Biomedical subjects

M A Dudley

Publications and source records attributed to M A Dudley.

At least 19 recordsLinked to original sources

PubMed2001

Oral IGF-I alters the posttranslational processing but not the activity of lactase-phlorizin hydrolase in formula-fed neonatal pigs.

To determine the cellular mechanism whereby oral insulin-like growth factor I (IGF-I) increases intestinal lactase-phlorizin hydrolase (LPH) activity, we studied 2-d-old pigs fed cow's milk formula (control, n = 5), formula + low IGF-I (0.5 mg/L; n = 6) or formula + high IGF-I (12.0 mg/L, n = 6) for 15 d. On d 15, intestinal protein synthesis and lactase processing were measured in vivo in fed pigs using a 6-h intravenous, overlapping infusion of multiple stable isotopes (2H(3)-Leu, 13C(1)-Leu, 13C(1)-Phe, 2H(5)-Phe, 13C(6)-Phe and 13C(9)-Phe). Morphometry and cell proliferation also were measured in the jejunum and ileum. Neither dose of IGF-I affected the masses of wet tissue, protein or DNA, or the villus height, cell proliferation or LPH-specific activity. Oral IGF-I decreased the synthesis and abundance of prolactase-phlorizin hydrolase (pro-LPH), but increased brush-border (BB)-LPH synthesis in the ileum. The BB-LPH processing efficiency was twofold to threefold greater in IGF-fed than in control pigs. In all pigs, villus height and the total mucosal and specific activity of LPH activity were greater in the ileum than in the jejunum, yet the synthesis of BB-LPH were significantly lower in the ileum than in the jejunum. We conclude that oral IGF-I increases the processing efficiency of pro-LPH to BB-LPH but does not affect LPH activity. Moreover, the posttranslational processing of BB-LPH is markedly lower in the ileum than in the jejunum.

Administration, Oral↗

PubMed2001

Intestinal protein and LPH synthesis in parenterally fed piglets receiving partial enteral nutrition and enteral insulinlike growth factor 1.

BACKGROUND: Providing partial enteral nutrition (PEN) supplemented with insulinlike growth factor-1 (IGF-1) to parenterally fed piglets increases lactase-phlorizin hydrolase (LPH) activity, but not LPH mRNA. The current aim was to investigate potential mechanisms by which IGF-1 up-regulates LPH activity. METHODS: Newborn piglets (n = 15) received 100% parenteral nutrition (TPN), 80% parenteral nutrition + 20% parenteral nutrition (PEN), or PEN + IGF-1 (1.0 mg. kg-1. d-1) for 7 days. On day 7, [2H3]-leucine was intravenously administered to measure mucosal protein and brush border LPH (BB LPH) synthesis. RESULTS: Weight gain, nutrient intake, and jejunal weight and length were similar among the treatment groups. Partial enteral nutrition alone increased mucosal weight, villus width and cross-sectional area, LPH activity, mRNA expression, and high mannose LPH precursor (proLPHh) abundance compared with TPN (P<0.05). Insulinlike growth factor-1 further increased mucosal weight, LPH activity, and LPH activity per unit BB LPH approximately twofold over PEN alone (P < 0.05) but did not affect LPH mRNA or the abundance of proLPHh (one of the LPH isoforms) or mature LPH. Isotopic enrichment of [2H3]-leucine in plasma, mucosal protein, and LPH precursors, and the fractional and absolute synthesis rates of mucosal protein and LPH were similar among the treatment groups. Insulinlike growth factor-1 treatment increased total mucosal protein synthesis (60%, P < 0.05) but not LPH synthesis compared with the other two groups. CONCLUSIONS: Because IGF-1 did not affect the fractional synthesis rate of either mucosal protein or LPH, the authors suggest that enteral IGF-1 increases mucosal protein mass and LPH activity by suppressing mucosal proteolytic degradation.

Animals↗

PubMed2001

Lactase synthesis is pretranslationally regulated in protein-deficient pigs fed a protein-sufficient diet.

The in vivo effects of protein malnutrition and protein rehabilitation on lactase phlorizin hydrolase (LPH) synthesis were examined. Five-day-old pigs were fed isocaloric diets containing 10% (deficient, n = 12) or 24% (sufficient, n = 12) protein. After 4 wk, one-half of the animals in each dietary group were infused intravenously with [(13)C(1)]leucine for 6 h, and the jejunum was analyzed for enzyme activity, mRNA abundance, and LPH polypeptide isotopic enrichment. The remaining animals were fed the protein-sufficient diet for 1 wk, and the jejunum was analyzed. Jejunal mass and lactase enzyme activity per jejunum were significantly lower in protein-deficient vs. control animals but returned to normal with rehabilitation. Protein malnutrition did not affect LPH mRNA abundance relative to elongation factor-1alpha, but rehabilitation resulted in a significant increase in LPH mRNA relative abundance. Protein malnutrition significantly lowered the LPH fractional synthesis rate (FSR; %/day), whereas the FSR of LPH in rehabilitated and control animals was similar. These results suggest that protein malnutrition decreases LPH synthesis by altering posttranslational events, whereas the jejunum responds to rehabilitation by increasing LPH mRNA relative abundance, suggesting pretranslational regulation.

Amino Acids↗

PubMed2001

Intestinal perfusion induces rapid activation of immediate-early genes in weaning rats.

C-fos and c-jun are immediate-early genes (IEGs) that are rapidly expressed after a variety of stimuli. Products of these genes subsequently bind to DNA regulatory elements of target genes to modulate their transcription. In rat small intestine, IEG mRNA expression increases dramatically after refeeding following a 48-h fast. We used an in vivo intestinal perfusion model to test the hypothesis that metabolism of absorbed nutrients stimulates the expression of IEGs. Compared with those of unperfused intestines, IEG mRNA levels increased up to 11 times after intestinal perfusion for 0.3-4 h with Ringer solutions containing high (100 mM) fructose (HF), glucose (HG), or mannitol (HM). Abundance of mRNA returned to preperfusion levels after 8 h. Levels of c-fos and c-jun mRNA and proteins were modest and evenly distributed among enterocytes lining the villi of unperfused intestines. HF and HM perfusion markedly enhanced IEG mRNA expression along the entire villus axis. The perfusion-induced increase in IEG expression was inhibited by actinomycin-D. Luminal perfusion induces transient but dramatic increases in c-fos and c-jun expression in villus enterocytes. Induction does not require metabolizable or absorbable nutrients but may involve de novo gene transcription in cells along the villus.

Animals↗

PubMed2000

Investigation of three doses of oral insulin-like growth factor-I on jejunal lactase phlorizin hydrolase activity and gene expression and enterocyte proliferation and migration in piglets.

In a previous study, oral IGF-I at 65 nM increased lactase phlorizin hydrolase (LPH) activity and villus height in piglets, however, the mechanisms were unknown. Herein, the response to a range of doses of IGF-I was investigated and we hypothesized that LPH and villus height would respond to oral IGF-I in a dose-dependent manner. Two 14-d experiments were conducted using cesarean-derived piglets. In experiment 1, piglets (n = 28) were fed formula containing 0, 33, 65, or 131 nmol/L (0, 0.25, 0.5, or 1.0 mg/L) recombinant human IGF-I. In experiment 2, 5'-bromodeoxyuridine was administered to piglets fed formula alone (n = 4) or containing 131 nmol/L IGF-I (n = 4). IGF-I did not affect body weight gain or intestinal weight or length. Jejunal villus height and LPH activity were significantly greater in piglets fed 131 nmol IGF-I/L than control piglets. Villus height and lactase activity in piglets fed the 33 and 65 nmol/L IGF-I doses were similar and intermediate between control and 131 nmol IGF-I/L. Jejunal mRNA expression and LPH polypeptide abundance were investigated in piglets receiving 0 or 131 nmol/L IGF-I. Steady state LPH mRNA abundance was significantly higher (p < 0.05) in IGF-I-treated piglets. The relative abundance of proLPH(h) was not significantly increased (p = 0.06) by IGF-I treatment. Mucosal DNA content and DNA synthesis were greater in piglets receiving 131 nmol IGF-I/L than control, however, enterocyte migration and mucosal protein content were unaffected. Thus, oral IGF-I increased jejunal LPH activity and LPH mRNA abundance and stimulated intestinal cell hyperplasia in normal piglets.

Animals↗

PubMed1998

Parenteral nutrition selectively decreases protein synthesis in the small intestine.

We investigated the effects of an elemental diet fed parenterally or enterally on total mucosal protein and lactase phlorizin hydrolase (LPH) synthesis. Catheters were placed in the stomach, jugular vein, and carotid artery of 12 3-day-old pigs. Half of the animals were given an elemental regimen enterally and the other half parenterally. Six days later, animals were infused intravenously with [2H3]leucine for 6 h and killed, and the midjejunum of each animal was collected for analysis. The weight of the midjejunum was 8 +/- 1.5 and 17 +/- 1.6 g in parenterally fed and enterally fed piglets, respectively. LPH activities (mumol.min-1.g protein-1) were significantly higher in parenterally vs. enterally fed piglets. Total small intestinal LPH activities were lower in parenterally vs. enterally fed animals. The abundance of LPH mRNA relative to elongation factor-1 alpha mRNA was not different between groups. The fractional synthesis rate of total mucosal protein and LPH was significantly lower in parenterally fed animals (67 +/- 7 and 66 +/- 7%/day, respectively) than in enterally fed animals (96 +/- 7 and 90 +/- 6%/day, respectively). The absolute synthesis rate (the amount of protein synthesized per gram of mucosa) of total mucosal protein was significantly lower in parenterally fed than in enterally fed piglets. However, the absolute synthesis rate of LPH was unaffected by the route of nutrient administration. These results suggest that the small intestine partially compensates for the effects of parenteral feeding by maintaining the absolute synthesis rate of LPH at the same levels as in enterally fed animals.

Animals↗

PubMed1998

Protein kinetics determined in vivo with a multiple-tracer, single-sample protocol: application to lactase synthesis.

Precise analysis of the kinetics of protein/enzyme turnover in vivo has been hampered by the need to obtain multiple tissue samples at different times during the course of a continuous tracer infusion. We hypothesized that the problem could be overcome by using an overlapping (i.e., staggered) infusion of multiple stable amino acid isotopomers, which would take the place of multiple tissue samples. We have measured, in pigs, the in vivo synthesis rates of precursor (rapidly turning over) and mature (slowly turning over) polypeptides of lactase phlorizin hydrolase (LPH), a model for glycoprotein synthesis, by using an overlapping infusion of [2H3]leucine, [13C1]leucine, [13C1]phenylalanine, [2H5]phenylalanine, [13C6]phenylalanine, and [2H8]phenylalanine. Blood samples were collected at timed intervals, and the small intestine was collected at the end of the infusion. The tracer-to-tracee ratios of each isotopomer were measured in the plasma and jejunal free amino acid pools as well as in purified LPH polypeptides. These values were used to estimate kinetic parameters in vivo using a linear steady-state compartmental model. The fractional synthesis rates of the high-mannose, complex glycosylated and mature brush-border LPH polypeptides, so determined, were 3.3 +/- 1.1%/min, 17.4 +/- 11%/min, and 0.089 +/- 0.02%/min, respectively. We conclude that this multiple-tracer, single-sample protocol is a practicable approach to the in vivo measurement of protein fractional synthesis rates when only a single tissue sample can be obtained. This method has broad application and should be particularly useful for studies in humans.

Amino Acids↗

PubMed1997

Effects of malnutrition on expression and activity of lactase in children.

BACKGROUND & AIMS: Many malnourished infants have reduced lactase specific activity in the small intestine. The aim of this study was to test the hypothesis that the hypolactasia of malnourished infants results from transcriptional suppression of lactase expression. METHODS: Biopsy specimens were studied from two groups of infants: 29 with malnutrition and 10 normally nourished controls with normal morphology and lactase activity. RESULTS: In malnourished infants, lactase messenger RNA (mRNA) was reduced to 32% and sucrase to 61% of normal. Lactase and sucrase enzyme proteins and activities were lower in malnourished infants, and partial villus atrophy was present. The genotype of adult hypolactasia was not present. CONCLUSIONS: Because the hypolactasia of malnourished children was associated with much lower lactase than sucrase mRNA abundance and because the epigenetic suppression, which accounted for the reduction of sucrase mRNA, was inadequate to explain the greater reduction of lactase mRNA, this study concludes that malnutrition suppresses lactase gene transcription or mRNA stability in infants. The reductions of lactase mRNA, distinct from those found in adults with genetic hypolactasia, explain the low lactase activities commonly found in malnourished infants.

Female↗

PubMed1997

Lactase phlorizin hydrolase synthesis is decreased in protein-malnourished pigs.

We have examined the effect of protein malnutrition on brush border (BB) lactase phlorizin hydrolase (LPH) synthesis in young pigs. Two groups of four 3-wk-old pigs were fed diets containing either 19 g soy protein, 63 g carbohydrate and 5 g fat per 100 g diet (a protein-sufficient diet) or 3 g soy protein, 85 g carbohydrate and 5 g fat per 100 g diet (a protein-deficient diet). After 8 wk of consuming the diets, pigs were infused intravenously with 2H3-leucine for 8 h, then killed. The jejunum was collected for measurement of lactase activity, LPH mRNA abundance and the rate of LPH post-translational synthesis. Lactase activities did not differ between groups (mean 8.1 +/- 1.2 micromol x min(-1) x g mucosa(-1)). LPH mRNA abundance relative to elongation factor-1alpha mRNA (the constitutive/reference mRNA) was significantly (P < 0.05) higher in well-nourished pigs (0.36 +/- 0.03%) than in protein-malnourished pigs (0.21 +/- 0.02%). The rate constants of BB LPH post-translational synthesis were also significantly higher in the well-nourished (103 +/- 9% x d(-1)) than in the protein-malnourished pigs (66 +/- 8% x d(-1)). Further, the absolute synthesis rate of BB LPH, a measure of the amount of enzyme synthesized per gram of tissue, was significantly higher in well-nourished than in protein-malnourished pigs (in arbitrary units, 892 +/- 90 vs. 450 +/- 34, respectively). Thus, protein malnutrition affects both LPH mRNA abundance and post-translational processing in young pigs.

Animals↗

PubMed1996

Lactase phlorhizin hydrolase turnover in vivo in water-fed and colostrum-fed newborn pigs.

We have estimated the synthesis rates in vivo of precursor and brush-border (BB) polypeptides of lactase phlorhizin hydrolase (LPH) in newborn pigs fed with water or colostrum for 24h post partum. At the end of the feeding period, piglets were anaesthetized and infused intravenously for 3h with L-[4-3H]- phenylalanine. Blood and jejunal samples were collected at timed intervals. The precursor and BB forms of LPH were isolated from jejunal mucosa by immunoprecipitation followed by SDS/PAGE, and their specific radioactivity in Phe determined. The kinetics of precursor and BB LPH labelling were analysed by using a linear compartmental model. Immunoisolated LPH protein consisted of five polypeptides [high-mannose LPH precursor (proLPHh), complex glycosylated LPH precursor (proLPHe), intermediate complex glycosylated LPH precursor (proLPH1i) and two forms of BB LPH]. The fractional synthesis rate (Ks) of proLPHh and proLPHc (approx. 5%/min) were the same in the two groups but the absolute synthesis rate (in arbitrary units, min-1) of proLPHh in the colostrum-fed animals was twice that of the water-fed animals. The Ks values of proLPHi polypeptides were significantly different (water-fed, 3.89%/min; colostrum-fed, 1.6%/min), but the absolute synthesis rates did not differ. The Ks of BB LPH was not different between experimental treatment groups (on average 0.037%/min). However, the proportion of newly synthesized proLPHh processed to BB LPH was 48% lower in colostrum-fed than in water-fed animals. We conclude that in neonatal pigs, the ingestion of colostrum stimulates the synthesis of proLPHh but, at least temporarily, disrupts the processing of proLPH polypeptides to the BB enzyme.

Amino Acid Sequence↗

PubMed1996

Severe protein deficiency and repletion alter body and brain composition and organ weights in infant pigs.

Three-wk-old genetically lean or obese pigs were used in two experiments to determine the changes in body composition, visceral organs and brain in response to severe protein deficiency. In Experiment 1, 16 obese pigs were fed an adequate (A, 21% protein, 3% fat) or a protein-deficient (D, 5% protein, 23% fat) diet for 7 wk. One-half of each group was killed at 7 wk, and the remainder of each group was fed the A diet for an additional 8 wk. At 7 wk, pigs fed D contained a higher percentage of fat than those fed A (P < 0.01); after 8-wk of repletion, body composition of the two groups was similar. Duodenum, jejunum, and ileum of the protein-deficient pigs had severely atrophic villi, submucosal edema, and atrophic muscle layers; after 8 wk of repletion, however, microscopic architecture of the gastrointestinal tract was restored to normal. Absolute cerebrum weight at 7 wk, but not after 8 wk repletion, in the pigs fed D were significantly less than in pigs fed A, indicating reduced brain cellularity after 7 wk of protein restriction, but not after 8 wk repletion. In Experiment 2, genetically obese (O, n = 8) and lean (L, n = 8) pigs consumed the A or D diet ad libitum for 10 wk. L and O pigs responded similarly to protein deficiency; D pigs were fatter than A pigs and plasma constituents, bone mineral content, bone mineral density and most organ weights revealed no interactions between diet and genotype. The pig model system used in these experiments enabled the isolation of protein deficiency from infectious disease, parasites and social environmental stimulation that may confound interpretation of human infant malnutrition experiments. The data suggest that genetically controlled body fatness is not a major determinant in the response of the infant pig to severe protein deficiency.

Animals↗

PubMed1996

The duration of medium-chain triglyceride feeding determines brush border membrane lipid composition and hydrolase activity in newly weaned rats.

To better understand the effect of dietary fat on intestinal brush border (BB) membranes in the young animal, we compared the effect of dietary medium-chain triglycerides (MCT) with that of monounsaturated and saturated long-chain triglycerides (LCT) on jejunal brush border membrane lipid composition and hydrolase activity in newly weaned rats. Twenty-day-old rat pups were divided into three groups and were weaned to diets containing 14% MCT + 6% soybean oil, 18% olive oil + 2% soybean oil, or 14% tallow + 6% soybean oil, and fed for 40 h or for 33 d. The diets were isonitrogenous and contained similar amounts of cholesterol and polyunsaturated fatty acids. Within 40 h, the fatty acid compositions of the brush border membranes were significantly different among treatment groups. These differences were maintained in rats fed for 33 d. No medium-chain fatty acids, but significantly greater amounts of polyunsaturated fatty acids, especially 18:2(n-6) and 20:4(n-6), were found in the brush border membranes of rats fed the medium-chain triglyceride diet. The cholesterol and phospholipid concentrations in the membranes were highest in rats fed the medium-chain triglyceride diet for 33 d. Rats fed that diet for 40 h had generally higher leucine aminopeptidase, sucrase and maltase activities compared with rats fed the olive oil or tallow diets. However, after 33 d of feeding, the differences between dietary treatment groups disappeared. This study demonstrates that, in the newly weaned rat pup, dietary medium-chain triglycerides and long-chain triglycerides rapidly affect the fatty acid composition of the brush border membrane. However, the changes in the hydrolase activities associated with the changes in the lipid composition of the membranes are transient.

Aging↗

PubMed1996

Orally administered lactoferrin increases hepatic protein synthesis in formula-fed newborn pigs.

Lactoferrin is a polypeptide which is abundant in colostrum; however, its biologic effect in the neonate is unknown. The objective was to determine the potentially anabolic effect of orally administered lactoferrin on visceral organ growth and protein synthesis in newborn pigs. We studied a total of 18 unsuckled newborn pigs from six litters. Three pigs from each litter were randomly assigned to one of three dietary treatment groups (n = 6) and bottle-fed (10 mL/h) formula, formula containing physiologic levels (1 mg/mL) of added bovine lactoferrin (bLF), or colostrum. After 24 h of feeding, we measured visceral organ protein synthesis in vivo using a flooding dose of [3H]phenylalanine. We also measured visceral organ protein and DNA mass, as well as intestinal hydrolase activities and villus morphology. Hepatic protein synthesis in pigs fed either formula containing bLF or colostrum was similar and in both groups was significantly higher than in pigs fed formula. Splenic protein synthesis was not significantly different in pigs fed either formula or formula containing bLF, but was significantly higher in colostrum-fed animals. There were no significant differences in small intestinal growth, protein synthesis, or hydrolase activities between newborn pigs fed formula, formula containing bLF, or colostrum. Our results demonstrate that feeding formula containing physiologic concentrations of added bLF increased hepatic protein synthesis in newborn pigs, suggesting that colostrumborne lactoferrin serves an anabolic function in neonates.

Administration, Oral↗

PubMed1994

Microanalysis of cholesterol, phospholipid, and medium- and long-chain fatty acids in biologic materials.

We have developed a procedure for the microanalysis of the cholesterol and phospholipid concentrations and the fatty acid (including medium-chain fatty acids) composition of total lipid and phospholipid. One-milligram lipid samples were extracted from jejunal brush border membranes isolated from rats fed a diet containing 14% medium-chain triglycerides. The samples were analyzed by gas chromatography using an autosampler. We were able to identify 17 fatty acids in the total lipid and in the phospholipid fractions. The cholesterol and phospholipid contents of the membranes were 270 +/- 21 and 298 +/- 35 nmol.mg protein-1, respectively; thus the cholesterol/phospholipid ratio was 0.92 +/- 0.05, a value typically reported for the rat. Medium-chain fatty acids were not detected in the brush border membranes. Therefore, to demonstrate that medium-chain fatty acids were not lost during analysis, we analyzed the rat diet, and brush border membrane samples to which medium-chain triglycerides had been added. Recovery of medium-chain fatty acids from brush border membrane samples was 95.7 +/- 2.6%. The percentage of medium-chain fatty acids detected in the diet was not significantly different from the percentage when the diet was prepared. In summary, our procedure enabled a complete lipid analysis of very small biologic specimens.

Animals↗

PubMed1994

Jejunal brush border hydrolase activity is higher in tallow-fed pigs than in corn oil-fed pigs.

We tested the effect of dietary fat on the lipid composition and hydrolase activity of jejunal brush border membranes in piglets. Eighteen 5-wk-old piglets were divided into three groups and for 4 wk fed either an unsaturated low fat diet (3.2% corn oil), an unsaturated high fat diet (17.2% corn oil) or a saturated high fat diet (2.2% corn oil + 15% tallow). Brush border membranes were prepared from the jejunal mucosa and analyzed for cholesterol, phospholipid and fatty acids. The activities of sucrase-isomaltase, lactase-phlorizin hydrolase, maltase-glucoamylase, aminopeptidase and alkaline phosphatase were measured. Lactase-phlorizin hydrolase isoforms were immunopurified and separated by SDS-PAGE, and their relative proportions were measured by densitometry. The activities of the disaccharidases and alkaline phosphatase, but not aminopeptidase, were greater in animals fed the saturated high fat diet than in animals fed the unsaturated high fat diet. The fatty acid composition of the membranes generally reflected the composition of the diet. Correlation analysis demonstrated that the phospholipid, fatty acid and cholesterol compositions of the membranes were associated with the differences in brush border hydrolase activity.

Animals↗

PubMed1994

Feeding colostrum rapidly alters enzymatic activity and the relative isoform abundance of jejunal lactase in neonatal pigs.

Our objective was to determine the short-term effects of feeding colostrum and mature milk on the enzyme activity and relative isoform abundance of lactase in neonatal pigs. We studied newborn pigs that were fed colostrum, mature milk and water for 6 h (Study 1). We also studied unfed pigs shortly after birth, and newborn pigs fed either colostrum or mature milk for 24 h (Study 2). The specific activity (micromol x min(-1) x g protein(-1)) of lactase in ajejunal mucosal homogenate and a purified membrane fraction was lower in pigs fed colostrum than in unfed newborns or those fed either milk or water. However, after 24 h, total jejunal lactase activity (micromol x mol(-1)), jejunal mass and protein content were higher in the colostrum- and milk-fed pigs than in the unfed newborns. In colostrum-fed pigs, the reduction in lactase specific activity after 6 h was associated with 1) a marked increase in the relative abundance of a 180-kDa protein, which was shown to be one of three pro-lactase isoforms, and 2) a lower relative abundance of the 160-kDa isoform, considered to be the mature form of the enzyme. Our evidence suggests that feeding either colostrum or mature milk increases total jejunal lactase activity. The reduction in both the specific activity and abundance of the mature isoform in conjunction with an increased relative abundance of the 180-kDa pro-lactase isoform suggests that feeding colostrum alters the post-translational processing of intestinal lactase in neonatal pigs.

Animals↗

PubMed1994

Brush-border disaccharidase synthesis in infant pigs measured in vivo with [2H3]leucine.

Conscious unrestrained piglets were fasted overnight and infused intravenously with [2H3]leucine for 6 h. Sucrase isomaltase and lactase phlorizin hydrolase were immunoprecipitated from jejunal mucosal membranes, and the immunoprecipitates were electrophoresed on polyacrylamide gels. Bands corresponding to the pro and mature isoforms of both enzymes were acid hydrolyzed. [2H3]leucine isotopic enrichment was measured by gas chromatography-mass spectrometry using negative chemical ionization. Plasma leucine reached isotopic steady state within 90 min. The isotopic enrichment of mucosal leucine was 73% of that of plasma leucine. The high mannose and complex glycosylated forms of prolactase were in isotopic equilibrium, and their isotopic enrichment was 94% of mucosal leucine. The fractional synthesis rates of total and membrane protein were 0.45 and 0.65 days-1, whereas the processing rates of mature lactase, sucrase, and isomaltase were 0.90, 0.23, and 0.21 days-1, respectively. Approximately 65% of the label in the sucrase isomaltase immunoprecipitate was in the complex glycosylated precursor, whereas 73% of the label in lactase phlorizin hydrolase was in the mature (160 kDa) form. We conclude that the low rate of brush-border sucrase synthesis reflects a slow rate at which the complex glycosylated precursor is processed to the brush-border form.

Animals↗

PubMed1993

In vivo sucrase-isomaltase and lactase-phlorizin hydrolase turnover in the fed adult rat.

We estimated in vivo turnover rates of sucrase-isomaltase and lactase-phlorizin hydrolase in adult rats. Fed animals received a primed continuous infusion of phenylalanine (300 microCi, 150 mumol Phe/100 g of body weight for 30 s, then 7.5 microCi, 3.75 mumol Phe/min for 10 to 140 min). Sucrase-isomaltase and lactase-phlorizin hydrolase were immunoprecipitated from jejunal mucosal membranes; isoforms were separated by SDS-polyacrylamide gel electrophoresis. Endoglycosidase H digestions and (for lactase-phlorizin hydrolase) N-terminal amino acid sequencing were performed on all isoforms. Specific radioactivity of prosucrase-isomaltase and prolactase-phlorizin hydrolase isoforms reached isotopic equilibrium by 60 and 90 min, respectively. Specific radioactivity of brush border sucrase and lactase did not reach steady state. The isotope kinetic, N-terminal amino acid sequencing, and endoglycosidase H digestion data suggested that one of the high molecular weight lactase isoforms is a dimer of mature lactase. Compartmental modeling of specific radioactivity demonstrated that mean intracellular residence time is 59 min for prosucrase-isomaltase isoforms and 68 min for prolactase-phlorizin hydrolase isoforms. Mean residence time in the brush border was 5.8 h for sucrase and 7.8 h for lactase. Fractional synthesis rates were 414%/day for sucrase and 307%/day for lactase. Thus, in vivo brush border sucrase and lactase turn over at similar rates in the adult rat.

Amino Acid Sequence↗