Immunocytochemical localization of lysosomal acid phosphatase in normal and "I-cell" fibroblasts

This study represents the first example of immunological localization of lysosomal acid phosphatase. The intracellular localization of lysosomal acid phosphatase was investigated with immunocytochemical methods at the light and electron microscopical level in cultured fibroblasts obtained from normal subjects and from a patient with I-cell disease. Double-labeling studies using fluorescence microscopy showed that acid phosphatase is present in the same organelles as other hydrolases. At the electron microscopic level in control fibroblasts acid phosphatase was found in the rough endoplasmic reticulum, lysosomes, at the plasma membrane, in vesicles just below the plasma membrane and in multivesicular bodies. This localization was comparable with that of other lysosomal enzymes tested (acid alpha-glucosidase, N-acetyl-beta-hexosaminidase, beta-galactosidase). Acid phosphatase labeling was mainly found in association with the lysosomal membrane and with membranous material present within the lysosome. In I-cell fibroblasts the label was present in the same subcellular organelles but always associated with membranous structures. We suggest that the association of acid phosphatase with membranes might explain the normal enzyme activity found in I-cell fibroblasts.     

Affinity cytochemical differentiation of glycoconjugates of small intestinal absorptive cells using Pisum sativum and Lens culinaris lectins

We studied the subcellular localization of glycoconjugates recognized by the garden pea and lentil lectins (Pisum sativum, PSA; Lens culinaris, LCA) in mature absorptive cells of duodenum and jejunum of fasted rats. PSA and LCA are mannose-, glucose-, and N-acetyl-glucosamine-recognizing lectins that bind with high affinity to fucosylated core regions of N-glycosidically linked glycans. The binding reactions were cytochemically demonstrated in a pre-embedment incubation system using peroxidase-labeled lectins. Both pea and lentil lectins bound with constituents of nuclear envelope and endoplasmic reticulum, cisternae of the Golgi apparatus, several Golgi-associated vesicles, lysosomes, and portions of the plasma membrane. PSA and LCA label was non-homogeneous in the endoplasmic reticulum; in the Golgi apparatus the reactions were most intense in the cis and medial cisternae of the stacks. For inhibition of the intense reactions apparent in the Golgi apparatus, in lysosomes, and at the plasma membrane, considerably higher concentrations of competitive sugars were necessary than for abolition of the endoplasmic reticulum label. This indicates that endoplasmic reticulum glycoconjugates bind at low affinities with pea and lentil lectins, and that high-affinity PSA/LCA-binding glycoconjugates, which may correspond to corefucosylated N-linked glycans, predominate in cis and medial Golgi cisternae, lysosomes, and at the plasma membrane.     

Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation

Neutrophil chemotaxis, phagocytosis, and oxygen-dependent microbicidal activity are initiated by interactions of stimuli with the plasma membrane. However, difficulties in neutrophil plasma membrane isolation have precluded studies on the precise structure or function of this cellular component. In this paper, a method is described for the isolation of representative human neutrophil plasma membrane vesicles, using nitrogen cavitation for cell disruption and a combination of differential centrifugation and equilibrium ultracentrifugation in Dextran gradients for membrane fractionation. Multiple biochemical markers and galactose oxidase-tritiated sodium borohydride surface labeling were employed to follow the yield, purity, and distribution of plasma membranes, nuclei, lysosomes, endoplasmic reticulum, mitochondria, and cytosol. According to these markers, neutrophil plasma membranes were exposed to minimal lysosomal hydrolytic enzymes and could be isolated free of other subcellular organelles. In contrast, disruption of neutrophils by mechanical homogenization resulted in > 20% lysosomal rupture and significant plasma membrane proteolysis. Electron microscopy demonstrated that plasma membranes isolated after nitrogen cavitation appeared to be sealed vesicles with striking homogeneity.     

Ultrastructural immunolocalization of apolipoprotein B within human jejunal absorptive cells

Apolipoprotein B (apoB) was localized by electron microscopy within absorptive cells of human jejunal biopsy specimens taken fasting and after micellar fat infusion. Nakane's double antibody immunoperoxidase technique was used to label apoB near open cut surfaces of 60-Micrometers fixed tissue slices sectioned by a Ralph knife in a Vibratome. In fasting tissue, apoB label was found within structurally intact peri-mitochondrial rough endoplasmic reticulum (RER) and within Golgi cisternae of absorptive cells covering the tips of jejunal villi. After fat infusion, apoB label was found adjacent to very low density lipoproteins (VLDL) and chylomicrons within apical smooth endoplasmic reticulum (SER). Less label was seen within RER than in fasting absorptive cells, and RER-SER connections containing apoB label were occasionally seen. Expanded Golgi vesicles and cisternae contained VLDL, chylomicrons, and apoB label. Vesicles containing chylomicrons and apoB label were occasionally visualized bordering the lateral plasma membrane in a configuration suggesting exocytosis. Specific apoB label was regularly seen within intercellular spaces and capillaries, but the in vivo significance of this Localization was problematical. These observations suggest that apoB is synthesized in RER, transfers to SER where it is incorporated into new VLDL and chylomicrons, and moves to Golgi cisternae and vesicles to be prepared for exocytosis through the plasma membrane.     

Association of isolated bovine kidney cortex peroxisomes with endoplasmic reticulum

Close lateral membrane associations of peroxisomes with endoplasmic reticulum are a common feature in bovine kidney cortex epithelial cells. Isolated highly purified peroxisome preparations from this tissue showed a remarkable and persistent copurification of peroxisomal marker enzymes with small amounts (5%) of the microsomal reference enzymes esterase and glucose-6-phosphatase. Contamination with mitochondrial and lysosomal markers was negligible. Ultrastructural examination of such preparations revealed a peculiar association of vesicles or short tubular segments with the peroxisomal membrane. Short electron dense crossbridges seemed to maintain their structural association. The cytochemical localization of glucose-6-phosphatase in peroxisome-associated membrane structures confirmed their derivation from endoplasmic reticulum. The metabolic significance of such structural peroxisome-endoplasmic reticulum associations is discussed.     

Localization of thyroglobulin antigenicity in rat thyroid sections using antibodies labled with peroxidase or (125)I-radioiodine

In the hope of localizing thyroglobulin within focullar cells of the thyroid gland, antibodies raised against rat thyroglobulin were labeled with the enzyme horseradish peroxidase or with (125)I-radioiodine. Sections of rat thyroids fixed in glutaraldehyde and embedded in glycol methacrylate or Araldite were placed in contact with the labeled antibodies. The sites of antibody binding were detected by diaminobenzidine staining in the case of peroxidase labeling, and radioautography in the case of 125(I) labeling. Peroxidase labeling revealed that the antibodies were bound by the luminal colloid of the thyroid follicles and, within focullar cells, by colloid droplets, condensing vacuoles, and apical vesicles. (125)I labeling confirmed these findings, and revealed some binding of antibodies within Golgi saccules and rough endoplasmic reticulum. This method provides a visually less distinct distribution than peroxidase labeling, but it allowed ready quantitation of the reactions by counts of silver grains in the radioautographs. The counts revealed that the concentration of label was similar in the luminal colloid of different follicles, but that it varied within the compartments of follicular cells. A moderate concentration was detected in rough endoplasmic reticulum and Golgi saccules, whereas a high concentration was found in condensing vacuoles, apical vesicles, and in the luminal colloid. Varying amounts of label were observed over the different types of colloid droplets, and this was attributed to various degrees of lysosomal degradation of thyroglobulin. It is concluded that the concentration of thyroglobulin antigenicity increases during transport from the ribosomal site of synthesis to the follicular colloid, and then decreases during the digestion of colloid droplets which leads to the release of the thyoid hormone.     

Developmental changes in localization of the main ganglioside during sea urchin embryogenesis

Ganglioside M5 (NeuGcalpha2-6Glcbeta1-1'Cer), the main ganglioside in sea urchin and sand dollar eggs, exists mainly in the endoplasmic reticulum and yolk granules in unfertilized eggs. To study the localization of ganglioside M5 after fertilization, early embryos were stained with an anti-ganglioside M5 monoclonal antibody. Using immunofluorescent and immunoelectron microscopy, intense label was observed in the outer surface and cytoplasm of embryos. These results indicate that ganglioside M5 was secreted during embryogenesis and localized in the extracellular matrix (ECM). When living embryos were incubated in sea water containing 7-nitrobenz-2-oxa-1,3-diazole labeled-ganglioside M5 (NBD-M5), the ECM and plasma membrane were strongly stained. Since the localization of NBD-M5 in the ECM was similar to that of extracellular M5, NBD-M5 was likely to be useful to examine the fate of extracellular ganglioside M5. Interestingly, NBD-M5 was incorporated in subcortical vesicles during embryogenesis, suggesting that the extracellular ganglioside M5 is transported into the cytoplasm. When fertilized eggs were incubated with NBD-M5 and tetramethylrhodamine dextran (a marker dye for endocytotic vesicles), colocalization of the dyes was observed in the vesicles. Thus, it was concluded that NBD-M5 in the ECM and/or plasma membrane was internalized in the cells by endocytosis, suggesting that extracellular M5 is transported from the ECM to endocytotic vesicles.     

Localization of lysosomal antigens in activated T-lymphocytes

Summary The lysosomal compartment has been examined in activated T-lymphocytes by immunogold electron microscopy and subcellular fractionation. Immunoprecipitation and sodium dodecyl sulphate-polyacrylamide gel, electrophoresis (SDS-PAGE) of radiolabelled extracts of the T-cells showed that they contained three antigens which are fundamental to normal lysosomal function: a representative lysosomal enzyme -glucuronidase, a lysosomal associated membrane protein (LAMP-1), and the cation-independent mannose 6-phosphate lysosomal enzyme targeting receptor (MPR). Immunogold labelling showed that -glucuronidase was present in the rough endoplasmic reticulum, the Golgi complex and Golgi-associated vesicles. The enzyme was also found to accumulate in distinct, non-Golgi organelles in which LAMP-1 was co-localized, probably lysosomes. LAMP-1 was also found in tubular elements of the golgi and in a complex of vesicles clustered near the nucleus where MPR was also present at high density.Fractionation of homogenates from lymphocytes on Percoll gradients revealed that -glucuronidase was distributed throughout the low density region containing rough endoplasmic reticulum, Golgi and plasma membrane components, and the high density region which contained only lysosomal activity. Multiple immunogold electron microscopy of the latter fraction showed the presence of homogenous vesicles which had large amounts of -glucuronidase within the lumen, LAMP-1 at the periphery and no MPR. These vesicles were probably mature lysosomes, arising from pre-lysosomal organelles enriched for LAMP-1 and MPR.     

Cellular localization of insulin-degrading enzyme in rat liver using monoclonal antibodies specific for this enzyme

Although insulin-degrading enzyme (IDE) has been implicated in the intracellular degradation of insulin, the cellular localization of this enzyme is still controversial. In the present study, we have examined the cellular localization of IDE in the rat liver by three different techniques using monoclonal antibodies. First, direct immunohistochemical staining of rat liver with one of the monoclonal antibodies revealed that IDE immunoreactivity mainly exists in parenchymal cells, especially in the vicinity of the portal tract and also in the epithelium of the bile duct under light microscopy. In the electron microscopic study, IDE immunoreactivity was found in the cytoplasm near the rough endoplasmic reticulum but not in the plasma membrane, nucleus, or mitochondria. Second, immunoblotting analysis of the subcellular fraction in rat liver showed that the monoclonal antibody specifically reacted with a single polypeptide in the cytosolic fraction, of apparent Mr 110,000, which was consistent with the Mr of IDE. However, a polypeptide band corresponding to IDE could not be observed in the plasma membrane, mitochondrial, or lysosomal fraction. Third, IDE was only detectable in the cytosolic fraction by sandwich radioimmunoassay using two monoclonal antibodies. These results all suggest that IDE is a cytosolic enzyme.     

Enzyme analysis and subcellular fractionation of human peripheral blood lymphocytes with special reference to the localization of putative plasma membrane enzymes

Human lymphocytes were isolated from defibrinated blood by Ficoll-Hypaque centrifugation with erythrocyte hypotonic lysis. Homogenates of mixed lymphocytes were subjected to analytical subcellular fractionation by sucrose gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their marker enzymes: cytosol (lactate dehydrogenase), plasma membrane (5′-nucleotidase), endoplasmic reticulum (neutral α-glucosidase), mitochondria (malate dehydrogenase), lysosomes (N-acetyl-β-glucosaminidase), peroxisomes (catalase). γ-Glutamyl transferase was exclusively localized to the plasma membrane. Leucine amino-peptidase, especially when assayed in the presence of Co²⁺, was also partially localized to the plasma membrane. Experiments with diazotized sulphanilic acid, a non-permeant enzyme inhibitor, showed that these plasma membrane enzymes are present on the cell surface. No detectable alkaline phosphatase was found in the lymphocytes. Acid phosphatase and β-glucuronidase were localized to lysosomes and there was some evidence for lysosomal heterogeneity. Leucine amino peptidase, optimal at pH 8.0, showed a partial localization to intracellular vesicles, possibly lysosomes, especially when assayed in the presence of EDTA. These studies provide a technique for determining the intracellular distribution of hitherto unassigned lymphocyte constituents and serve as a basis for investigating the cell pathology of lymphocytic disorders.     

Intracellular distribution of Sindbis virus membrane proteins in BHK-21 cells infected with wild-type virus and maturation-defective mutants.

The association of Sindbis virus proteins with cellular membranes during virus maturation was examined by utilizing a technique for fractionating the membranes of BHK-21 cells into three subcellular classes, which were enriched for rough endoplasmic reticulum, smooth endoplasmic reticulum, and plasma membrane. Pulse-chase experiments with wild-type (strain SVHR) virus-infected cells showed that virus envelope proteins were incorporated initially into membranes of the rough endoplasmic reticulum and subsequently migrated to the smooth and plasma membrane fractions. Large amounts of capsid protein were associated with the plasma membrane fraction even at the earliest times postpulse, and relatively little was found associated with the other membranes, suggesting a rapid and preferential association of nucleocapsids with the plasma membrane. We also examined the intracellular processing of the proteins of two temperature-sensitive Sindbis virus mutants in pulse-chase experiments at the nonpermissive temperature. Labeled virus proteins of mutant ts-20 (complementation group E) first appeared in the rough endoplasmic reticulum and were then transported to the smooth and plasma membrane fractions, as in wild-type (strain SVHR) virus-infected cells. In cells infected with ts-23 (complementation group D), the pulse-labeled virus proteins appeared initially in the rough membrane fraction and were transported to the smooth membrane fraction, but only limited amounts reached the plasma membrane. Thus, in ts-23-infected cells, the transport of the virus-encoded proteins from the smooth membranes seemed to be defective. In both ts-20- and ts-23-infected cells the envelope precursor polypeptide PE2 was not processed to E2, and no label was incorporated into free virus at the nonpermissive temperature.     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.     

Glycoproteins of the lysosomal membrane

Three glycoprotein antigens (120, 100, and 80 kD) were detected by mono- and/or polyclonal antibodies generated by immunization with highly purified rat liver lysosomal membranes. All of the antigens were judged to be integral membrane proteins based on the binding of Triton X-114. By immunofluorescence on normal rat kidney cells, a mouse monoclonal antibody to the 120-kD antigen co-stained with a polyclonal rabbit antibody that detected the 100- and 80-kD antigens as well as with antibodies to acid phosphatase, indicating that these antigens are preferentially localized in lysosomes. Few 120-kD-positive structures were found to be negative for acid phosphatase, suggesting that the antigen was not concentrated in organelles such as endosomes, which lack acid phosphatase. Immunoperoxidase cytochemistry also showed little reactivity in Golgi cisternae, coated vesicles, or on the plasma membrane. Digestion with endo-beta-N-acetylglucosaminidase H (Endo H) and endo-beta-N-acetylglucosaminidase F (Endo F) demonstrated that each of the antigens contained multiple N-linked oligosaccharide chains, most of which were of the complex (Endo H-resistant) type. The 120-kD protein was very heavily glycosylated, having at least 18 N-linked chains. It was also rich in sialic acid, since neuraminidase digestion increased the pI of the 120-kD protein from less than 4 to greater than 8. Taken together, these results strongly suggest that the glycoprotein components of the lysosomal membrane are synthesized in the rough endoplasmic reticulum and terminally glycosylated in the Golgi before delivery to lysosomes. We have provisionally designated these antigens lysosomal membrane glycoproteins lgp120, lgp100, lgp80.     

Phosphomannosyl-enzyme receptors in rat liver. Subcellular distribution and role in intracellular transport of lysosomal enzymes

beta-Hexosaminidase B purified from human fibroblast secretions was used as a ligand to study phosphomannosyl-enzyme receptors in membranes from rat tissues. Enzyme binding to rat liver membranes was saturable, competitively inhibited by mannose 6-phosphate, not dependent on calcium, and destroyed by prior treatment of the hexosaminidase with either alkaline phosphatase or endoglycosidase H. Most (90%) of the phosphomannosyl-enzyme receptors were found in endoplasmic reticulum, Golgi apparatus, and lysosomes; 9.5% in the plasma membrane, and less than 1% in nuclei and mitochondria. Receptors were vesicle-enclosed in all fractions except plasma membrane. Receptors in the endoplasmic reticulum apparently were occupied by endogenous ligands, but most receptors in lysosomes and plasma membrane were unoccupied. Most of the endogenous beta-hexosaminidase was in lysosomes and was released from vesicles by detergent treatment. Displacement of the residual receptor-bound endogenous beta-hexosaminidase (mostly in endoplasmic reticulum and Golgi apparatus) from detergent-treated membranes by mannose 6-phosphate released high uptake enzyme with properties expected for phosphomannosyl-enzymes. Mannose 6-phosphate-inhibitable enzyme receptor activity was found in nine rat organs and correlated roughly with their lysosomal enzyme content. These data support a general model for lysosomal enzyme transport in which the phosphomannosyl-enzyme receptor acts as a vehicle for delivery of newly synthesized acid hydrolases from the endoplasmic reticulum to lysosomes.     

A cytochemical ultrastructural study of the lysosomal system of different species of malaria parasites

We have used ultrastructural techniques in different malarial species to demonstrate a lysosomal system. First, we have tried to localize acid phosphatase, a typical lysosomal label. Its activity was localized in the endoplasmic reticulum and in endocytic vesicles, and in dense-cored vesicles near the digestive vacuoles, especially in Plasmodium falciparum (FCR3 strain). Then, we have studied the different cellular compartments of the malarial parasite by the zinc iodide-osmium tetroxide technique that heavily contrasted the cellular compartments of the parasite. This experiment led to the observation of a profound rearrangement of the endoplasmic reticulum, especially in P. berghei. A very atypical but functional Golgi apparatus was demonstrated in all the growing stages of the parasite and lysosome-like vesicles were observed, showing a structure very similar to those of the coated vesicles of a true Golgi complex. The presence of these organelles are in favor of the existence of a lysosomal system and of the endogenicity of some enzymes involved hemoglobin degradation.     

Gp96/GRP94 is a putative high density lipoprotein-binding protein in liver

We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization: it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed.     

Immunocytochemical localization of P170 at the plasma membrane of multidrug-resistant human cells

P170 (P-glycoprotein) is a membrane protein found in high levels in multidrug-resistant cultured cell lines. We have localized this protein using monoclonal antibody MRK16 by immunofluorescence and electron microscopy in the multidrug-resistant human carcinoma cell line KB-C4. The P170 determinant recognized by antibody MRK16 was found on drug-resistant KB-C4 cells, but not on parental drug-sensitive KB-3-1 cells. The determinant was present on the external surface of the plasma membrane and on the luminal side of Golgi stack membranes. P170 was excluded from coated pits at the plasma membrane and absent from endocytic vesicles and lysosomes. This determinant was detected only in small amounts in the endoplasmic reticulum. The high protein concentration of P170 in the plasma membrane is consistent with a role of this protein as a drug efflux pump at the cell surface.     

Isolation of a vesicular intermediate in the cell-free transfer of membrane from transitional elements of the endoplasmic reticulum to Golgi apparatus cisternae of rat liver

Preparations enriched in part-smooth (lacking ribosomes), part-rough (with ribosomes) transitional elements of the endoplasmic reticulum when incubated with ATP plus a cytosol fraction responded by the formation of blebbing profiles and approximately 60-nm vesicles. The 60-nm vesicles formed resembled closely transition vesicles in situ considered to function in the transfer of membrane materials between the endoplasmic reticulum and the Golgi apparatus. The transition elements following incubation with ATP and cytosol were resolved by preparative free-flow electrophoresis into fractions of differing electronegativity. The main fraction contained the larger vesicles of the transitional membrane elements, while a less electronegative minor shoulder fraction was enriched in the 60-nm vesicles. If the vesicles concentrated by preparative free-flow electrophoresis were from material previously radiolabeled with [3H]leucine and then added to Golgi apparatus immobilized to nitrocellulose, radioactivity was transferred to the Golgi apparatus membranes. The transfer was rapid (T1/2 of about 5 min), efficient (10-30% of the total radioactivity of the transition vesicle preparations was transferred to Golgi apparatus), and independent of added ATP but facilitated by cytosol. Transfer was specific and apparently unidirectional in that Golgi apparatus membranes were ineffective as donor membranes and endoplasmic reticulum vesicles were ineffective as recipient membranes. Using a heterologous system with transition vesicles from rat liver and Golgi apparatus isolated from guinea pig liver, coalescence of the small endoplasmic reticulum-derived vesicles with Golgi apparatus membranes was demonstrated using immunocytochemistry. Employed were polyclonal antibodies directed against the isolated rat transition vesicle preparations. When localized by immunogold procedures at the electron microscope level, regions of rat-derived vesicles were found fused with cisternae of guinea pig Golgi apparatus immobilized to nitrocellulose strips. Membrane transfer was demonstrated from experiments where transition vesicle membrane proteins were radioiodinated by the Bolton-Hunter procedure. Additionally, radiolabeled peptide bands not present initially in endoplasmic reticulum appeared following coalescence of the derived vesicles with Golgi apparatus. These bands, indicative of processing, required that both Golgi apparatus and transition vesicles be present and did not occur in incubated endoplasmic reticulum preparations or on nitrocellulose strips to which no Golgi apparatus were added.(ABSTRACT TRUNCATED AT 400 WORDS)     

Studies on the subcellular localization and role of glutathione-insulin transhydrogenase in rat liver

₁₂₅I-insulin was shown to be internalized in vivo to a discrete population of low-density membranes (ligandosomes), distinct from the Golgi, endoplasmic reticulum, plasma membrane, and lysosomes. However, analytical subcellular fractionation shows that glutathione-insulin transhydrogenase is localized to the endoplasmic reticulum. Measurement of the specific enzyme activity of glutathione-insulin transhydrogenase showed no differences between normal, diabetic, and hyperinsulinaemic rats. These results suggest that glutathione-insulin transhydrogenase is not directly involved in the subceltular processing of receptor-bound internalized insulin.     

Characterization of ATP-driven calcium uptake in renal basal-lateral and renal endoplasmic reticulum membrane vesicles

ATP-driven calcium uptake was studied in basal-lateral membranes and in microsomal fractions, isolated from pig kidney cortex. The uptake is strongly enhanced in conditions where calcium inside the vesicles is precipitated by oxalate (5 mM) or phosphate (40 mM). Both anions were equally effective for the stimulation of calcium uptake in the microsomes but oxalate was less effective than phosphate in the basal-lateral membrane fraction. The active calcium pumps in the renal basal-lateral and microsomal fractions are different transport ATPases characterized by phosphorylated intermediates of 135 kDa and 115 kDa respectively. The subcellular distribution of the 135 kDa and 115 kDa phosphointermediates, reflects the distribution of typical marker enzymes for the basal-lateral membrane and for the endoplasmic reticulum. The calmodulin binding to the 135 kDa polypeptide as estimated by 125I-labelled calmodulin overlay, can be used as a specific marker for the basal-lateral plasma membrane calcium pump.