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Studies were made of the effects of pre- and post-weaning undernutrition and/or protein deficiency on intestinal phytase and phosphatase activities in albino rats and reversibility of the same by subsequent dietary rehabilitation. Neonatal undernutrition induced by rearing the pups in litters of 16 caused a marked decrease in alkaline phytase activity (as compared to those reared in litters of 8), while acid phytase activity decreased to a lesser extent and acid and alkaline phosphatase activities did not change. When neonatally undernourished rats were subsequently continued on a 4 or a 20% protein diet in restricted amounts (2.5 g/day) for 6 weeks the decreases in the alkaline phytase activity but not in that of acid phytase were further aggravated. Acid and alkaline phosphatases were not influenced by these treatments either. On dietary rehabilitation of these rats for subsequent 6 weeks on a 20% protein diet (ad libitum) acid and alkaline phytase activities of intestine recovered partially. These studies indicate the importance of alkaline phytase activity as a marker of intestinal maturation and is also suggestive of interrelationships between nutrition, intestinal development and its alkaline phytase activity.

Recent advances in nutritional and biochemical research have substantiated the importance of inositol as a dietary and cellular constituent. The processes involved in the metabolism of inositol and its derivatives in mammalian tissues have been characterized both in vivo and at the enzyme level. Biochemical functions elucidated for phosphatidylinositol in biological membranes include the mediation of cellular responses to external stimuli, nerve transmission, and the regulation of enzyme activity through specific interactions with various proteins. Inositol deficiency in animals has been shown to produce an accumulation of triglyceride in liver, intestinal lipodystrophy, and other abnormalities. The metabolic mechanisms giving rise to these latter phenomena have been extensively studied as a function of dietary inositol. Altered metabolism of inositol has been documented in patients with diabetes mellitus, chronic renal failure, galactosemia, and multiple sclerosis. A moderate increase in plasma and nerve inositol levels by dietary supplementation has been suggested as a means of treating diabetic neuropathy, although excessively high levels, such as are found in uremic patients, may be neurotoxic. A thorough consideration of the biochemical functions of inositol and a further characterization of various diseases with the aid of appropriate animal models may suggest a possible role for inositol and other dietary components in their prevention and treatment

Rat intestine was found to show a distinct acid phytase activity (pH optimum 4.7) in addition to that of an alkaline phytase (pH optimum 8.0). The phytase and phosphatase activities were found to differ in their developmental pattern and responded differentially to some inhibitors. Thus the two activities seem to be due to two independent enzymes and are not the activity of a nonspecific phosphatase as has been suspected formerly.

Soybean phytase (myo-inositol-hexakisphosphate phosphohydrolase; EC 3.1.3.8) was purified from 10-day-old germinating cotyledons using a four-step purification scheme. Phytase was separable from the major acid phosphatase present, and stained as a minor band of the three acid phosphatases detectable by activity staining after gel electrophoresis. The purified enzyme exhibited two closely migrating bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of approximately 59 and 60 KDa. The molar extinction coefficient of the enzyme at 280 nm was estimated to be 7.5 X 10(4) M-1 cm-1. The isoelectric point of phytase, as judged by the elution profile on chromatofocusing, was about 5.5. The enzyme was totally absorbed to a Procion Red HE3B column and eluted as a single protein component at a salt concentration of 250-300 mM. The enzyme possessed a high affinity for phytic acid (apparent Km = 48 microM), and was strongly inhibited by phosphate (apparent Ki = 18 microM), vanadate, and fluoride. Characteristic of other plant phytases, the pH and temperature optima were 4.5-4.8 and 55 degrees C, respectively.

The cause of marked inhibitory effect of bran on absorption of dietary nonheme iron was studied in man by double-radioiron technique. Washing bran with hydrochloric acid but not with water removed inhibitory factor(s). Inhibition was almost restored by reconstituting phytate level. Removal of phytates in bran by endogenous phytase significantly increased absorption of iron. Removing, by washing with water, phosphates formed from phytates during enzymatic dephytinization led to a bran fraction with only a small remaining inhibitory effect on iron absorption. Half the iron in bran is in the form of monoferric phytate, which is well-absorbed. When potassium and magnesium phytates were added in amounts present in bran, the same inhibitory effect on iron absorption was seen. Although there appear to be other factors in bran that partly explain the inhibition, phytates are the main cause of the inhibitory effect of bran on iron absorption.

Soluble plutonium complexing agents in foodstuffs have been identified. These have been labelled to high specific activity with 238Pu and their gut uptake determined in rats. The range of values found for naturally-occurring complexes was between 0.03% for oxalate and 0.1% for phytate. However, reasons are advanced for believing that the enhancement of uptake in rats fed the phytate complex would not be expected to occur in man. These results are not taken, therefore, as suggesting that there should be any change in the gut uptake factor of 0.05% currently recommended by the National Radiological Protection Board for plutonium contained in foodstuffs eaten by humans.

Human intestinal phytases play a minor role in phytate degradation. However, endogenous phytases in bran have a major effect on phytate hydrolysis. The amount of endogenous phytase activity varies with the method of processing of the fiber.

1. The effects of phosphorus deprivation on phytate digestibility, phosphorus utilization and intestinal phytase (EC 3.1.3.8) and alkaline phosphatase (EC 3.1.3.1) in rats were investigated. 2. P deprivation was achieved by giving rats a diet containing 3 g P/kg and resulted in hypophosphataemia, hypercalcaemia, hypercalciuria, and lower levels of P absorbed and retained, and calcium retained. 3. Rats adapted to P deprivation by increasing the digestion of total dietary-P and phytate-P. 4. Levels of intestinal alkaline phosphatase and alkaline phytase were not different between the two treatment groups. 5. P deprivation in the rats given the marginal-P diet may be a result of a lower absorption of total dietary-P or increased absorption of inositol phosphates formed during the enzymatic hydrolysis of phytate which are not readily utilized by the rat. 6. These results suggest that intestinal phytase and alkaline phosphatase do not play a role in the adaptive increase in phytate digestibility by rats given marginal-P diets. The adaptation may result from enhanced phytase or alkaline phosphatase synthesis by the gastrointestinal microflora stimulated by a lower level of P in the digesta.

1. The activities of alkaline phosphatase (EC 3.1.3.1) and phytase (EC 3.1.3.8) were similarly distributed in the small intestine of rats. Regional differences in activity were reflected by similar differences in the capacity of ligated intestinal segments to hydrolyse phytate in vivo. Activities were greatest in the duodenum and lowest in the terminal ileum. 2. Specific activities of both enzymes were tenfold greater in the brush border fraction of duodenal mucosa compared with entire mucosal homogenates. 3. Brush-border alkaline phosphatase and phytase activities required both magnesium and zinc ions for maximal activity. 4. Zn deficiency induced by feeding a diet low in Zn (0.5 mg Zn/kg) caused similar reductions in activity of both enzymes. 5. Zn deficiency induced by feeding diets marginally adequate in Zn (12 mg/kg) and phytate (10 g/kg) caused reductions in alkaline phosphatase, phytase activities and phytate hydrolysis in vivo. 6. It is suggested that phytase activity is a manifestation of alkaline phosphatase and the significance of this in relation to phytate-induced in Zn deficiency is discussed.

Aspergillus ficuum phytase was covalently immobilized on Fractogel TSK HW-75 containing 2-oxy-l-alkylpyridinium salts. A packed-bed bioreactor was constructed with the immobilized phytase. An HPLC ion-exchange method was used to analyze the enzymatic products of the bioreactor. Immobilized fungal phytase was able to hydrolyze myo-inositol Hexa-, penta-, tetra-, tri-, and diphosphates. When the substrate solution was recirculated for 5 hr in the bioreactor about 50% inorganic orthophosphate was released and myo-inositol-diphosphate and mono-phosphate were the only remaining products.

Purified Aspergillus ficuum phytase's partial primary structure and amino acid and sugar composition were elucidated. Determination of kinetic parameters of the enzyme at different pH values and temperatures indicated no significant alteration of the Km for phytate while the Kcat was affected. The enzyme was able to release more than 51% of the total available Pi from phytate in a 3.0 hr assay at 58 degrees C, but the Kcat dropped to 15% of the initial rate. Substrate selectivity studies revealed phytate to be the preferred substrate. The pH optima of phytase was 5.0, 4.0, and 3.0 for phytate, ATP, and polyphosphate, respectively. The enzyme had varied sensitivity towards cations. While Ca++ and Fe++ produced no effect on the catalytic rate of the enzyme, Cu+, Cu++, Zn++, and Fe were found to be inhibitory. Mn++ was observed to enhance enzyme activity by 33% at 50 microM. Known inhibitors of acid phosphatases e.g. L (+)-tartrate, phosphomycin, and sodium fluoride had no effect on enzyme activity.

Alkaline phosphatase, highly purified from bovine intestinal mucosa, has significant hydrolytic activity against phytate and CaATP. Phytase and CaATPase activities require quite different assay conditions than those which are optimal for conventional alkaline phosphatase substrates such as 4-nitrophenyl phosphate. We have used affinity chromatography and antibody recognition to demonstrate that the phytase and CaATPase activities are not due to contaminating enzymes, but are intrinsic activities of intestinal alkaline phosphatase. All of the phytase and CaATPase activities present in crude extracts of bovine intestinal mucosa can be accounted for by alkaline phosphatase. Apparently neither phytase nor CaATPase exist in this tissue as independent enzymes. Specific substrates which require assay conditions quite different from the conventional 4-nitrophenyl phosphate substrate may account for the physiological function of "alkaline phosphatase."

At least two-thirds of the phosphorus ingested by pigs is in the form of phytates. Two intestinal vitamin D-sensitive enzymes, alkaline phosphatase and phytase, might be involved in phytate-P digestion. The effects of dietary vitamin D upon the two intestinal phosphatases and P utilization in pigs fed a high phytate-P diet are reported here. Fourteen vit. D-depleted pigs (25-hydroxy vit. D: about 2 ng/ml plasma) were divided into two groups fed the same basal diet containing 0.6% P (of which 80% was phytic) and 0.6% Ca. One group (+D) was supplemented for 5 weeks with vit. D (1 000 IU D3/kg diet) and the other (-D) received none. P absorption and retention was two times higher in +D pigs than in -D animals (balance technique), and tibia X-ray pictures showed a lower bone density with the -D diet than with the +D diet. Surprisingly, vit. D supplementation had no effect on either of the mucosal enzymes (phytase and alkaline phosphatase). It may be concluded that vit. D improves phytate-P absorption via a mechanism which does not involve an increase in the activity of the intestinal phosphatases.

An enzyme which liberates Pi from myo-inositol hexaphosphate (phytic acid) was shown to be present in culture filtrates of Bacillus subtilis. It was purified until it was homogeneous by ultracentrifugation, but it still showed two isozymes on polyacrylamide gel electrophoresis. The enzyme differed from other previously known phytases in its metal requirement and in its specificity for phytate. It had a specific requirement for Ca2+ for its activity. The enzyme hydrolyzed only phytate and had no action on other phosphate esters tested. This B. subtilis phytase is the only known phytate-specific phosphatase. The products of hydrolysis of phytate by this enzyme were Pi and myo-inositol monophosphate. The enzyme showed optimum activity at pH 7.5. It was inhibited by Ba2+, Sr2+, Hg2+, Cd2+, and borate. Its activity was unaffected by urea, diisopropylfluorophosphate, arsenate, fluoride, mercaptoethanol, trypsin, papain, and elastase.

The effects of dietary phosphorus and sulphaguanidine levels, and sex differences on: (a) phytate digestibility, (b) calcium and P utilization, (c) the activities of alkaline phosphatase (EC 3.1.3.1), alkaline phytase (EC 3.1.3.8) and acid phosphatase (EC 3.1.3.2) in the intestinal mucosa of male and female rats were investigated. There was a linear increase in femur ash, Ca and P contents and the maximum force withstood by the fresh femurs as dietary P level was increased from 1.5 to 3.0 to 4.5 g/kg diet. The apparent digestibilities of Ca, P and phytate-P decreased as the level of P in the diet increased. Rats given the diets with 1.5 or 3.0 g P/kg were hypercalciuric and hypophosphaturic compared with rats receiving 4.5 g P/kg diet. The level of Ca retained was similar for all treatments. The level of P retained increased as the dietary P level increased. This suggests that P deprivation was a result of inadequate amounts of P retained and not due to the absorption of inositol phosphates formed during the enzymic hydrolysis of phytate. The addition of sulphaguanidine increased phytate digestibility without changing the activities of acid and alkaline phosphatase or alkaline phytase of the intestinal mucosa. This suggests that these enzymes did not play a role in the increase in phytate digestibility. However, dietary sulphaguanidine enhanced phytate digestibility, suggesting that alterations in the diet which modify either the composition or metabolism of the gastrointestinal microflora may be beneficial in enhancing the in vivo hydrolysis of phytate. Differences between males and females are reported and discussed.

Two types of extracellular acid phosphatases are synthesized by Aspergillus ficuum NRRL 3135: a nonspecific orthophosphoric monoester phosphohydrolase (EC 3.1.3.2) with an optimum pH of 2.0, and an enzyme with restricted specificity, a mesoinositol-hexaphosphate phosphohydrolase (EC 3.1.3.8; phytase) with an optimum pH of 5.5. Although the pH 5.5 enzyme is termed a phytase, both enzymes hydrolyze phytin. Synthesis of the enzymes is repressed by high orthophosphate concentrations in the fermentation medium. The highest total level for each enzyme is synthesized in low orthophosphate medium. In high orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme. In low orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme during the early stages of growth, but the reverse occurs after 5 days. The enzymes are differentiated by heat denaturation at acid and alkaline pH levels. They are separated into two distinct fractions on Sephadex G-100 followed by carboxymethylcellulose column chromatography. This indicates that the two enzymes are structurally different. The K(m) for both enzymes is 1.25 mm when calcium phytate is the substrate. Orthophosphate competitively inhibits the pH 2.0 (K(i) = 1.1 x 10(-2)m) but not the pH 5.5 phosphatase. Neither enzyme is denatured by 50% (w/v) urea or inhibited by 0.01 m tartrate. Thus, they differ from human prostatic phosphatase.

The effect of phosphate on the production of phosphatases by Aspergillus awamori var. kawachii was studied. In a high phosphate medium, little phosphatase was produced, and the phosphatase activity was predominately for beta-glycerophosphate. In a low phosphate medium, the production of phosphatase was increased and activity for glucose-6-phosphate predominated. Medium containing 1 mg of phosphorus per 100 ml was optimal, and the amount of phosphatase produced in this medium was about 200 times that produced in a high phosphate medium. By means of column chromatography on diethylaminoethyl cellulose, the phosphatase produced in the high phosphate medium was found to be eluted mainly at fraction e; the phosphatase of the low phosphate medium was separated into fractions a, b, c, and d. Thus, the phosphatase fractions produced in the low phosphate medium were different from those of the high phosphate medium. Since no specific effect on the production of esterases was observed when various phosphate esters were used as substrates, the enzymes of phosphate metabolism appear to be activated by nonspecific phosphate sources.

The fungus Aspergillus ficuum NRRL 3135 is known to produce an extracellular nonspecific orthophosphoric monoester phosphohydrolase (EC 3.1.3.2) with a pH optimum of 2.0, as well as an extracellular myo-inositol hexaphosphate phosphohydrolase (EC 3.1.3.8; phytase) with pH optima of 2.0 and 5.5. Both these enzymes are also known to hydrolyze myo-inositol hexaphosphate. The pentaphosphates liberated in the first step of this hydrolysis have been isolated and identified by ion-exchange chromatography and optical rotation. The nonspecific orthophosphoric monoester phosphohydrolase produces a single pentaphosphate, d-myo-inositol-1,2,4,5,6-pentaphosphate, whereas the phytase, at both pH 2.0 and 5.5, produces a mixture of two pentaphosphates. The major component of this mixture is d-myo-inositol-1,2,4,5,6-pentaphosphate and the other is d-myo-inositol-1,2,3,4,5-pentaphosphate. Thus the pathways of dephosphorylation of myo-inositol hexaphosphate by these two enzymes differ from that of wheat-bran phytase which forms l-myo-inositol-1,2,3,4,5-pentaphosphate.