Amino acid transport by the helicoidal colon of the new-born pig

The proximal colon of the new-born pig maintains a stable short-circuit current which is partly dependent upon the presence of methionine. This interaction between methionine and short circuit current shows Michaelis- Menten knetics with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ of 0.24 mM and a $\text{V}$ of 27 μA·cm^?2. The net flux of methionine to the serosal surface of proximal colons also shows a hyperbolic relation to the external concentration of methionine ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{0.38}\text{mM}$; $\text{V 10.4}\text{nmol}\text{·}\text{cm}{}^{\mathrm{?2}}\text{·}\text{min}{}^{\mathrm{?1}}$). The proximal colon concentrates methionine within its epithelium giving a mucosal to medium ratio of $\text{11.2 ± 1.9}$ (90 min incubation in 1 mM methionine).The ability of the colon to transport methionine across and concentrate methionine within its mucosa is maintained for at least 24 h after birth. Colonic transport of amino acids could be physiologically important in the pig, where the immediate post-natal transfer of immune globulins has been shown to cause a temporary inhibition of normal intestinal function.     

Characterization of glucose transport by cultured rabbit kidney proximal convoluted and proximal straight tubule cells

Rabbit kidney proximal convoluted tubule (RPCT) and proximal straight tubule (RPST) cells were independently isolated and cultured. The kinetics of the sodium-dependent glucose transport was characterized by determining the uptake of the glucose analog alpha-methylglucopyranoside. Cell culture and assay conditions used in these experiments were based on previous experiments conducted on the renal cell line derived from the whole kidney of the Yorkshire pig (LLC-PK1). Results indicated the presence of two distinct sodium-dependent glucose transporters in rabbit renal cells: a relatively high-capacity, low-affinity transporter (V(max) = 2.28 +/- 0.099 nmoles/mg protein min, Km = 4.1 +/- 0.27 mM) in RPCT cells and a low-capacity, high-affinity transporter (V(max) = 0.45 +/- 0.076 nmoles/mg protein min, K(m) = 1.7 +/- 0.43 mM) in RPST cells. A relatively high-capacity, low-affinity transporter (V(max) = 1.68 +/- 0.215 nmoles/mg protein min, Km = 4.9 +/- 0.23 mM) was characterized in LLC-PK1 cells. Phlorizin inhibited the uptake of alpha-methylglucopyranoside in proximal convoluted, proximal straight, and LLC-PK1 cells by 90, 50, and 90%, respectively. Sodium-dependent glucose transport in all three cell types was specific for hexoses. These data are consistent with the kinetic heterogeneity of sodium-dependent glucose transport in the S1-S2 and S3 segments of the mammalian renal proximal tubule. The RPCT-RPST cultured cell model is novel, and this is the first report of sodium-dependent glucose transport characterization in primary cultures of proximal straight tubule cells. Our results support the use of cultured monolayers of RPCT and RPST cells as a model system to evaluate segment-specific differences in these renal cell types.     

Effect of E. coli heat-stable enterotoxin on colonic transport in guanylyl cyclase C receptor-deficient mice

We studied the functional importance of the colonic guanylyl cyclase C (GCC) receptor in GCC receptor-deficient mice. Mice were anesthetized with pentobarbital sodium, and colon segments were studied in Ussing chambers in HCO3- Ringer under short-circuit conditions. Receptor-deficient mouse proximal colon exhibited similar net Na+ absorption, lower net Cl- absorption, and a negative residual ion flux (J(R)), indicating net HCO3- absorption compared with that in normal mice. In normal mouse proximal colon, mucosal addition of 50 nM Escherichia coli heat-stable enterotoxin (STa) increased the serosal-to-mucosal flux of Cl- (J(s-->m)(Cl)) and decreased net Cl- flux (J(net)(Cl)) accompanied by increases in short-circuit current (I(sc)), potential difference (PD), and tissue conductance (G). Serosal STa had no effect. In distal colon neither mucosal nor serosal STa affected ion transport. In receptor-deficient mice, neither mucosal nor serosal 500 nM STa affected electrolyte transport in proximal or distal colon. In these mice, 1 mM 8-bromo-cGMP produced changes in proximal colon J(s-->m)(Cl) and J(net)(Cl), I(sc), PD, G, and J(R) similar to mucosal STa addition in normal mice. We conclude that the GCC receptor is necessary in the mouse proximal colon for a secretory response to mucosal STa.     

Electrical PD,short-circuit current and fluxes of Na and Cl across avian intestine

In vitro measurements were made of transmural potential difference (PD), short-circuit current (Isc), resistance and unidirectional fluxes of 22Na and 36Cl across the duodenum, jejunum, ileum and colon of normal sodium-replete domestic fowl (Gallus domesticus). The PD ranged from about 1 mV across the duodenum to 8 mV across the colon while the Isc was, respectively, 2.8 and 64 microA X cm-2. The jejunum and ileum exhibited values between these extremes. Unidirectional fluxes (under short-circuit conditions) of Na and Cl were lowest across the duodenum where there was no evidence of active transport of these ions. Unidirectional fluxes of Na and Cl were less across the jejunum than across the ileum or colon. A net active transport of Na (but not Cl) was observed in the ileum (= 106% of the Isc) and colon (= 50% of Isc). The possible physiological significance of these observations in the domestic fowl are discussed and are compared to that of a mammal, the rabbit.     

Calcium secretion in canine tracheal mucosa

Calcium (Ca) affects many cellular functions of the respiratory tract mucosa and might alter the viscoelastic properties of mucus. To evaluate Ca homeostasis in a respiratory epithelium we investigated transport of Ca by the canine tracheal mucosa. Mucosal tissues were mounted in Ussing-type chambers and bathed with Krebs-Henseleit solution at 37 degrees C. Unidirectional fluxes of 45Ca were determined in tissues that were matched by conductance and short-circuit current (SCC). Under short-circuit conditions there was a significant net Ca secretion of 1.82 +/- 0.36 neq . cm-2 . h-1 (mean +/- SE). Under open-circuit conditions, where the spontaneous transepithelial potential difference could attract Ca toward the lumen, net Ca secretion increased significantly to 4.40 +/- 1.14 compared with 1.54 +/- 1.17 neq . cm-2 . h-1 when the preparation was short-circuited. Addition of a metabolic inhibitor, 2,4-dinitrophenol (2 mM in the mucosal bath), decreased tissue conductance and SCC and slightly decreased the unidirectional movement of Ca from submucosa to lumen. Submucosal epinephrine (10 microM) significantly enhanced Ca secretion by 2.0 +/- 0.63 neq . cm-2 . h-1. Submucosal ouabain (0.1 mM) failed to inhibit Ca secretion. The data suggest that canine tracheal mucosa secretes Ca; this secretory process is augmented by epinephrine or by the presence of a transepithelial potential difference as found under in vivo conditions.     

Active hexose transport across cultured human Caco-2 cells: characterisation and influence of culture conditions

Human Caco-2 cells (passage 80 to 100) were seeded onto collagen-coated Millipore filter assemblies and these were maintained in culture either (a) floated on the surface of the medium or (b) submerged within the body of the medium. Structural and functional assessments were made over a 30-day period. After seeding, all cells assumed a flattened, squamous configuration and rapidly became confluent. Cells submerged within the medium formed polarised monolayers with well developed junctional complexes, abundant apical microvilli and increasing levels of alkaline phosphatase activity. Cells grown floated on the surface of the medium formed complex multilayers in which polarisation was confined to the surface layer. Junctional complexes and apical microvilli were similar to those seen in submerged monolayers but alkaline phosphatase activities were higher. Transepithelial electrical resistance increased rapidly from day 1, as the layers became confluent. Electrical resistance was higher and short-circuit current and potential differences were lower across monolayers than across multilayers. After 10 days in culture, the addition of D-glucose to the apical bathing solution, of all cell layers, caused a rapid rise in short-circuit current and potential difference. These changes were sodium-dependent and phlorizin-sensitive. Galactose and 3-O-methylglucose induced similar changes and the affinity constants for these hexoses ranked in the order reported for rat jejunum (Km glucose 2.44 +/- 0.52 mM; Km galactose 8.05 +/- 1.33 mM; Km 3-O-methylglucose 22.0 +/- 5.2 mM). Culture conditions had a marked effect on hexose maximum transport rates (glucose Vmax: submerged 2.94 +/- 0.20 microA/cm2; floated 9.94 +/- 0.82 microA/cm2, P less than 0.05) but affinity constants were unchanged. Apical to basolateral mannitol fluxes, used as an index of paracellular permeability, decreased from day 1 to day 5 and then remained steady. Fluxes across monolayers and multilayers were not significantly different. We conclude that sodium-dependent hexose transport occurs in cultured Caco-2 cell layers grown on permeable supports. Culture conditions, however, have a marked effect on both cell layer structure and function, and should be an important factor when considering Caco-2 cells as an in vitro model of enterocyte function.     

Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.     

Seasonal changes in sodium transport and amiloride sensitivity in the isolated intestine of the earthworm, Lumbricus terrestris (L.)

Temporal changes in sodium flux rates and the electrical properties of two regions of the intestine appear to occur in a yearly cycle. In RIIIA, the anterior portion of the mid-intestine, the short-circuit current in January and April preparations is 60.2 and 10.2 microA cm-2, respectively and the net sodium fluxes are 1.50 and 1.24 mu Eq cm-2 hr-1, respectively. In RIIIB, the posterior portion of the mid-intestine, the short-circuit current in January and April preparations is 50.7 and 27.9 microA cm-2, respectively, while the net sodium fluxes are 1.78 and 0.59 mu Eq cm-2 hr-1, respectively. Sodium transport in RIIIB is inhibited by amiloride (10(-4)M) in January preparations but is refractory to amiloride (less than or equal to 10(-3)M) in April preparations.     

Effect of nitric oxide on electrolyte transport across the porcine proximal colon

The effect of nitric oxide (NO) on ion transport in the porcine proximal colon was investigated in slide-stripped epithelia mounted in Ussing chambers. The serosal addition of the NO-donors sodium nitroprusside (SNP, 0.5 mM) or S-nitroso-N-acetylpenicillamine (SNAP, 0.5 mM) induced a steep increase of short-circuit current ( I(sc)). The stimulatory effect of SNP on I(sc) could not be blocked by piroxicam or tetrodotoxin. Potassium channel inhibitors (quinidine, tetraethylammonium or barium) added serosally reduced the SNP- or SNAP-induced increases of I(sc). In chloride-free solutions, the SNP-induced increase of I(sc) was smaller than in chloride-containing solutions. Cl(- )and Na(+) flux measurements demonstrated that SNP diminished Cl(-) and Na(+) net absorption. Pre-treatment with barium was able to block the inhibitory effect of SNP on NaCl net absorption totally. NO effects on paracellular pathways were assessed by measuring flux rates of [(14)C]-D-mannitol. SNP did not change unidirectional D-mannitol flux rates. In conclusion, NO inhibits NaCl net absorption in the proximal colon of pigs by acting directly on the enterocyte. The antiabsorptive (and/or prosecretory) effect of NO depends on a functional basolateral potassium conductance.     

Unidirectional fluxes of short-chain fatty acids across segments of the large intestine in pig,sheep and pony compared with guinea pig

Unidirectional fluxes of short-chain fatty acids across pig, sheep and pony caecum, proximal and distal colon were studied under short-circuit current conditions in Ussing chambers. Findings are compared with results from guinea pig. Marked species differences are apparent; highest mucosal-to-serosal fluxes of acetate, propionate and butyrate were seen in guinea pig, lower values in pig and smallest fluxes in sheep and pony. Segmental differences between caecum, proximal and distal colon exist mainly in guinea pig and are less developed in pig, sheep and pony. Inhibition of Na⁺/H⁺ exchange by amiloride added to the mucosal solution decreased the mucosal-to-serosal fluxes of short-chain fatty acids clearly in guinea pig caecum and proximal colon, and very little in distal colon. This effect was somewhat less pronounced in pig caecum and distal colon, in caecum and distal colon of sheep and caecum of the pony. In pig, sheep and pony proximal colon and pony distal colon no significant inhibition was observed. Inhibition of the K⁺-H⁺ ATPase by addition of ouabain to the mucosal solution diminished mucosal-to-serosal fluxes of short-chain fatty acids in the guinea pig distal colon extensively. No comparable inhibition was seen in any of the other segments in the animals studied.     

Functional activities of the colon of the desert gerbil (Gerbillus cheesmani)

The effects of starvation and undernourishment on the potential differences (pd in mV), basal short-circuit current (Isc in microA/cm(2)), resistance (R in omega) and glucose-dependent short-circuit current (Isc in microA/cm(2)) across stripped sheets of proximal, mid and distal colon of the gerbil (Gerbillus cheesmani) were investigated. The effects of replacing sodium chloride by lithium chloride, replacing chloride in Krebs buffer by gluconate and removing bicarbonate from bathing buffers were also investigated. Starvation (4 days, water ad lib) and undernourishment (50% control food intake for 21 days) had no significant changes on pd and R of the three regions of stripped colon. Starvation increased the basal Isc in the proximal and the mid-colon only while undernourishment increased the basal Isc of three regions of the colon. In addition, starvation and undernourishment increased the glucose-dependent Isc in the three regions. Replacing sodium chloride by lithium chloride caused a slight decrease in the basal Isc of proximal and mid colon taken from starved animals. In undernourished gerbils, although there was a slight decrease in basal Isc of proximal and mid colon the big decrease was observed in Isc of the distal colon. Replacing chloride by gluconate had no effect on the Isc of the different regions of colon taken from fed and starved animals but decreased the Isc of the three regions of undernourished animals. The absence of bicarbonate reduced the Isc of proximal and mid colon taken from starved gerbils and those of three regions taken from undernourished animals. The results of the present study suggest that the Isc of proximal and mid colon from starved gerbils could result from active sodium transport together with bicarbonate secretion while the Isc of the three regions taken from undernourished gerbils results from active sodium absorption together with both chloride and bicarbonate secretion.     

Active transport of magnesium across the isolated midgut of Hyalophora cecropia.

1. The 28Mg-measured net flux of magnesium from lumen-side to haemolymph-side of the isolated and short-circuited midgut was 1.97 +/- 0.28 mu-equiv cm(-2) /(-1) in 8 mM-Mg2+. 2. The magnesium-influx shows a delay before the tracer steady-state is attained, indicating the existence of a magnesium-transport pool equivalent to 6.7 mu-equiv/g wet weight of midgut tissue. 3. Magnesium depresses the short-circuit current produced the midgut but not the potassium transport, the depression being equal to the rate of magnesium transport. 4. Magnesium transport yields a linear Lineweaver-Burk plot with an apparent Km of 34 mM-Mg2+ and an apparent Vmax of 14.9 mu-equiv cm(-1) /(-1). 5. Magnesium is actively transported across the midgut and contributes to the regulation of the haemolymph magnesium concentration in vivo.     

Evidence of multiple operational affinities for D-glucose inside the human erythrocyte membrane.

1. The Michaelis-Menten parameters of labelled D-glucose exit from human erythrocytes at 2 degrees C into external solution containing 50 mM D-galactose were obtained. The Km is 3.4 +/0 0.4 mM, V 17.3 +/- 1.4 MMOL . 1(-1) cell water . min-1 for this infinite-trans exit procedure. 2. The kinetic parameters of equilibrium exchange of D-glucose at 2 degrees C are Km = 25 +/- 3.4 mM, V 30 +/- 4.1 mmol . 1(-1) cell water . min-1. 3. The Km for net exit of D-glucose into solutions containing zero sugar is 15.8 +/- 1.7 mM, V 9.3 +/- 3.3 mmol . 1(-1) cell water . min-1. 4. This experimental evidence corroborates the previous finding of Hankin, B.L., Lieb, W.R. and Stein, W.D. [(1972) Biochim. Biophys. Acta 255, 126--132] that there are sites with both high and low operational affinities for D-glucose at the inner surface of the human erythrocyte membrane. This result is inconsistent with current asymmetric carrier models of sugar transport.     

Investigations on the sodium dependence of bile acid fluxes in the isolated perfused rat liver.

At [Na+]o = 118 mM the concentrative transfer of cholic and taurocholic acid from the perfusate into the isolated rat liver displays saturation kinetics (taurocholate: V = 299 nmol-min-1-g-1, Km = 61 muM; Cholate: V=327 nmol-min-1-g-1, Km = 436 muM). Perfusion with an isotonic sodium-free medium did not change the feature of a carrier-mediated transport but did markedly reduce V without affecting Km (taurocholate: V = 65 nmol-min-1-g-1, Km = 78 muM; cholate: V = 104 nmol-min-1-g-1, Km = 354 muM). It was experimentally assured that the observed reduction of bile salt uptake was not a consequence of regurgitation of bile salts or due to an excessive intracellular accumulation during cholestasis in the sodium-free state. The rate of taurocholate efflux is very low when compared with the rapid rate of the uptake. A stimulatory action of extracellular sodium on this pathway was also observed. Inhibition of the (Na+ + K+)-ATPase by 1 mM ouabain resulted in a decrease of bile salt uptake. Activation of the enzyme by potassium readmission to a K+-deprived liver enhanced bile salt uptake. The immediate response to alteration of the enzyme activity suggests a close association of a fraction of bile acid active transport with the sodium pump.     

Methionine transport in Salmonella typhimurium: evidence for at least one low-affinity transport system.

The systems which transport methionine in Salmonella typhimurium LT2 have been studied. Fourteen mutants, isolated by three different selection procedures, had similar growth characteristics and defects in the specific transport process showing a Km of 0.3 microM for L-methionine, and therefore lack the high-affinity, metP transport system. The sites of mutation in four of the mutants were shown by P1-mediated transduction to be linked (0.3 to 1.1%) with a proline marker located at unit 7 on the S. typhimurium chromosome. The high-affinity system was subject to both repression and transinhibition by methionine, and it may also be regulated by the metJ and metK genes. There appeared to be at least two additional transport systems with relatively low affinities for methionine in the metP763 mutant strain, with apparent Km values for methionine of 24 microM and approximately 1.8 mM. The latter system, with a very low affinity for methionine, was inhibited by leucine. In addition, methionine inhibited leucine transport, suggesting that one of the low-affinity methionine transport systems may actually be a leucine transport system.     

Renal transport of taurine in luminal membrane vesicles from rabbit proximal tubule

The uptake of taurine by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule was examined. In pars convoluta, the transport of taurine was characterized by two Na(+)-dependent (Km1 = 0.086 mM, Km2 = 5.41 mM) systems, and one Na(+)-independent (Km = 2.87 mM) system, which in the presence of an inwardly directed H(+)-gradient was able to drive the transport of taurine into these vesicles. By contrast, in luminal membrane vesicles from pars recta, the transport of taurine occurred via a dual transport system (Km1 = 0.012 mM, Km2 = 5.62 mM), which was strictly dependent on Na+. At acidic pH with or without a H(+)-gradient, the Na(+)-dependent flux of taurine was drastically reduced. In both kind of vesicles, competition experiments only showed inhibition of the Na(+)-dependent high-affinity taurine transporter in the presence of beta-alanine, whereas there was no significant inhibition with alpha-amino acids, indicating a beta-amino acid specific transport system. Addition of beta-alanine, L-alanine, L-proline and glycine, but not L-serine reduced the H(+)-dependent uptake of taurine to approx. 50%. Moreover, only the Na(+)-dependent high-affinity transport systems in both segments specifically required Cl-. Investigation of the stoichiometry indicated 1.8 Na+: 1 Cl-: 1 taurine (high affinity), 1 Na+: 1 taurine (low affinity) and 1 H+: 1 taurine in pars convoluta. In pars recta, the data showed 1.8 Na+: 1 Cl-: 1 taurine (high affinity) and 1 Na+: 1 taurine (low affinity).     

Effects of NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) on chloride transport in intestinal tissues and the T84 cell line.

NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) has been reported to block Cl- channels in isolated rabbit nephrons with high potency (IC50 = 80 nM). The effects of this compound on Cl(-)-mediated transport processes in intestinal tissues have been studied using agonist-stimulated short-circuit current (T84) in Ussing chamber experiments and 36Cl- fluxes in monolayers of a colonic cell line (T84). NPPB inhibited PGE1-stimulated Isc in rabbit distal colon and ileum at concentrations in the range 20 to 100 microM. However, NPPB at the same concentrations also inhibited glucose-stimulated Isc in rabbit ileum, suggesting that its effects were not restricted to those on Cl- transport. Consistent with this, exposure of rabbit distal colon to 100 microM NPPB was found to reduce endogenous ATP levels by 69%, implying that, at these concentrations, NPPB could impair active transport processes by an effect on cellular energy metabolism. Clear evidence for a direct effect of NPPB on epithelial chloride channels was found in studies on Cl- fluxes in T84 cell monolayers. NPPB inhibited VIP-stimulated Cl- uptake into T84 cells with an IC50 of 414 microM. NPPB (1 mM) also inhibited Cl- efflux from pre-loaded cells confirming its effect as a weak Cl- channel blocker in this system.     

The human OCTN1 (SLC22A4) reconstituted in liposomes catalyzes acetylcholine transport which is defective in the mutant L503F associated to the Crohn's disease

The organic cation transporter (OCTN1) plays key roles in transport of selected organic cations, but understanding of its biological functions remains limited by restricted knowledge of its substrate targets. Here we show capacity of human OCTN1-reconstituted proteoliposomes to mediate uptake and efflux of [(3)H]acetylcholine, the Km of transport being 1.0mM with V(max) of 160nmol?mg(-1)protein?min(-1). OCTN1-mediated transport of this neurotransmitter was time-dependent and was stimulated by intraliposomal ATP. The transporter operates as uniporter but translocates acetylcholine in both directions. [(3)H]acetylcholine uptake was competitively inhibited by tetraethylammonium, γ-butyrobetaine and acetylcarnitine, and was also inhibited by various polyamines. Decreasing intraliposomal ATP concentrations increased OCTN Km for acetylcholine, but V(max) was unaffected. Evaluation of the acetylcholine transporter properties of a variant form of OCTN1, the Crohn's disease-associated 503F variant, revealed time course, Km and V(max) for acetylcholine uptake to be comparable to that of wild-type OCTN1. Km for acetylcholine efflux was also comparable for both OCTN1 species, but V(max) of OCTN1 503F-mediated acetylcholine efflux (1.9nmol?mg(-1)protein?min(-1)) was significantly lower than that of wild-type OCTN1 (14nmol?mg(-1)protein?min(-1)). These data identify a new transport role for OCTN1 and raise the possibility that its involvement in the non-neuronal acetylcholine system may be relevant to the pathogenesis of Crohn's disease.     

A synthesis of acylphosphonic acids and of 1-aminoalkylphosphonic acids: the action of pyruvate dehydrogenase and lactate dehydrogenase on acetylphosphonic acid.

Acylphosphonic acids, R-CO-PO(OH)2, have been synthesized by the steps [formula: see text] of which the last is new and provides a mild method for de-esterifying acylphosphonic acids. Their reductive amination gives a simple way of making 1-aminoalkylphosphonic acids. Acetylphosphonic acid inhibited NAD+ reduction by pyruvate with the pyruvate dehydrogenases from Escherichia coli and Bacillus stearothermophilus. The inhibition was competitive with pyruvate, with Ki of 6 microM for the E. coli enzyme (pyruvate Km 0.5 mM) and one of 0.4 mM of the B. stearothermophilus enzyme (pyruvate Km 0.1 mM). Acetylphosphonate and its monomethyl ester are substates for pig heart lactate dehydrogenase, with Km values of 15 mM and 10 mM respectively (pyruvate Km 0.05 mM) and specificity constants one thousandth that for pyruvate.     

Continuous spectrophotometric assays for beta-glucosidases acting on the plant glucosides L-picein and prunasin.

The neutral pH optimum beta-glucosidases of mammalian liver and almonds are each capable of hydrolyzing a number of plant glucosides, including L-picein (p-hydroxyacetophenone-beta-D-glucoside) and prunasin (D-mandelonitrile-beta-D-glucoside). Taking advantage of the marked differences in the spectra of the substrate/product pairs of L-picein/p-hydroxyacetophenone and prunasin/mandelonitrile, we have devised spectrophotometric assays that permit the continuous monitoring at pH 7.0 of p-hydroxyacetophenone (piceol) release from L-picein by guinea pig hepatic cytosolic beta-glucosidase and mandelonitrile from prunasin by almond beta-glucosidase. When L-picein hydrolysis was monitored at 320 nm and prunasin at 282 nm, the molar absorption coefficients determined for their products, namely piceol and mandelonitrile, were 3200 and 1360 M-1 cm-1, respectively. The kinetic parameter Km and Vmax values obtained using these spectrophotometric procedures for the guinea pig liver cytosolic beta-glucosidase acting on L-picein were 0.88 mM and 5.29 x 10(5) units/mg protein and for the almond beta-glucosidase acting on prunasin, Km 1.1 mM and Vmax 5.24 x 10(6) units/mg protein. These values agreed well with previously reported values obtained using less convenient, discontinuous assay procedures.