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Articles

Dietary Biotin Intake Modulates the Pool of Free and Protein-Bound Biotin in Rat Liver¹

Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences and the College of Agricultural and Life Sciences, University of Florida, Gainesville, FL 32611-0370

³To whom correspondence should be addressed. E-mail: rjmc{at}gnv.ifas.ufl.edu.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The current studies were undertaken to analyze the relationships among dietary biotin intake, hepatic free biotin and hepatic protein-bound biotin in rats. The biotin status of rats was manipulated through dietary intervention to model moderate biotin deficiency, adequacy, supplementation and pharmacologic biotin supplementation (0, 0.06, 0.6 and 100 mg/kg, respectively). Urinary biotin excretion was directly related to biotin intake, but no difference between biotin-adequate and biotin-supplemented rats was detected. In contrast, plasma biotin was directly and significantly regulated by biotin intake at every intake level. A hepatic free biotin pool was directly demonstrated in these studies, and like plasma, its size was directly related to dietary biotin intake. The relationship between dietary biotin intake and protein-bound biotin was also analyzed. Moderate biotin deficiency markedly decreased the abundance of each biotinylated polypeptide in rat liver. Biotin supplementation did not significantly elevate the abundance of biotinylated pyruvate, propionyl CoA, methylcrotonyl CoA or acetyl CoA carboxylase 1. The abundance of biotinylated acetyl CoA carboxylase 2, however, was significantly higher in biotin-supplemented rats. Pharmacologic biotin intake significantly reduced the abundance of biotinylated propionyl CoA and methylcrotonyl CoA carboxylase. These results indicate the following: 1) moderate biotin deficiency reduces free and protein bound biotin; 2) biotin intakes in rats that mimic the currently recommended daily value (DV) do not result in full protein biotinylation; and 3) pharmacologic supplementation may reduce the abundance of functional carboxylases.

KEY WORDS: • vitamin • biotin • carboxylase • deficiency • supplementation • rats

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Biotin is present in organisms in two distinct forms, i.e., unassociated (free) and protein bound. In the protein-bound form, biotin is covalently bound to the -amino group of lysine residues occurring in a specific amino acid motif (1) . In contrast, free biotin exists as a distinct pool in several compartments, including urine, plasma and tissue. The free biotin undergoes substantial metabolism in lower as well as higher organisms (2 3 4 5 6) . In urine, the biotin and its associated metabolites are all free due to normal glomerular filtration of protein. The majority (90%) of serum biotin is also free, with the remainder either reversibly or covalently bound (7 ,8) . Indirect evidence for an intracellular pool of free biotin has been demonstrated in several ex vivo systems, including cultured hepatocytes and peripheral blood mononuclear cells (9 ,10) . The free biotin pool in the tissues of intact animals has not been previously characterized.

Biotin deficiency markedly reduces urinary biotin excretion, with onset beginning 2–3 wk after consumption of a biotin-free diet (11) . In a similar manner, biotin deficiency effectively reduces serum and plasma biotin levels (11 12 13) . The depleting effect of biotin deficiency on tissue biotin levels, including brain, liver and pancreas, has also been analyzed in several studies (14 15 16) . As might be expected, consumption of a biotin-supplemented diet or administration of biotin markedly elevates urinary biotin excretion as well as serum or plasma levels in both humans and rodents (17 ,18) . Biotin supplementation also raises the concentration of the biotin metabolites, including biotin sulfoxide and bisnorbiotin (19) . To our knowledge, the effect of dietary biotin intake on the free pool of biotin in tissues has not been previously investigated.

The relationship between dietary biotin intake and protein-bound biotin has also been analyzed. Biotinylation is required for the carboxylase function, and therefore conditions that reduce protein biotinylation reduce enzymatic activity (11 ,15 ,16) . It has been proposed that during biotin deficiency, a pool of apocarboxylases is present because the administration of biotin to biotin-deficient rats results in a rapid restoration of the abundance of biotinylated polypeptides (20) .

We sought to determine whether alterations in dietary biotin intake regulate tissue biotin pools in a manner similar to plasma and urine. In this report, the free pool of biotin in the liver and its relationship to protein biotinylation were analyzed separately in biotin-deficient, biotin-adequate, biotin-supplemented and pharmacologically supplemented rats. The results have implications for understanding the relationship between dietary biotin intake and the maintenance of biotin-dependent cellular function in higher organisms.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Materials.

Male Sprague-Dawley rats were obtained from Harlan (Indianapolis, IN). Purified biotin-free rodent diet based on a modified AIN76A formulation was obtained from Research Diets (New Brunswick, NJ). The composition of the diet is as follows: spray-dried egg white (20%), cornstarch (15%), sucrose (50%), cellulose (5%), corn oil (5%), AIN76A salt and vitamin mix (without biotin) and choline bitartrate (0.2%). d-Biotin, protease inhibitor cocktail and o-phenylenediamine dihydrochloride were purchased from Sigma (St. Louis, MO); avidin-horseradish peroxidase (Avidin-HRP)⁴ was purchased from Pierce Chemical Company (Birmingham, AL); 96-well microtiter plates (Nunc Maxisorp) and bovine serum albumin (BSA) were purchased from Fisher Scientific (Pittsburgh, PA). Enhanced chemiluminescence (ECL)-Plus reagent was purchased from Amersham-Pharmacia (Piscataway, NJ). Biotinylated BSA was synthesized by mixing 50 mL of 10 g/L BSA in ice-cold 0.1 mol/L NaHCO ₃ (pH 7.5) with 5 mL of a 12 g/L N-hydroxysuccinimide ester (NHS-biotin) in dimethyl sulfoxide overnight at 4°C. The mixture was dialyzed for 48 h with gentle stirring at 4°C.

Animals and dietary treatments.

In this series of experiments, we used a standard diet commonly used for the analysis of dietary components formulated by the American Institute of Nutrition (AIN76A) (21) . Although similar to diets used in other studies on biotin nutriture, this diet differs substantially from those used earlier in terms of carbohydrate source and amount, fatty acid composition and some vitamins and minerals (11 12 13 ,20 ,22 ,23) . This diet has been modified to include spray-dried egg white as its sole protein source. Avidin protein in egg white binds 1.44 mg biotin/kg of purified diet, inhibiting biotin absorption (24) . The level of dietary biotin designated in these studies represents biotin in excess of the binding capacity of the dietary egg white avidin. Male Sprague-Dawley strain rats (n = 20), 50–74 g initial weight, were housed individually in hanging wire-bottomed cages in an environmentally controlled room with constant temperature (22°C) and a 12-h light:dark cycle. Rats were randomly assigned to an AIN 76A-based egg white powdered diet containing one of the following biotin concentrations (n = 5/group): 0 mg biotin/kg diet (deficient), 0.06 mg/kg (adequate), 0.6 mg/kg (supplemented) and 100 mg/kg (pharmacologic). After a 4-d acclimation period during which all rats consumed the 0.6 mg biotin/kg diet, rats were randomly assigned to groups and given free access to highly purified water for 3 wk. Body weight and food consumption were measured three times weekly for 21 d. Rats were placed into metabolic chambers 24 h before the end of the study to allow the discrete collection of urine for biotin analysis. Rats were anesthetized under halothane vapor and killed by exsanguination. All procedures were approved by the University of Florida Animal Care and Use Committee.

Sample preparation.

Blood was withdrawn using an EDTA-coated syringe to inhibit clotting and centrifuged at 10,000 x g for 10 min to collect plasma. For the competitive assay of plasma biotin, samples were ultrafiltered using a 5000 nominal molecular weight cut-off (NMWCO) filter (Millipore, Bedford, MA) as previously described (25) . Liver (300 mg) was removed and homogenized in 5 volumes of ice-cold homogenization buffer (300 mmol/L mannitol, 10 mmol/L HEPES, pH 7.2, 1 mmol/L EDTA and protease inhibitor cocktail) and centrifuged at 200,000 x g for 30 min at 4°C. The supernatant (soluble fraction) was also ultrafiltered using a 5000 NMWCO filter before the competitive binding assay of biotin. The pellet, representing the total membrane fraction, was resuspended in homogenization buffer to a final concentration of 40 g/L. All samples were immediately frozen in a mixture of dry ice and isopropanol and stored at -80°C until needed.

Competitive binding assay of biotin.

The measurement of biotin in urine, plasma and liver was performed with a coupled HPLC/competitive binding assay as previously described with minor modifications (25) . The reversed-phase column used was a Sphereclone 250 x 4.6 mm (Phenomenex, Torrance, CA), and the biotin-containing chromatography fractions were dried under a stream of nitrogen before the assay.

Synthesis of Avidin–AlexaFluor 430.

NeutrAvidin, an isoelectrically neutral and deglycosylated form of avidin, was conjugated to the succinimidyl ester form of AlexaFluor 430. NeutrAvidin, 10 g/L in 50 mmol/L sodium bicarbonate, pH 8.3, was mixed 5:1 (v/v) with a solution of AlexaFluor 430 succinimidyl ester, 10 g/L, in dimethyl sulfoxide. The mixture was slowly mixed on a vortexer for 1 h at room temperature. Unconjugated dye was removed by size exclusion chromatography over a DG-10 column (Bio-Rad, Hercules, CA) equilibrated in PBS (20 mmol/L sodium phosphate, pH 7.2, 150 mmol/L NaCl). Equal fractions (1 mL) were collected and the peaks with the highest absorbance at 280 nm combined. Sodium azide (0.2 g/L) was added for preservation, and the conjugate was stored at 4°C protected from light.

Detection and quantification of biotinylated proteins.

Using the avidin blotting technique, we specifically detected five distinct proteins in the liver, corresponding to the five carboxylase enzymes acetyl CoA carboxylase (ACC) isoforms 1 and 2, pyruvate carboxylase (PC), propionyl CoA carboxylase (PCC) and methylcrotonyl CoA carboxylase (MCC). The specificity of this detection was confirmed through competition with excess biotin (data not shown). Two slightly different techniques were used in this analysis, i.e., direct fluorescent avidin blotting for PC, PCC and MCC, and enzyme-linked horseradish peroxidase–avidin conjugate for detection of ACC isoforms 1 and 2. The enzyme-linked method was used for the last-mentioned detection due to the sensitivity limit of the direct fluorescent avidin blotting.

The concentration of protein in liver samples was determined as previously described (26) . Equal amounts of liver total membrane fractions (0.1 mg for the analysis of ACC1 and ACC2, 0.05 mg for all other carboxylases) were resolved by 10% SDS-PAGE and run overnight at 40 V. The gel was electroblotted to polyvinyldifluoride (PVDF; Immobilon-P, Millipore) for 2.5 h at 12 V as previously described (27) . The blot was then washed in three changes of methanol and allowed to air dry. For the detection of ACC isoforms 1 and 2, the blot was incubated in 5 g/L nonfat dry milk (NFDM) in Tris-buffered saline (20 mmol/L Tris, pH 7.4, 150 mmol/L NaCl) with 0.05% (v/v) Tween 20 (TBS-T) and a 1:750 dilution of Neutralite avidin–horseradish peroxidase conjugate for 1 h. The PVDF was washed three times in TBS-T without NFDM. A chemiluminescent substrate that also exhibits fluorescent properties (ECL-Plus) was then applied to the blot and allowed to stand for 5 min. The reaction was then stopped by placing the blot back into TBS-T. For the detection of PC, PCC and MCC, the blot was incubated in TBS-T/0.5% NFDM containing avidin–AlexaFluor 430 conjugate for 45 min at room temperature on an orbital shaker. Biotinylated bands on both blots were then detected and emitted fluorescence quantified on a Storm fluorescent optical scanner as described by the manufacturer (Amersham-Pharmacia). Several control experiments were performed to ensure that measurement was in the linear range of detection (data not shown).

Statistical analysis.

Results are expressed as means ± SEM. The significance of differences (P < 0.05) was tested by one-way ANOVA with Newman-Keuls post test. In some cases, the data were log transformed to achieve acceptable homogeneity of variance.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Effect of dietary biotin intake on rat growth and food intake.

At the observed rate of food intake, rats consumed 0 (deficient), 3.69 (adequate), 36.9 (supplemented) or 6150 (pharmacologic) nmol biotin/d. The dietary biotin level in the AIN76A modified diet had no significant effect on either growth or food intake (Fig. 1 ). Additionally, no outward signs of biotin deficiency were found in the rats consuming the biotin-free diet. Toxicity was not evident in pharmacologically supplemented rats.

View larger version (16K): [in this window] [in a new window]   Figure 1. Body weight (upper panel) and food intake (lower panel) of rats fed various levels of biotin. Open symbols, 0 mg biotin/kg; light gray symbols, 0.06 mg biotin/kg; dark gray symbols, 0.6 mg biotin/kg; black symbols, 100 mg biotin/kg. Results are expressed as means ± SEM, n = 5.

Rats consuming the biotin-free diet for 21 d excreted significantly less biotin in the urine compared with biotin-adequate rats (P < 0.05, 21 ± 10 and 168.4 ± 82 nmol/h, respectively) (Fig. 2 ,upper panel). Rats consuming the diet containing a pharmacologic level of biotin had a greatly increased urinary excretion rate (P < 0.001, 35090 ± 59 nmol/h). There was no significant difference in the urinary biotin excretion rate between biotin-adequate and biotin-supplemented rats (P < 0.05, 168.4 ± 82.6 and 206.2 ± 36.4 nmol/h, respectively).

View larger version (24K): [in this window] [in a new window]   Figure 2. Urinary biotin excretion (upper panel), plasma biotin (middle panel) and free biotin in the soluble fraction of liver (lower panel) in rats consuming varying levels of dietary biotin. Data are means ± SEM, n = 5. Bars with different letters differ, P < 0.05.

The concentration of hepatic free biotin, normalized to wet organ weight, was 0.032 ± 0.007 nmol/g tissue in biotin-adequate rats (Fig. 2 , lower panel). Biotin-deficient rats exhibited a 78% reduction in free biotin (P < 0.05, 0.006 ± 0.002 nmol/g tissue). Hepatic free biotin did not differ between biotin-adequate and biotin-supplemented rats (0.032 ± 0.007 and 0.061 ± 0.014 nmol/g tissue). Pharmacologically supplemented rats had a 44-fold elevation in free liver biotin concentration compared to biotin-adequate rats (P < 0.01, 1.414 ± 0.138 nmol/g tissue).

Effect of dietary biotin intake on protein biotinylation.

We next assessed the effect of dietary biotin intake on the relative abundance of biotinylated polypeptides using avidin blotting. Rats consuming biotin-free diets had less of the biotinylated forms of all carboxylases after 21 d (P < 0.05) (Fig. 3 ). The magnitude of the reduction ranged from 50% for ACC isoforms 1 and 2 to 60% for PC, PCC and MCC (Fig. 4 ). The abundance of ACC isoform 2 was significantly elevated by 40% in biotin-supplemented rats compared to biotin-adequate rats (P < 0.05). There was no difference in the abundance of biotinylated PC, PCC, MCC, and ACC1 between biotin-adequate and biotin-supplemented rats. Pharmacologic biotin supplementation significantly reduced the abundance of biotinylated ACC2, PCC and MCC (P < 0.05).

View larger version (46K): [in this window] [in a new window]   Figure 3. Effect of dietary biotin intake on the abundance of biotinylated polypeptide in rats consuming varying levels of dietary biotin. An equal amount of rat liver total membrane fraction was resolved on either a 5% (ACC1, ACC2 blot) or 10% (PC, PCC, MCC blot) SDS-PAGE gel and transferred to PVDF as described in the Materials and Methods section. ACC1 and ACC2 were then detected using avidin–HRP and ECL-Plus as described, and PC, PCC, and MCC were detected using avidin–AlexaFluor 430 as described. Abbreviations: ACC, acetyl CoA carboxyalse; PC, pyruvate carboxylase; PCC, propionyl CoA carboxylase; MCC, methylcrotonyl CoA carboxylase; PVDF, polyvinyldifluoride; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence.

View larger version (27K): [in this window] [in a new window]   Figure 4. Quantification of abundance of biotinylated polypeptides in rats of varying biotin status. The fluorescence of each detected biotinylated polypeptide was quantified using the Storm optical scanner and ImageQuant software. PC, pyruvate carboxylase; PCC/MCC, propionyl CoA carboxylase and methylcrotonyl CoA carboxylase; ACC1, acetyl CoA carboxylase isoform 1; ACC2, acetyl CoA carboxylase isoform 2. All data are expressed in arbitrary units as means ± SEM, n = 5. Bars with different letters differ, P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The experimental diets chosen in this study model several relevant states of biotin nutriture. Dietary biotin intake had no effect on growth rate or food intake, suggesting that severe deficiency was absent. This is in contrast to earlier studies in which loss of growth was observed relatively early in the dietary manipulation, and may represent an improvement in the use of the AIN76A diet design (11 ,12 ,36) . In these experiments, rats consuming the 0.06 mg biotin/kg diet exhibited an intake of 400 µg/d for a 70-kg individual, approximating the daily value (DV) of 300 µg. It should be noted that an important inconsistency exists between the DV and the current dietary reference intake of 30 µg/d for adults. A biotin-supplemented condition was obtained in rats consuming the 0.6 mg/kg diet, or 10 times the DV. This level represents a dietary intake easily obtained in self-selected biotin supplements or in the treatment of biotin-responsive disorders, such as biotinidase deficiency or multiple carboxylase deficiency (37 38 39) . To determine whether pharmacologic doses of biotin might lead to adverse effects on physiology, some rats were given a diet containing 100 mg/kg, a dietary level that is the highest intake yet tested.

Despite the lack of outward signs of deficiency, biochemical measurements clearly indicated a change in biotin status. We confirmed previous studies that demonstrated a direct relationship between dietary biotin intake and urinary biotin excretion (6 ,11) . Urinary biotin excretion was significantly depressed in moderately biotin-deficient rats, suggesting the induction of homeostatic mechanisms attempting to conserve biotin pools. There was no difference in the urinary excretion of biotin in rats consuming either the adequate or supplemented diets, suggesting that biotin pools were not yet sufficiently large to spill over into the urine. Alternatively, biotin reabsorption capacity in the kidney might not be saturated, preventing an increase in urinary excretion. This was an unexpected finding because the adequate diet in our studies corresponded to a relatively high human intake, and the supplemented diet represented a 10-fold increase over that level. It is possible that, consistent with earlier studies in humans, excess biotin was catabolized to bisnorbiotin or biotinsulfoxide (18) . Biotin catabolites were not measured in these studies. We observed the expected large increases in urinary biotin excretion and plasma biotin concentrations in rats consuming the pharmacologic doses of biotin, in agreement with human studies that demonstrate a rapid and large excretion of biotin in excess of required levels (18 ,19 ,40) .

Plasma biotin pools were similarly influenced by dietary biotin intake. Moderate biotin deficiency was remarkably effective in reducing plasma biotin levels, suggesting that in this limited case, plasma biotin was a useful index of biotin status. In fact, plasma biotin might be argued to be a better marker of biotin status in this study because a significant elevation of plasma biotin was observed in biotin-supplemented rats, in contrast to urinary biotin excretion for which no such difference was found. Whether such a conclusion can be extended to biotin-supplemented humans is unclear because during biotin deficiency, urinary biotin excretion appears to be a more reliable indicator of biotin status (41) . Plasma biotin was significantly elevated in pharmacologically supplemented rats compared with all other dietary groups, demonstrating that very high circulating biotin concentration did not elicit any outward adverse effects on physiology.

The soluble fraction of liver, which in our experiments reasonably represents the cytosolic cellular fraction, also demonstrated a free biotin pool responsive to dietary biotin intake. The intracellular biotin pool in biotin-deficient rats was 20% that in biotin-adequate rats. Like plasma biotin, it was possible to greatly expand the hepatic free biotin pool possibly available for protein biotinylation or other proposed functions of biotin (42) . Although high plasma biotin concentrations may have contaminated the liver samples and falsely elevated the biotin level, the ratio of plasma to liver free biotin was very small in all dietary groups, suggesting that contamination was not contributing substantially to the liver biotin measurement.

After establishing the biotin status of each dietary group, the effect of biotin intake on the abundance of biotinylated polypeptides was analyzed. An important assumption in this type of analysis is that the abundance of the polypeptide in question is not altered in abundance by the dietary manipulation. We also assert that changes in the abundance of biotinylated polypeptides are due to the biotin deficiency per se, rather than changes in the apoprotein form of the polypeptide. Although we cannot rule out the latter possibility because Western blot analysis of each carboxylase was not performed, our assumption is supported by the following findings: 1) biotin deficiency does not alter the abundance of carboxylase mRNA, and 2) in biotin-deficient rats, large pools of apocarboxylases are present that are immediately available for biotinylation upon the return of biotin to the system (20) . Post-transcriptional regulation of PCC and MCC has been proposed, but the relevant studies did not directly analyze the amount of PCC or MCC polypeptide (20) . Biotin-deficient rats, even those not exhibiting outward signs of deficiency, have a significantly reduced abundance of the biotinylated form of all carboxylases. This demonstrates that loss of carboxylase activity is occurring before overt signs of biotin deficiency emerge. Biotin-adequate rats, consuming close to the suggested DV, appear to demonstrate full biotinylation of PC, MCC, PCC and ACC1 because biotin-supplemented rats did not have significantly more biotinylated carboxylases. Interestingly, we found a significant difference in the abundance of biotinylated ACC2 between biotin-adequate and biotin-supplemented rats, consistent with an earlier report of higher sensitivity of ACC biotinylation to dietary biotin supply (23) . We find this observation to be of interest because it suggests that in rats consuming near the DV for biotin, the function of ACC2 may not be maximal. A recent report provides strong evidence that ACC2 is localized to the outer mitochondrial membrane, where it is anchored to the membrane by an additional amino terminal membrane-spanning sequence (43) . This places the enzymatic domain of ACC2 facing the cytosolic side. A proposed function for ACC2 in this orientation is the production of cytosolic malonyl CoA, which is a potent allosteric inhibitor of the carnitine acyltransferase, the carrier by which fatty acids enter the mitochondria for ß-oxidation. Therefore, the function of ACC2 may be to suppress fatty acid oxidation in certain tissues. The physiologic or functional implications for incomplete biotinylation of ACC2 under presumably adequate biotin intake can of course not be inferred from our studies. Several other studies have clearly demonstrated alterations in fatty acid metabolism in biotin-deficient rats, but have usually focused on fatty acid biosynthesis rather than ß-oxidation (44 45 46 47 48 49 50 51 52 53 54 55) . Another unexpected finding of this study was the effect of pharmacologic intake of dietary biotin on protein biotinylation. We observed that in pharmacologically supplemented rats, the abundance of some biotinylated carboxylases was significantly reduced. Whether this represents a physiologically relevant detrimental effect of pharmacologic biotin intake has yet to be determined.

Changes in biotin status have been demonstrated to affect a range of metabolic processes, from changes in carboxylase activity to changes in the expression of nonbiotin-dependent enzymes such as glucokinase, ornithine transcarbamolase and phosphoenolpyruvate carboxykinase (13 ,56 57 58 59) . The sensitivity of this regulation has not been addressed, but the possibility that marginal biotin status at the cellular level regulates gene expression cannot be ruled out and is, in fact, supported by this work because marginal deficiency and supplementation resulted in altered tissue free biotin pools. Finally, the use of rats as a model for biotin metabolism in humans has been validated (60 ,61) ; thus, our data suggest that our current knowledge and recommendations concerning biotin intake and the maintenance of biotin function may not be in agreement and require further investigation.

	FOOTNOTES

 
¹ Supported by the Florida Agricultural Experiment Station and approved for publication as Journal Series no. R-08063.

² These authors contributed equally to this study.

⁴ Abbreviations used: ACC, acetyl CoA carboxyalse; BSA, bovine serum albumin; DV, daily value; ECL, enhanced chemiluminescence; HRP, horseradish peroxidase; MCC, methylcrotonyl CoA carboxylase; NFDM, nonfat dry milk; NMWCO, nominal molecular weight cut-off; PC, pyruvate carboxylase; PCC, propionyl CoA carboxylase; PVDF, polyvinyldifluoride; TBST, Tris-buffered saline with Tween 20.

Manuscript received April 8, 2001. Initial review completed May 3, 2001. Revision accepted June 6, 2001.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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