beta immunoreactivity followed the order IIB > IIX > IIA > I. This gradient parallels the ATPase activity associated to MHC isoforms, indicating that the expression of beta enolase in myofibres is finely regulated as a function of energetic requirements. By contrast, variations in alpha immunolabelling intensity appeared independent of fibre types. Longitudinal muscle sections exhibited a striated pattern of alpha immunoreactivity. Confocal microscopy analyses demonstrated that alpha was localised at the M band. Most beta immunoreactivity was diffuse all over the sarcoplasm. However, some beta immunoreactivity was striated and localized at both Z and M bands. Thus, betabeta enolase could participate to multi-enzyme complexes present at the I band, and involved with local ATP production. Our results support the notion that isozymes differ in their ability to interact with other macromolecules, thus segregating to different subcellular sites where they would respond to specific functional demands.     

A new esterase locus, Es-19, in the rat]

A new esterase polymorphism was identified in epididymal homogenates from inbred rat strains by polyacrylamide gel electrophoresis. The inbred rat strains showed either fast (A) or slow (B) bands. Strain distributions of the phenotypes differed from those of other esterase loci. Genetic analyses revealed that the polymorphism is controlled by codominant alleles (Es-19a and Es-19b) and is not linked to linkage groups, I, II, IV, V, VI, XIII of the rat.     

Mapping of nucleoside phosphorylase (Np-1) and esterase 10 (Es-10) on mouse chromosome 14

A method for detecting two alleles at Np-1 (nucleoside phosphorylase) and three alleles at Es-10 (esterase 10) from mouse blood by cellulose acetate electrophoresis is described. The allelic constitution at these loci for 44 inbred strains and stocks was determined. The location of Np-1 on chromosome 14 was established by backcross experiments in which alleles at Np-1 and Robertsonian translocations were segregating. Es-10 was shown to be linked to Np-1, and the following genetic map of Chr 14 was constructed: centromere-(8.9±4.0 cM)-[Np-1, Wc]-(10.2±1.9 cM)-Es-10-(15.5±3.7 cM)-s. The homologous human loci, NP and ES-D, are not linked.This work was supported by Contract E(11-1)-3267 with the Energy Research and Development Administration, by Contracts NO1-ES4-2156 and NO1-ES4-2159 with the National Institute of Environmental Health Sciences, and by Grants GM 19656 and GM 20919 from the National Institute of General Medical Sciences. D. A. K. was a participant in the 1975 Summer Program for College, Graduate, and Medical Students, which was supported, in part, by the Clark Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Protein domains, catalytic activity, and subcellular distribution of mouse NTE-related esterase

A mammalian family of lipid hydrolases, designated “patatin-like phospholipase domain containing (PNPLA)” recently has attracted attention. NTE-related esterase (NRE) as a member of PNPLA is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE). Mouse NRE (mNRE) has a predicted amino-terminal transmembrane region (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. In the current study, we described the expression of green fluorescent protein (GFP)-tagged constructs of mNRE and mutant proteins lacking the specific protein domains. Esterase assays indicated that neither the TM nor R-domain was essential for mNRE esterase activity, but the TM significantly contributed to its activity. Subcellular distribution showed that mNRE was anchored in ER via its TM domain and that its C-domain was associated with ER. Furthermore, experiments involving proteinase treatment revealed that most of mNRE molecule was exposed on the cytoplasmic face of ER membranes. Collectively, our results for the first time revealed the protein domains, catalytic activity, and subcellular location of mNRE and a simplified model for mNRE was proposed.     

Expression,subcellular localisation,and possible roles of dipeptidyl peptidase 9 (DPP9) in murine macrophages

Dipeptidyl peptidase 9 (DPP9) is a peptidase of the DPPIV gene family, and its role in immune responses has been reported. In this study, we compared the messenger RNA expression profile of DPP9 to that of the related DPP8 and DPPIV in murine haematopoietic and lymphatic tissues. A similar order of expression levels was observed for all 3 peptidases: peritoneal macrophages < bone marrow < spleen ≤ lymph nodes. Also, we examined the subcellular localisation of DPP9 and its possible role(s) in J774 cell line of macrophage origin. DPP9 was dominantly expressed intracellularly. DPPIV‐like enzymatic activity was mostly present in cytoplasm, but also in cell membranes and organelles/vesicles. Decreased expression of DPP9 was observed upon activation of J774 cells by combined treatment with interferon gamma and lipopolysaccharide. Changes induced by DPP9 gene silencing in J774 cells suggest possible role of DPP9 in regulation of proliferation and activation status. The colocalisation of DPP9 with endocytosed DQ‐OVA demonstrated in endosomes of J774 cells might suggest the role of DPP9 in peptide processing within endosomal/vesicular compartment.     

Effects of delta9-tetrahydrocannabinol on ATPases in mouse brain subcellular fractions.

The effect of (?)-Δ⁹-THC on the activities of Mg²⁺?, Na⁺?K⁺? and Mg²⁺Ca²⁺-ATPases were studied in mouse brain subcellular fractions. $\text{In vitro}$treatment with Δ⁹-THC produced a dose dependent stimulation of Mg²⁺ ATPase in the crude mitochondrial fraction and its subfractions and a dose-related inhibition of this activity in the microsomal fraction. Na⁺-K⁺- and Mg²⁺-Ca²⁺-ATPase activities were inhibited in a dose-related manner in all subcellular fractions studied.