Rabbit red blood cell hexokinase:intracellular distribution during reticulocytes maturation

Summary The intracellular localization and isozyme distribution of hexokinase were studied during rabbit reticulocyte maturation and aging. In reticulocytes 50% of the enzyme was particulate while in the mature erythrocytes all the hexokinase activity was soluble. The bound enzyme co-sediments with mitochondria and by column chromatography it was found to be hexokinase Ia. The cytosol of reticulocytes contains hexokinase Ia (38%) and hexokinase Ib (62%) while the mature erythrocytes contain only hexokinase Ia. The amount of bound hexokinase decreases very quickly during cell maturation and aging as was shown by following in vivo reticulocyte maturation or by analysis of hexokinase compartmentation in cells of different ages, obtained by density gradient ultracentrifugations. A role for this intracellular distribution of hexokinase is suggested.     

Purification, properties, and immunochemical localization of a receptor for intrinsic factor-cobalamin complex in the rat kidney

High levels of receptor for intrinsic factor-cobalamin (vitamin B12) were detected in human, canine, and rat kidneys. The ratio of specific activity (picomoles/mg of protein) for kidney relative to intestine was 116, 20, and 797, respectively, in these species. The receptor was purified about 3000-fold from 200 g of rat kidney with a recovery of 16% and exhibited a single band on nondenaturing gel electrophoresis. Quantitative amino acid analysis of the receptor gave a value of 457,310 g of amino acid/mol of intrinsic factor-cobalamin binding activity. The pure receptor revealed an Mr of 430,000, as assessed by filtration with Bio-Gel A-5m. Treatment with papain resulted in the production of active monomers of Mr to about 205,000-210,000. Electrophoresis in the presence of sodium dodecyl sulfate confirmed the monomer Mr to be 230,000. The monomer receptor did not reveal the presence of any further subunits upon reductive alkylation. Following cyanogen bromide cleavage the kidney receptor revealed three peptides of Mr 115,000, 60,000, and 54,000. The pI of these peptides was 5.17, 6.17, and 6.17, respectively. Western blot analysis using antiserum raised to the receptor demonstrated a protein with an Mr of 175,000 and 230,000 for intestinal and kidney membrane receptors, respectively. Immunologically, the rat kidney receptor was identical to the rat ileal receptor but was distinct from the canine ileal receptor. Ultrastructural localization revealed the presence of the receptor in the apical surface membrane of proximal tubular cells of the kidney and absorptive cells of the ileum. The kidney is the best source for obtaining this receptor in reasonable quantities.     

Lectin binding to the porcine and human ileal receptor of intrinsic factor-cobalamin

The purified porcine recpptor for the intrinsic factor-cobalamin complex bound to concanavalin A, lentil lectin and wheat germ lectin covalently coupled to Sepharose and was eluted with the corresponding soluble sugars. In contrast, human intrinsic factor bound efficiently to concanavalin A, to some extent to lentil lectin, but only slightly to wheat germ agglutinin. The binding of IF-Cbl to the receptor was inhibited when the receptor was pre-incubated with soluble wheat germ aglutinin, with an inhibition constant estimated to be 1.9 mol/l. After transfer of the purified receptor from SDS-PAGE to Immobilon, ligand blotting of the purified receptor with iodinated lectin showed that concanavalin A and lentil lectin bound to three (75, 56 and 43 kDa) components but that wheat germ agglutinin bound only to the 75 kDa component. These results showed that the subunit of the receptor could bind to wheat germ agglutinin, resulting in an inhibition of its binding with intrinsic factor. Both binding sites of intrinsic factor and of wheat germ agglutinin could be located near to each other.     

Interaction of receptor for intrinsic factor-cobalamin complex with synthetic and brush-border lipids

The intestinal receptor for intrinsic factor-cobalamin complex integrates preferentially with cationic liposomes in such a way that its ligand binding sites are exposed to proteases and to antibody to the receptor. After integration the receptor could be solubilized from the liposomes by detergents and chaotropic salts. Kinetically, the brush-border and the liposome-bound receptor behaved similarly, and Triton X-100 had no effect either on Ka or Vmax, suggesting the absence of any effects of a lipid domain on ligand binding activity. However, acidic phospholipids caused inhibition when added to the receptor-ligand assay system in the absence of liposomes.     

Immunocytochemical localization of intracellular antigens with SEM

Summary A method for the localization of intracellular antigens with a scanning electron microscope using peroxidase-labelling antibodies is described. A search for a hydrogen donor which may be deposited at the sites of antigen by enzymatic action and emit secondary electrons or generate backscatter electrons was made. It was found that when 4-chloro-1-naphthol was used, the peroxidase deposited reaction product which resulted in a strong secondary electron emission at the site of antigen. With this method, the presence of luteinizing hormone in secretion granules and other cytoplasmic structures of gonadotropic cells was demonstrated. The level of detection of intracellular antigens with this method is not as high as that detectable with light microscopical examination of the same specimens, that is, more reaction product at the site of antigen is required to be detectable with scanning electron microscopy than with light microscopy. In spite of the lack of high sensitivity, the intracellular antigens may be localized with the method described.     

Defective brush-border expression of intrinsic factor-cobalamin receptor in canine inherited intestinal cobalamin malabsorption

Ligand binding activity of intrinsic factor-cobalamin receptor (IFCR) was determined in homogenates and isolated brush-border membranes (BBM) of ileum and kidney from dogs exhibiting simple autosomal recessive inheritance of selective cobalamin malabsorption (Fyfe, J. C., Giger, U., Hall, C. A., Jezyk, P. F., Klumpp, S. A., Levine, J. S., and Patterson, D. F. (1991) Pediatr. Res. 29, 24-31). IFCR activity of affected dog ileal homogenates was 3-4-fold higher than normal whereas IFCR activity in affected dog kidney homogenates was one-tenth of normal. The recovery of IFCR activity in the BBM of ileum and renal cortex of affected dogs was 30- and 20-fold less than normal, respectively. The dissociation constant (Kd) for intrinsic factor-cobalamin was similar in BBM of both tissues and was the same in affected and normal dogs. In the affected dog ileal BBM, activities of alkaline phosphatase and sucrase-isomaltase and vesicular transport of glucose and Na(+)-taurocholate were normal. Immunoblots showed no IFCR cross-reactive material in the ileal or renal BBM of affected dogs. IFCR purified by affinity chromatography from kidney of both normal and affected dogs had an Mr = 230,000. However, amino acid analysis revealed that the affected dog IFCR had more lysine than the normal, and protease cleavage of the purified IFCRs revealed different peptide maps. Asparagine-linked oligosaccharides of both proteins were sensitive to peptide N-glycosidase F cleavage, but only the affected dog IFCR was endoglycosidase H sensitive. These results suggest that cobalamin malabsorption in this canine family is caused by inefficient BBM expression of IFCR due to a mutation of IFCR and its retention in an early biosynthetic compartment.     

Synthesis and brush border expression of intrinsic factor-cobalamin receptor from rat renal cortex.

The main objective of the current study was to investigate the factors that affect brush border membrane expression of intrinsic factor-cobalamin receptor (IFCR). Because of high levels of IFCR expression (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449) in the rat kidney, we have studied the synthesis and expression of IFCR using rat cortical slices in culture. The IFCR activity in the renal apical brush border was maximum from rats between the age of 20-24 days and about 75% of the activity was lost from the isolated apical surface membranes following culture of cortical slices with nonradioactive intrinsic factor-cobalamin. However, the membrane IFCR activity recovered to 100 or 75%, respectively, when the slices were cultured with intrinsic factor-cobalamin mixed with either leupeptin or chloroquine. When these lysosomotropic agents were added during the metabolic labeling of the cortical slices with trans-35S-label neither the synthesis nor the amount of [35S]IFCR transported to the apical membrane was inhibited. However, with the addition of colchicine, the apical membrane expression of [35S]IFCR was inhibited by 75-80%. Metabolic labeling of cortical slices with trans-35S-label and immunoprecipitation of the Triton X-100 extract from the total, internal, and apical membranes revealed the presence of a 230-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With either continuous or pulse-chase labeling of the cortical slices, the amount of 230-kDa [35S]IFCR recovered in the apical membrane did not exceed 10-15% of the total labeled receptor synthesized. Based on these and our recent studies (Seetharam, S., Dahms, N., Li, N., Ramanujam, K.S., and Seetharam, B. (1991) Biochem. Biophys. Res. Commun. 177, 751-756), we propose that rat renal IFCR is synthesized as a single polypeptide chain of 220 kDa and is transported slowly to the apical membrane during which four or five N-linked oligosaccharides are processed to the complex type. Moreover, the brush border expression of IFCR is regulated by the biosynthetic and not by the endocytic pathway.     

Functional expression of intrinsic factor-cobalamin receptor by renal proximal tubular epithelial cells.

Previous studies from our laboratory (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489-4494) have identified and isolated a 230-kDa receptor from rat and canine kidney which binds with high affinity [57Co]cyanocobalamin (Cbl) complexed to gastric intrinsic factor (IF). Although these studies have identified a renal receptor which binds intrinsic factor-cobalamin (IFCR), it is not known whether the binding is specific for IF-Cbl and whether renal cells internalize [57Co]Cbl bound to IF and transport [57Co]Cbl across the cell. Using a variety of renal cells, our results show that IF-[57Co]Cbl binding activity is detected in proximal tubular-derived epithelial cells from opossum (OK) and porcine kidney (LLC-PK1) but not in distal tubular-derived cells from canine kidney cells (MDCK). Metabolic labeling studies with Tran 35S-label confirmed the presence of a 230-kDa IFCR in OK and LLC-PK1 cells. Cell surface labeling and binding studies demonstrated that IFCR is targeted to the apical membrane. This apical expression of IFCR in OK cells is inhibited by the microtubule-disruptive drugs, colchicine and nocodazole. Opossum kidney cells when grown on culture inserts are polarized and transport [57Co]Cbl only when bound to IF and not to other Cbl binders. Furthermore, the transport of [57Co]Cbl occurred unidirectionally from the apical to the basolateral surface. Treatment of cells with colchicine or nocodazole inhibited the surface binding of IF-[57Co]Cbl as well as the transcytosis of [57Co]Cbl by 70-75%. IFCR retained intracellualarly by incubation of cells with colchicine or nocodazole is degraded by leupeptin-sensitive proteases. Based on these results, we suggest that proximal tubular-derived epithelial cells transport [57Co]Cbl bound to IF in a saturable way via receptor-mediated endocytosis.     

Binding of cobalamin analogs to intrinsic factor-cobalamin receptor and its prevention by R binder

It is now known that nonphysiological cobalamin analogs exist in the gastrointestinal tract, but their metabolic behavior is unclear. In this study, [57Co]cobinamide was used to study its affinity to hog intrinsic factor-cobalamin (IF-Cbl) receptor which has no species specificity against human IF-Cbl receptor, and its relation to human saliva R binder. Cobinamide was prepared from [57Co]cyanocobalamin and separated by paper chromatography. Human IF-Cbl complex was bound to IF-Cbl receptor but free cyanocobalamin was not. Although R binder-cobinamide was not bound to the IF-Cbl receptor, free cobinamide was bound to the IF-Cbl receptor to a significant extent (about one-half of IF-cyanocobalamin binding to the IF-Cbl receptor). We then investigated the binding of cobinamide to R binder and trypsin-treated R binder. Association constant of cobinamide binding to the IF-Cbl receptor was 1.0 X 10(9) M-1 which was much lower than that of cobinamide binding to trypsin-treated R binder and to untreated R binder. Further study indicated that cobinamide binding to the IF-Cbl receptor was blocked by the addition of R binder and also by trypsin-treated R binder. We conclude that one of the roles of R binder is to prevent binding of free cobalamin analogs to the IF-Cbl receptor in the gut.     

Immunocytochemical localization of glucocorticoid receptor in rat skin

The aim of the present study was to map immunohistochemically the distribution of the glucocorticoid receptor (GR) in rat skin. Nuclear GR-like immunoreactivity (LI) was found in both epidermis and dermis. In the epidermis, the basal cell layer showed an intense immunoreaction; the lower part of the spinous layer was also labelled. In the dermis, the fibroblasts as well as the sweat glands, sebocytes and adipocytes were GR-immunoreactive (IR). In the root sheath of the hair follicle the staining was most intensive in the outer layer. The endothelial cells comprising the smooth muscle cells of the blood vessels, as well as the arrector pili muscle, showed GR-LI. In the peripheral nerves, the immunoreaction was localized to the nuclei of the Schwann cells and in the perineurial fibroblasts. Mast cells did not show nuclear GR-LI. Based on our immunocytochemical findings that several cell types of the skin are GR-IR, the variable physiological and pharmacological effects of glucocorticoids are easier to understand.     

Immunocytochemical localization of ubiquitin-activating enzyme in the cell nucleus

Ubiquitin-activating enzyme, "E1", is the first enzyme in the pathway leading to formation of ubiquitin-protein conjugates. We present immunocytochemical evidence that Ubiquitin-activating enzyme is concentrated in the cell nucleus. This finding points to the nucleus as the major site of action of this enzyme. Since ubiquitin itself is not similarly compartmentalized, this result suggests a high level of ubiquitin conjugate formation in the nucleus with a rapid turnover of ubiquitin conjugates.     

Immunocytochemical localization of myosin in rabbit liver cell

It has been established that the liver cell possesses its own myosin which resembles other non-muscle myosins in subunit composition and in its dependence of actin-activated Mg2+-ATPase activity on light chain phosphorylation (Ueno T, Sekine T: Biochem Int, 1987;15:1205). We have raised a specific antibody against rabbit liver cell myosin. Immunoblot analysis has shown that the purified antibody reacts only with the heavy chain of liver cell myosin. The antibody did not react with rabbit skeletal muscle myosin or with smooth muscle myosin extracted from rabbit intestinal wall. Cryostat liver sections analyzed by indirect immunofluorescence microscopy showed a characteristic polygonal staining pattern, indicating that myosin is concentrated close to the plasma membrane, particularly in the region of bile canaliculi. Myosin therefore appears to be localized in the area where actin filaments are also abundant.     

Immunocytochemical localization of the epidermal growth factor receptor in mouse testis

The distribution of the epidermal growth factor receptor (EGFR) in mouse testis was ascertained by immunocytochemical methodology using a polyclonal antibody (RK2) shown previously to recognize the cytoplasmic domain of the human (A431 cells), murine (Swiss 3T3 cells), and chicken (CK 109 cells) EGFR. Initial studies performed to determine the usefulness of this antibody as a probe of the murine EGFR in testis employed two murine cell lines, TM4 and MA10, of Sertoli cell and Leydig cell origin, respectively, in which a physiological response of EGF and specific binding of iodinated EGF has been demonstrated. Western blotting in membrane preparations of TM4 and MA10 revealed only one prominent band at 170 kDa. Immunocytochemical localization in TM4 and MA10 cells illustrated a plasma membrane distribution of the receptor. Western blotting of membrane fractions prepared from testis also revealed a specific band at 170 kDa. In the intact testis, the EGFR was immunolocalized specifically in Leydig cells and Sertoli cells only. These results suggest that the involvement of EGF action in spermatogenesis may occur at the level of the somatic components of the testes, principally in the Leydig and Sertoli cells.     

Immunocytochemical localization of the receptor for asialoglycoprotein in rat liver cells

We used high-resolution immunocytochemistry on ultrathin frozen sections labeled with colloidal gold to study the subcellular distribution of the asialoglycoprotein receptor in rat liver. The receptor was localized along the entire hepatocyte plasma membrane, including the bile capillary membrane, but was scarce intracellularly. Sinusoidal lining (Kupffer) cells and blood cells showed no immunoreactivity. In liver cells of rats injected with 1 to 100 micrograms of asialoorosomucoid (ASOR) 2-15 min before tissue fixation, endocytotic internalization of receptors at the blood front was conspicuous. At all times in this interval, receptor was present in approximately 100-nm vesicles and larger vacuoles adjacent to the sinusoidal plasma membrane. No other significant intracellular receptor was noted during the 15-min exposure to ASOR; in particular, lysosomes and Golgi complex were not labeled. Our observations, in combination with data from the literature which demonstrate that, under these conditions, the ligand is transferred further to the Golgi complex-lysosome region, suggest that the receptor and ligand are dissociated in the vicinity of the plasma membrane, after which the receptor rapidly returns to the cell surface.     

雌激素Beta受体在大鼠脑内表达的免疫组化定位研究

为了探讨雌激素作用于神经系统的机理,采用硫酸镍铵增强显色的免疫组化SP法研究了新的雌激素受体（ER－β）在成年雌雄大鼠脑内的分布。研究证实ER－β免疫阳性物质主要位于神经元的细胞核内,但在个别脑区也可在胞浆甚至突起内检测到。最强的ER－β免疫阳性信号见于前嗅核、大脑皮质、小脑浦肯野细胞、斜角带垂直部、蓝斑和三叉神经运动核等部位;中等强度的染色见于隔内侧核、杏仁外侧核、黑质、中央灰质等部位;较弱的阳性反应见于下丘脑与杏仁复合体的部分核团。在一些部位还存在表达水平甚至细胞内定位模式的性别差异,如前庭上核内的表达只见于雌性;雄性大鼠三叉神经运动核内ER－β蛋白主要表达于胞浆内,细胞核为阴性;而在雌性大鼠该部位ER－β蛋白主要位于细胞核等。以上结果表明ER－β蛋白在大鼠脑内分布广泛并具有一定的性别差异,在与学习记忆有关的脑区如大脑皮质和基底前脑内有很高的表达,提示在脑组织内雌激素可能通过ER－β这一新的信号途径发挥多种重要的调控作用,如学习记忆等。     

Alterations in distribution of surface and intracellular antigens during epididymal maturation of rat spermatozoa

The surface membrane of mammalian spermatozoa is known to undergo considerable conformational and organizational changes during epididymal maturation. However, much less is known about remodelling of intracellular membranes. In this communication we have used specific immunological markers to study the behavior of several antigens both on and within rat spermatozoa as they mature in the epididymis. Four monoclonal antibodies (McAbs) designated 5B1, 1B5, 2D6, and 1B6 were used to probe testicular and caput and cauda epididymal spermatozoa by indirect immunofluorescence and immunogold labeling techniques. None of the McAbs bound to testicular spermatozoa; in all cases, they became reactive only on spermatozoa which had reached the caput epididymis. McAb 5B1 was restricted to the outer acrosomal membrane (OAM) of the acrosomal cap domain. The epitope first appeared on antigen(s) with molecular mass (Mr) of approximately 200 kDa in immature spermatozoa, but later in mature spermatozoa the antigen(s) had Mr of approximately 160 kDa. The antigen(s) recognized by 1B5 McAb on the other hand was initially distributed over the OAM of the entire acrosomal domain (cap + equatorial segment), but during maturation it became progressively more restricted in area until in cauda spermatozoa only the anterior tip of the OAM bound the McAb. McAb 2D6 also bound to the entire OAM and acrosomal contents of caput spermatozoa, but, unlike 5B1 and 1B5 McAbs, reactivity was transient. That is, staining was first detected in caput spermatozoa but then disappeared in corpus and cauda spermatozoa. In contrast to all of the above, 1B6 McAb bound to the surface membrane overlying the entire head domain of caput spermatozoa, but during maturation it became restricted to the postacrosomal domain. These results indicate that, in addition to remodeling of the surface membrane during epididymal maturation, extensive processing of intracellular membrane antigens also takes place and that it is very active within the acrosome. The nature of these intracellular processing events remains to be elucidated, but they may have important consequences for membrane fusion and cell recognition phenomena during fertilization.