scholarly journals Bilateral ureteral obstruction induces early downregulation and redistribution of AQP2 and phosphorylated AQP2

2011 ◽  
Vol 301 (1) ◽  
pp. F226-F235 ◽  
Author(s):  
Lene Stødkilde ◽  
Rikke Nørregaard ◽  
Robert A. Fenton ◽  
Guixian Wang ◽  
Mark A. Knepper ◽  
...  
Keyword(s):  
Ureteral Obstruction ◽  
Cellular Localization ◽  
Collecting Duct ◽  
Intracellular Localization ◽  
Water Channel ◽  
Degradation Pathway ◽  
Salt Transport ◽  
Apical Surface ◽  
Altered Expression ◽  
Early Endosomes

Bilateral ureteral obstruction (BUO) is characterized by impairment of urine flow from the kidneys and altered expression of specific membrane proteins in the kidney involved in regulation of renal water and salt transport. Importantly, 24-h BUO reduces the abundance of the collecting duct water channel aquaporin-2 (AQP2) and AQP2 phosphorylated at serine 256 (AQP2pS256). To investigate the mechanism behind downregulation of AQP2 in BUO, rats were subjected to BUO and examined after 2, 6, 12, and 24 h. Q-PCR and immunoblotting showed significantly decreased AQP2 mRNA expression after 2-h BUO and decreased abundance of total AQP2 after 12 and 24 h. In parallel, immunohistochemistry showed weaker labeling of AQP2 at the apical surface of inner medullary collecting ducts (IMCD) compared with controls. The abundance of AQP2pS256 was significantly reduced from 6-h BUO and was confirmed by immunohistochemistry. Importantly, immunoblotting showed reduced abundance of AQP2pS261 after 12- and 24-h BUO mimicking total AQP2. Immunohistochemistry demonstrated early changed intracellular localization of AQP2pS261 in BUO, and colocalization studies showed redistribution from the apical membrane to early endosomes and lysosomes. In conclusion, BUO induces a very early regulation of AQP2 both at the level of abundance and on cellular localization. AQP2 and AQP2 phosphorylated at ser261 redistribute to more intracellular localizations and colocalize with the early endosomal marker EEA1 and the lysosomal marker cathepsin D, suggesting that early downregulation of AQP2 could in part be caused by degradation of AQP2 through a lysosomal degradation pathway.

scholarly journals Rab7 involves Vps35 to mediate AQP2 sorting and apical trafficking in collecting duct cells

2020 ◽  
Vol 318 (4) ◽  
pp. F956-F970 ◽  
Author(s):  
Wei-Ling Wang ◽  
Shih-Han Su ◽  
Kit Yee Wong ◽  
Chan-Wei Yang ◽  
Chin-Fu Liu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Small Gtpase ◽  
Apical Plasma Membrane ◽  
Water Reabsorption ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Vacuolar Protein

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for osmotic water reabsorption by kidney collecting ducts. In response to vasopressin, AQP2 traffics from intracellular vesicles to the apical plasma membrane of collecting duct principal cells, where it increases water permeability and, hence, water reabsorption. Despite continuing efforts, gaps remain in our knowledge of vasopressin-regulated AQP2 trafficking. Here, we studied the functions of two retromer complex proteins, small GTPase Rab7 and vacuolar protein sorting 35 (Vps35), in vasopressin-induced AQP2 trafficking in a collecting duct cell model (mpkCCD cells). We showed that upon vasopressin removal, apical AQP2 returned to Rab5-positive early endosomes before joining Rab11-positive recycling endosomes. In response to vasopressin, Rab11-associated AQP2 trafficked to the apical plasma membrane before Rab5-associated AQP2 did so. Rab7 knockdown resulted in AQP2 accumulation in early endosomes and impaired vasopressin-induced apical AQP2 trafficking. In response to vasopressin, Rab7 transiently colocalized with Rab5, indicative of a role of Rab7 in AQP2 sorting in early endosomes before trafficking to the apical membrane. Rab7-mediated apical AQP2 trafficking in response to vasopressin required GTPase activity. When Vps35 was knocked down, AQP2 accumulated in recycling endosomes under vehicle conditions and did not traffic to the apical plasma membrane in response to vasopressin. We conclude that Rab7 and Vps35 participate in AQP2 sorting in early endosomes under vehicle conditions and apical membrane trafficking in response to vasopressin.

Aquaporin 2 is a vasopressin-independent, constitutive apical membrane protein in rat vas deferens

2000 ◽  
Vol 278 (4) ◽  
pp. C791-C802 ◽  
Author(s):  
Anna L. Stevens ◽  
Sylvie Breton ◽  
Corinne E. Gustafson ◽  
Richard Bouley ◽  
Raoul D. Nelson ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Cellular Localization ◽  
Vas Deferens ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Sperm Concentration ◽  
Apical Plasma Membrane ◽  
Principal Cells ◽  
Aquaporin 2

Aquaporin 2 (AQP2), the vasopressin-regulated water channel, was originally identified in renal collecting duct principal cells. However, our recent description of AQP2 in the vas deferens indicated that this water channel may have extra-renal functions, possibly related to sperm concentration in the male reproductive tract. In this study, we have examined the regulation and membrane insertion pathway of AQP2 in the vas deferens. The amino acid sequence of vas deferens AQP2 showed 100% identity to the renal protein. AQP2 was highly expressed in the distal portion (ampulla) of the vas deferens, but not in the proximal portion nearest the epididymis. It was concentrated on the apical plasma membrane of vas deferens principal cells, and very little was detected on intracellular vesicles. Protein expression levels and cellular localization patterns were similar in normal rats and vasopressin-deficient Brattleboro homozygous rats, and were not changed after 36 h of dehydration, or after 3 days of vasopressin infusion into Brattleboro rats. AQP2 was not found in apical endosomes (labeled with Texas Red-dextran) in vas deferens principal cells, indicating that it is not rapidly recycling in this tissue. Finally, vasopressin receptors were not detectable on vas deferens epithelial cell membranes using a [3H]vasopressin binding assay. These data indicate that AQP2 is a constitutive apical membrane protein in the vas deferens, and that it is not vasopressin-regulated in this tissue. Thus AQP2 contains targeting information that can be interpreted in a cell-type-specific fashion in vivo.

scholarly journals Mouse model of type II Bartter's syndrome. II. Altered expression of renal sodium- and water-transporting proteins

2008 ◽  
Vol 294 (6) ◽  
pp. F1373-F1380 ◽  
Author(s):  
Carsten A. Wagner ◽  
Dominique Loffing-Cueni ◽  
Qingshang Yan ◽  
Nicole Schulz ◽  
Panagiotis Fakitsas ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Statistical Significance ◽  
Salt Transport ◽  
Postnatal Life ◽  
Thick Ascending Limb ◽  
Type Ii ◽  
Bartter’S Syndrome ◽  
Wild Type ◽  
Bartter's Syndrome ◽  
Altered Expression

Bartter's syndrome represents a group of hereditary salt- and water-losing renal tubulopathies caused by loss-of-function mutations in proteins mediating or regulating salt transport in the thick ascending limb (TAL) of Henle's loop. Mutations in the ROMK channel cause type II antenatal Bartter's syndrome that presents with maternal polyhydramnios and postnatal life-threatening volume depletion. We have developed a colony of Romk null mice showing a Bartter-like phenotype and with increased survival to adulthood, suggesting the activation of compensatory mechanisms. To test the hypothesis that upregulation of Na+-transporting proteins in segments distal to the TAL contributes to compensation, we studied expression of salt-transporting proteins in ROMK-deficient ( Romk−/−) mice. Plasma aldosterone was 40% higher and urinary PGE2 excretion was 1.5-fold higher in Romk−/− compared with wild-type littermates. Semiquantitative immunoblotting of kidney homogenates revealed decreased abundances of proximal tubule Na+/H+ exchanger (NHE3) and Na+-Pi cotransporter (NaPi-IIa) and TAL-specific Na+-K+-2Cl−-cotransporter (NKCC2/BSC1) in Romk−/− mice, while the distal convoluted tubule (DCT)-specific Na+-Cl− cotransporter (NCC/TSC) was markedly increased. The abundance of the α-,β-, and γ-subunits of the epithelial Na+ channel (ENaC) was slightly increased, although only differences for γ-ENaC reached statistical significance. Morphometry revealed a fourfold increase in the fractional volume of DCT but not of connecting tubule (CNT) and collecting duct (CCD). Consistently, CNT and CD of Romk−/− mice revealed no apparent increase in the luminal abundance of the ENaC compared with those of wild-type mice. These data suggest that the loss of ROMK-dependent Na+ absorption in the TAL is compensated predominately by upregulation of Na+ transport in downstream DCT cells. These adaptive changes in Romk−/− mice may help to limit renal Na+ loss, and thereby, contribute to survival of these mice.

P2Y2 receptor mRNA and protein expression is altered in inner medullas of hydrated and dehydrated rats: relevance to AVP-independent regulation of IMCD function

2005 ◽  
Vol 288 (6) ◽  
pp. F1164-F1172 ◽  
Author(s):  
Bellamkonda K. Kishore ◽  
Carissa M. Krane ◽  
R. Lance Miller ◽  
Huihui Shi ◽  
Ping Zhang ◽  
...  
Keyword(s):  
Blot Analysis ◽  
Expression Profiles ◽  
Collecting Duct ◽  
Water Channel ◽  
Prostaglandin E ◽  
P2y2 Receptor ◽  
Sprague Dawley ◽  
Altered Expression ◽  
Enhanced Production ◽  
Hydration And Dehydration

Arginine vasopressin (AVP), acting through a cAMP second messenger system, regulates osmotic water permeability ( Pf) of the collecting duct. In the collecting duct, the activities of cAMP and phosphonositides (PI) are mutually inhibitory. The P2Y2 receptor (P2Y2-R) is a G protein-coupled extracellular nucleotide receptor associated with PI signaling pathway. Previously, we showed that P2Y2-R is expressed in inner medullary collecting duct (IMCD) of rat, and its agonist (ATP/UTP) activation decreased AVP-induced Pf and resulted in enhanced production of prostaglandin E2. Hydrated and dehydrated states are associated with alterations in the circulating levels of AVP, expression and/or subcellular distribution of AVP-regulated aquaporin-2 water channel in IMCD and thus Pf of IMCD. We hypothesized that altered expression and/or signaling via P2Y2-R may also modulate IMCD function in these conditions. Sprague-Dawley rats were subjected to dehydration by water deprivation (48 h) or hydration (48 or 96 h) by providing sucrose water. Hydration or dehydration resulted in marked alterations in mRNA expression (Northern blot analysis and real-time RT-PCR) and protein abundance (Western blot analysis) of P2Y2-R, with hydrated rats showing significantly higher levels compared with dehydrated rats. Sequential hydration and dehydration experiments also revealed that the regulated expression profiles of P2Y2-R mRNA and protein are discordant. Conversely, the expression of V2-R mRNA remained unaltered during hydration and dehydration. Because virtually all renal cells release ATP in a regulated fashion, the observed alterations in P2Y2-R expression in the inner medulla in hydrated and dehydrated states may constitute a novel mechanism of purinergic modulation of IMCD function.

Bilateral ureteral obstruction downregulates expression of vasopressin-sensitive AQP-2 water channel in rat kidney

1996 ◽  
Vol 270 (4) ◽  
pp. F657-F668 ◽  
Author(s):  
J. Frokiaer ◽  
D. Marples ◽  
M. A. Knepper ◽  
S. Nielsen
Keyword(s):  
Ureteral Obstruction ◽  
Marked Reduction ◽  
Renal Excretion ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Water Channel ◽  
Free Water ◽  
Slow Recovery ◽  
Free Water Clearance ◽  
Urological Disorders

Polyuria after release of bilateral ureteral obstruction (BUO) is frequently seen in patients with urological disorders. In this study, we examined the effect of BUO and release of BUO on the expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2) in rat kidney. Ureters were obstructed for 24 h in all experiments, and BUO was either not released or released for 24 or 48 h or 7 days. Each group of experimental rats were matched with sham-operated controls. One kidney was used for membrane fractionation and immunoblotting, whereas the contralateral was fixed for immunocytochemistry. Immunoblotting demonstrated a significant reduction in AQP-2 expression in inner medullar during 24 h of BUO to 26 +/- 8% (P < 0.001). Release of BUO was associated with immediate onset of a predominant osmotic-dependent polyuria. Forty-eight hours after release of BUO, the reduction in AQP-2 expression persisted (19 +/- 8%, P < 0.001), concurrent with a marked nonosmotic postobstructive polyuria, as determined by a significant reduction in free-water clearance (-50 +/- 7 vs. -85 +/- 10 microliters.min-1.kg-1, P < 0.05). Immunofluorescence and immunoelectron microscopy confirmed the reduced levels of AQP-2 in collecting duct principal cells. Seven days after release, the renal excretion of water and electrolytes had almost normalized. However, the downregulation of AQP-2 was not partly reversed (49 +/- 14%, P < 0.001), and, consistent with this, the urinary concentrating capacity was significantly reduced 7 days after release to a 18-h period of thirst. This strongly suggests that the persistent downregulation of AQP-2 is the cause of the slow recovery in concentration capacity. In conclusion, BUO and release of BUO were associated with a marked reduction in expression of AQP-2, coincident with the development and maintenance of postobstructive polyuria. Thus reduced AQP-2 levels may represent an important factor in the slow recovery from postobstructive diuresis.

Altered expression profile of transporters in the inner medullary collecting duct of aquaporin-1 knockout mice

2005 ◽  
Vol 289 (1) ◽  
pp. F194-F199 ◽  
Author(s):  
Ryan G. Morris ◽  
Shinichi Uchida ◽  
Heddwen Brooks ◽  
Mark A. Knepper ◽  
Chung-Lin Chou
Keyword(s):  
Expression Profile ◽  
Knockout Mice ◽  
Collecting Duct ◽  
Water Channel ◽  
Major Protein ◽  
Altered Expression ◽  
Aquaporin 1 ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct ◽  
Concentrating Defect

Aquaporin-1 is the major protein responsible for transport of water across the epithelia of the proximal tubule and thin descending limbs. Rapid water efflux across the thin descending limb is required for the normal function of the countercurrent multiplier mechanism. Therefore, urinary concentrating capacity is severely impaired in aquaporin-1 knockout (AQP1 −/−) mice. Here, we have investigated the long-term consequences of deletion of the AQP1 gene product by profiling abundance changes in transporters expressed in the inner medullas of AQP1 (−/−) mice vs. heterozygotes [AQP1 (+/−)], which have a normal concentrating capacity. Semiquantitative immunoblotting demonstrated marked suppression of two proteins strongly expressed in the inner medullary collecting duct (IMCD): UT-A1 (a urea transporter) and AQP4 (a basolateral water channel). Furthermore, the urea permeability of the IMCD was significantly reduced in AQP1 (−/−) mice. In contrast, there was increased expression of three proteins normally expressed at higher levels in the cortical collecting duct (CCD) than in IMCD: AQP3 (another basolateral water channel) and the epithelial sodium channel subunits β-ENaC and γ-ENaC. Changes in expression of these proteins were confirmed by immunocytochemistry. Messenger RNA profiling (real-time RT-PCR) revealed changes in UT-A1, β-ENaC, γ-ENaC, and AQP3 transcript abundance that paralleled the changes in protein abundance. Thus, from the perspective of transport proteins, the IMCDs of AQP1 (−/−) mice have a significantly altered phenotype. To address whether these changes are specific to AQP1 (−/−) mice, we profiled IMCD transporter expression in a second knockout model manifesting a concentrating defect, that of ClC-nK1, a chloride channel in the ascending thin limb important for urinary concentration. As in the AQP1 knockout mice, ClC-nK1 (−/−) mice showed decreased expression of UT-A1 and increased expression of β-ENaC and γ-ENaC vs. WT controls. In conclusion, the expression profile of IMCD transporters is markedly altered in AQP1 −/− mice and this manifestation is related to the associated concentrating defect.

Altered expression of urea transporters in response to ureteral obstruction

2004 ◽  
Vol 286 (6) ◽  
pp. F1154-F1162 ◽  
Author(s):  
Chunling Li ◽  
Janet D. Klein ◽  
Weidong Wang ◽  
Mark A. Knepper ◽  
Søren Nielsen ◽  
...  
Keyword(s):  
Ureteral Obstruction ◽  
Urinary Tract Obstruction ◽  
Collecting Duct ◽  
Free Water ◽  
Protein Abundance ◽  
Water Reabsorption ◽  
Plasma Urea ◽  
Altered Expression ◽  
Vasa Recta ◽  
Transporter Expression

Urea plays an important role in the urinary concentrating capacity. Renal inner medullary (IM) urea transporter expression was examined in rats with bilateral (BUO) or unilateral ureteral obstruction (UUO). BUO (24 h) was associated with markedly increased plasma urea (42.4 ± 1.0 vs. 5.2 ± 0.2 mmol/l) and a significant decease in expression of UT-A1 (28 ± 8% of sham levels), UT-A3 (45 ± 11%), and UT-B (70 ± 8%). Immunocytochemistry confirmed downregulation of UT-A1 and UT-A3 in IM collecting duct and UT-B in the descending vasa recta. Three days after release of BUO, UT-A1, UT-A3, and UT-B remained significantly downregulated (UT-A1: 37 ± 6%; UT-A3: 25 ± 6%; and UT-B: 10 ± 5% of sham levels; P < 0.05) concurrent with a persistent polyuria and a marked reduction in solute-free water reabsorption (115 ± 11 vs. 196 ± 8 μl·min−1·kg−1, P < 0.05). Moreover, 14 days after release of BUO, total UT-A1, UT-A3, and UT-B remained significantly decreased compared with sham-operated controls and urine urea remained reduced (588 ± 43 vs. 1,150 ± 94 mmol/l). Consistent with increased levels of plasma urea 24 h after onset of UUO (7.4 ± 0.3 vs. 4.8 ± 0.3 mmol/l), the protein abundance of UT-A1, UT-A3, and UT-B in IM was markedly reduced in the obstructed kidney, which was confirmed by immunocytochemistry. In the nonobstructed kidney, the expression of urea transporters did not change. In conclusion, reduced expression of UT-A1, UT-A3, and UT-B levels in both BUO and UUO rats suggests that urea transporters play important roles in the impaired urinary concentrating capacity in response to urinary tract obstruction.

scholarly journals Intracellular localization of the P21rho proteins.

1992 ◽  
Vol 119 (3) ◽  
pp. 617-627 ◽  
Author(s):  
P Adamson ◽  
H F Paterson ◽  
A Hall
Keyword(s):  
Plasma Membrane ◽  
Cellular Localization ◽  
Biological Function ◽  
Intracellular Localization ◽  
Heterologous Proteins ◽  
Ras Proteins ◽  
Rho Proteins ◽  
Early Endosomes ◽  
Chimeric Molecules ◽  
Palmitoylation Site

The three mammalian ras proteins associated specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-rasB) or a palmitoylation site (p21Ha-ras and p21N-ras). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-rasB or P21N-ras/Ha-ras. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins, rap1, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21rho, the prototype of this family are known; the COOH termini of p21rhoA and p21rhoC resemble p21K-rasB with a polybasic domain, whereas p21rhoB resembles p21N-ras/Ha-ras with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21ras proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21rhoA and p21rhoC is cytosolic whereas p21rhoB is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p21rho proteins cycle on and off the plasma membrane and this may have important implications for their biological function.

Candesartan prevents long-term impairment of renal function in response to neonatal partial unilateral ureteral obstruction

2007 ◽  
Vol 292 (2) ◽  
pp. F736-F748 ◽  
Author(s):  
Sukru Oguzkan Topcu ◽  
Michael Pedersen ◽  
Rikke Nørregaard ◽  
Guixian Wang ◽  
Mark Knepper ◽  
...  
Keyword(s):  
Ureteral Obstruction ◽  
Collecting Duct ◽  
Water Channel ◽  
Free Water ◽  
Unilateral Ureteral Obstruction ◽  
Obstructive Nephropathy ◽  
Sham Operation ◽  
Ang Ii ◽  
Functional Changes

Angiotensin II (ANG II) plays an important role in the development of obstructive nephropathy. Here, we examined the effects of the ANG II receptor type 1 (AT1R) blockade using candesartan on long-term renal molecular and functional changes in response to partial unilateral ureteral obstruction (PUUO). Newborn rats were subjected to severe PUUO or sham operation (Sham) within the first 48 h of life. Candesartan was provided in the drinking water (10 mg·kg−1·day−1) from day 21 of life until 10 wk of age. Renal blood flow (RBF) was evaluated by MRI, glomerular filtration rate (GFR) was measured using the renal clearance of 51Cr-EDTA, and the renal expression of Na-K-ATPase and the collecting duct water channel aquaporin-2 (AQP2) was examined by immunoblotting and immunocytochemistry. At 10 wk of age, PUUO significantly reduced RBF (0.8 ± 0.1 vs. 1.6 ± 0.1 ml·min−1·100 g body wt−1; P < 0.05) and GFR (37 ± 16 vs. 448 ± 111 μl·min−1·100 g body wt−1; P < 0.05) compared with Sham. Candesartan prevented the RBF reduction (PUUO+CAN: 1.6 ± 0.2 vs. PUUO: 0.8 ± 0.1 ml·min−1·100 g body wt−1; P < 0.05) and attenuated the GFR reduction (PUUO+CAN: 265 ± 68 vs. PUUO: 37 ± 16 μl·min−1·100 g body wt−1; P < 0.05). PUUO was also associated with a significant downregulation in the expression of Na-K-ATPase (75 ± 12 vs. 100 ± 5%, P < 0.05) and AQP2 (52 ± 15 vs. 100 ± 4%, P < 0.05), which were also prevented by candesartan (Na-K-ATPase: 103 ± 8 vs. 100 ± 5% and AQP2: 74 ± 13 vs. 100 ± 4%). These findings were confirmed by immunocytochemistry. Consistent with this, candesartan treatment partly prevented the reduction in solute free water reabsorption and attenuated fractional sodium excretion in rats with PUUO. In conclusion, candesartan prevents or attenuates the reduction in RBF, GFR and dysregulation of AQP2 and Na-K-ATPase in response to congenital PUUO in rats, suggesting that AT1R blockade may protect the neonatally obstructed kidney against development of obstructive nephropathy.

The Immuno-Electron Microscopic Localization of the Mo-Fe Protein Component of Nitrogenase in Cells of Azotobacter Vinelandii

1973 ◽  
Vol 31 ◽  
pp. 574-575
Author(s):  
J. T. Stasny ◽  
R. C. Burns ◽  
R. W. F. Hardy
Keyword(s):  
Cellular Localization ◽  
Direct Evidence ◽  
Azotobacter Vinelandii ◽  
Intracellular Localization ◽  
Protein Component ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Fe Protein ◽  
Intracytoplasmic Membranes

Structure-functlon studies of biological N2-fixation have correlated the presence of the enzyme nitrogenase with increased numbers of intracytoplasmic membranes in Azotobacter. However no direct evidence has been provided for the internal cellular localization of any nitrogenase. Recent advances concerned with the crystallizatiorTand the electron microscopic characterization of the Mo-Fe protein component of Azotobacter nitrogenase, prompted the use of this purified protein to obtain antibodies (Ab) to be conjugated to electron dense markers for the intracellular localization of the protein by electron microscopy. The present study describes the use of ferritin conjugated to goat antitMo-Fe protein immunoglobulin (IgG) and the observations following its topical application to thin sections of N2-grown Azotobacter.

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