Properties of larval and imaginal membrane-bound digestive enzymes from Trichosia pubescens

Trichosia pubescens larval midgut ceca cells display in their plasma membranes α-glucosidases (M_r 95,000; pH_o 5.5; K_m 5.7 mM; K_i for TRIS 8.9 mM), trehalases (M_r 69,000; pH_o 5.3; K_m 0.92 mM; K_i for TRIS 57 mM), and aminopeptidases (M_r 95,000; pH_o 8.7; K_m 0.19 mM) which are solubilized by Triton X-100. The enzymes were purified by electrophoresis and used to raise antibodies in a rabbit. T. pubescens imaginal midgut cells display in their plasma membranes an α-glucosidase (M_r 156,000; pH_o 5.8; K_m 2.3 mM; K_i for TRIS 0.2 mM), a trehalase (M_r 93,000; pH_o 5.5; K_m 0.72 mM; K_i for TRIS 45.5 mM), and an aminopeptidase (M_r 210,000; pH_o 9.0; K_m 0.47 mM). Antiserum produced against the larval enzymes shows no precipitation arc when tested by double immunodiffusion or by immunoelectrophoresis with Triton X-100-solubilized membrane proteins from imaginal midguts. Otherwise, a similar test showed that larval midgut cecal enzymes and larval ventriculus enzymes display complete immunological identity. The data suggest that, despite the fact the larval and imaginal aminopeptidase, α-glucosidase, and trehalase probably have similar functions, the genes coding for them in larvae and imagoes must differ.     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Ultrastructural and biochemical aspects of digestion in the imagoes of the flyRhynchosciara americana

The midgut of adultRhynchosciara americana Wiedemann (Diptera: Sciaridae) displays, in contrast to the midguts of other adult Diptera, two caeca connected to a ventriculus. All midgut cells exhibit long apical microvilli, and narrow and ramified basal channels with openings to the underlying space. These morphological features are thought to be involved in the absorption of nutrients from food. Enzymatic assays inR. americana adults revealed that amylase occurs in salivary glands and midgut, whereas aminopeptidase, α-glucosidases and trypsin occur only in the midgut, mainly in the ventriculus. There is a soluble (Mr 105000) and a membrane-bound aminopeptidase (solubilized form, Mr 110000). Soluble α-glucosidase inactivates easily and could not be characterized, whereas membrane-bound α-glucosidases were resolved after solubilization into three molecular species (Mr 186000, 105000 and 84000) with different substrate specificities. The activities of trypsin (pH optimum 9.0), which was inhibited completely by soybean trypsin inhibitor, and of amylase (pH optimum 5.5), were not sufficiently high to be further characterized. The data support the assertion thatR. americana adults are able, to a limited extent, to digest and absorb starch and proteins, in addition to nectar sugars. The results, supported by published data, suggest that there is an inverse correlation between the digestive enzyme activities and midgut absorptive surface in insects which has nectar as a major food.     

Consumption of food and spatial organization of digestion in the cassava hornworm, Erinnyis ello

Fifth-instar Erinnyis ello larvae eat 2.1 times their own weight per day of Euphorbia pulcherrima leaves, with a coefficient of digestibility of 45% and an efficiency of food conversion into tissue of 25%. The food takes about 150 min to go through the gut. Midgut contents have a pH of 9.3–9.8, depending on the region. Cellulase is absent from the gut in E. ello. Significant gut hydrolase activities are found only in midgut. Amylase and trypsin occur in the midgut tissue and contents and in regurgitated material, whereas aminopeptidase, α-glucosidase, β-glucosidase and trehalase are found in major amounts in the midgut tissue, in minor amounts in the midgut contents and are absent from regurgitated material. The results support the hypothesis that digestion starts in the endoperitrophic space under the action of amylase and trypsin and is largely completed in the ectoperitrophic space through the catalytic action of several oligomer and dimer hydrolases. Involvement of a membrane-bound aminopeptidase in the terminal digestion of oligopeptides cannot, at present, be excluded. The finding that less than 7% of the total amylase and trypsin are excreted, after a time identical to the passage time of the food bolus, leads to the proposal for the existence of some mechanism by which those enzymes are recovered from the undigested food before it is excreted.     

Plasma membrane-associated amylase and trypsin: Intracellular distribution of digestive enzymes in the midgut of the cassava hornworm,Erinnyis ello

Differential centrifugation of homogenates of midgut cells prepared in isotonic solutions has been carried out and hydrolase and enzyme marker activities have been determined in the isolated fractions. α- and β-Glucosidase and trehalase seem to occur loosely associated with large structures, from which they are set free by homogenizing in water. They are also found in the cytosolic fraction. Aminopeptidase activity follows that of alkaline phosphatase, whereas that of amylase and trypsin occur mainly among particulate fractions. Part of the amylase present in the particulate fractions seems to correspond to soluble amylase bound by membranes. The enrichment factor for alkaline phosphatase and aminopeptidase in microvilli purified from midgut cells is 4 and for amylase 1.5 Amylase and trypsin are only partly released from a membrane fraction after several washings in different media, including ultracentrifugation in a discontinuous glycerol gradient. About 50% of the amylase and trypsin are solubilized from the membranes by treatment with Triton X-100. The results support the proposal that intermediate and final digestion in Erinnyis ello occur under the action of glycocalyx-associated hydrolases (α- and β-glucosidase and trehalase) and of plasma membrane-bound enzymes (aminopeptidase, and perhaps also amylase and trypsin).     

A quantitative study of some digestive enzymes in the rabbitfish, Siganus canaliculatus and the sea bass, Lates calcarifer

Digestive enzyme distribution and activity in the digestive tracts of the rabbitfish, Siganus canaliculatus and the sea bass, Lates calcarifer were studied. Quantitative determinations of digestive enzymes in the guts of both fishes showed that they were capable of digesting carbohydrates and proteins in their diet. The carbohydrases, amylase, laminarinase, maltase, sucrase and trehalase were detected in the rabbitfish; their activities being mainly in the stomach, intestine and pyloriccaeca. Amylase, maltase, trehalase and chitinase activities were recorded in the gut of the sea bass, primarily in the intestine and the pyloriccaeca. Their activities were significantly lower than those in the rabbitfish. Proteases (pepsin, chymotrypsin, elastase, leucine aminopeptidase and trypsin) were found in both the rabbitfish and the sea bass. Pepsin activity however, was higher in the sea bass; while trypsin and chymotrypsin activities were higher in the rabbitfish. The activities of the various digestive enzymes in both fishes are discussed in relation to their feeding habits.     

Invertases in young and mature leaves of Citrus sinensis

The sucrose catabolic enzymes acid invertase (EC 3.2.1.26) and alkaline invertase (EC 3.2.1.27) were studied in young and mature Citrus sinensis leaf tissue. In young, expanding leaves (60 % final length) soluble acid invertase activity predominated, while soluble alkaline invertase activity predominated in mature leaves. The acid and alkaline invertase activities were separated on Sephadex G-200. The acid invertase had an M_r of approximately 60 000, pH maximum of 4.5 and apparent K_m of 3.3 mM sucrose. The alkaline invertase had an M_r of approximately 200 000, pH maxima of 6.8 and an apparent K_m of 20 mM sucrose. Alkaline invertase was strongly inhibited by 10 mM Tris while acid invertase was not. Possible physiological roles for the two invertases are discussed.     

The physiological role of the peritrophic membrane and trehalase: Digestive enzymes in the midgut and excreta of starved larvae of Rhynchosciara

Amylase, cellulase, trehalase, aminopeptidase and trypsin were determined using the midgut and trehalose using the haemolymph of starved and of subsequently fed larvae of Rhynchosciara americana. Midgut trehalase activity decreases steadily during starvation and increases again on feeding, whereas haemolymph trehalose titres remain constant, suggesting that trehalase is a true digestive enzyme. The decrease in amylase, cellulase and trypsin activity in the midgut during starvation is of the same order as that recovered from the excreta. Since this finding is exactly what one would expect if enzyme production stops in response to starvation, this supports the hypothesis that synthesis that synthesis of these enzymes is controlled. The excretion rate of amylase, cellulase and trypsin is very low in comparison to their activity inside the peritrophic membrane and the travel time of the food bolus through the gut. It is proposed that the peritrophic membrane separates two extracellular sites for digestion as an adaptation to conserve secreted enzymes. This could be accomplished by the existence of an endo-ectoperitrophic circulation of the enzymes involved in the initial attack on the food and by restricting to the ectoperitrophic fluid the enzymes which participate only in intermediary digestion of food.     

α-Glucosidase activity in haemolymph of the American cockroach, Periplaneta americana

α-Glucosidase activity of whole haemolymph has been investigated in adult males of the American cockroach, Periplaneta americana. Two electrophoretically distinguishable enzymes capable of hydrolysing α-glucosidic linkages are present in the serum component of the haemolymph, and one of these hydrolyses trehalose. Trehalase activity is also present in haemocytes, and the haemocyte enzyme shares an identical electrophoretic mobility and similar pH sensitivity with the serum trehalase. Furthermore, both enzymes are inhibited to the same extent by sodium ethylene diamine tetracetate (EDTA); thus it is suggested that the same enzyme may be responsible for trehalase activity in the two components. The K_m of EDTA-inhibited trehalase is 3·3 mM and this value is reduced to 1·8 mM upon activation of the enzyme by calcium ions. The properties of the trehalase are discussed in light of the possible rôle of the enzyme in regulating haemolymph trehalose and glucose concentrations.     

Trehalase transformation in silkworm midgut during metamorphosis

Summary The particulate trehalase from silkworm larval midgut was effectively solubilized by repeated freezing and thawing, and by incubation with snake venom and non-ionic detergents (Lubrol PX and WX and Triton X-100). With solubilization the activity was enhanced and the activation behaviour was dependent upon the developmental stage of silkworms, being highest (up to about 3-fold) at the spinning stage.When chromatographed on DEAE-cellulose columns separately, the enzyme solubilized by freezing and thawing and the soluble trehalases from feeding larval midgut were respectively eluted as single peaks, P I and P II. However, both P I and P II trehalases were demonstrated after solubilization of the particulate fraction from feeding larvae with Triton X-100, or after treatment of the midgut of spinning larvae by freezing and thawing.The apparent molecular weights of P I and P II trehalases as estimated by Sephadex G-200 chromatography were about 70,000 and 140,000, respectively. The optimum pH was 6.0 for P I and about 5.0 for P II trehalase. TheK _m values were about 1.0 mM for P I trehalase and 0.30 mM for P II trehalase.These results suggest that in feeding larval midgut there are two types of trehalase which are distinguishable from each other by intracellular localization, protein nature and kinetic properties. Furthermore, when the midgut undergoes metamorphosis, the P I enzyme found predominantly in feeding stages seems to be transformed to the P II enzyme via an intermediate form (Ppt-P II) with transitional properties.     

Partial purification and characterization of the major midgut proteases of grass grub larvae (Costelytra zealandica,Coleoptera: Scarabaeidae)

The major proteases of the grass grub (Costelytra zealandica) larval midgut have been identified, partially purified and characterized. Identification was made initially on the basis of hydrolysis of synthetic substrates (blocked and partially blocked esters and amides of specific amino acids), thus classifying the activities into different classes of endo- and exopeptidases. A range of inhibitors specific to different classes of proteases were used to confirm the presence of trypsin, chymotrypsin, elastase, leucine aminopeptidase and carboxypeptidases A and B and to establish the absence of thiol- and metallo-endopeptidases. The dominant endopeptidase in the midgut is trypsin, which is present in four forms, distinguishable by net charge, but indistinguishable either in terms of Michaelis-Menten parameters (K_m and k_cat) or in molecular weight (23,000). The pH optimum lies between pH 9–10. Leucine aminopeptidase has a molecular weight of 91,000 and a pH optimum at pH 8.0. Carboxypeptidase A has a molecular weight of 43,000 and a pH optimum at pH 8.5. All enzymes retained substantial activity at pH 7.0–7.1, the pH of the midgut lumen, where the bulk of the activity was located. Protease levels in the hindgut (or fermentation sac) were 1–5% of those in the midgut. The range of enzymes appears sufficient for complete breakdown of ingested protein.     

Spatial organization of digestion in the larval and imaginal stages of the sciarid fly Trichosia pubescens

Aminopeptidase, amylase, cellulase and trypsin are found in major amounts in the midgut lumen, whereas alkaline phosphatase, cellobiase, α-glucosidase, maltase and trehalase occur mainly in the midgut tissue of Trichosia pubescens larvae. Cellulase and a part of the amylase seem to be derived from the fungi the larvae eat. Based on the molecular weights of the enzymes which pass and of those which do not pass through the peritrophic membrane, it is possible to estimate the peritrophic membrane pores as having diameters of 7.5–8.0 nm. Purification and assays of microvillar enzymes from different larval midgut regions suggest that alkaline phosphatase, cellobiase, α-glucosidase, maltase and trehalase are bound to the plasma membrane chiefly of midgut caeca cells. The results support the hypothesis that digestion starts in the endoperitrophic space under the action of amylase, cellulase and trypsin, goes on in the ectoperitrophic space by amylase, cellulase and aminopeptidase and is completed through the catalytic action of plasma membrane-bound hydrolases. The data lead to the conclusion that the spatial organization in T. pubescens larvae is identical to that of another Sciarid fly (Rhynchosciara americana) despite the finding that the midgut trehalase is bound to the plasma membrane in T. pubescens and soluble in R. americana. With metamorphosis salivary amylase appears, α-glucosidase, trehalase and maltase increase, and the other midgut hydrolases decrease or even disappear. This is in accordance with the fact that the larvae feed on decaying plants and fungi and the imagoes feed on nectar.     

Purification and properties of α-amino--caprolactam racemase from Achromobacter obae

We have purified a unique enzyme, α-amino--caprolactam racemase 945-fold from an extract of Achromobacter obae by Octyl—Sepharose CL-4B and Thiopropyl—Sepharose 6B and some other chromatographies. The purified enzyme was found homogeneous by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a monomeric structure with M_r 50 000 and a sedimentation coefficient (s_20,w) of 4.28 S. The enzyme contains pyridoxal 5'-phosphate as a coenzyme. The pH optimum for the enzyme activity is 9.0. D- and L-α-amino--caprolactams are the only substrates. The K_m values for the D- and L-isomers are, 8 and 6 mM, respectively.     

Spatial organization of digestion,secretory mechanisms and digestive enzyme properties in Pheropsophus aequinoctialis (Coleoptera: Carabidae)

Aminopeptidase (soluble form M_r 110,000), carboxypeptidase A (soluble form M_r 47,000), maltase (a dimer composed of two identical M_r 60,000 subunits) and trypsin (two charge isomers with M_r 34,000) are found in major amounts in the crop and midgut tissue, whereas amylase (a trimer of three identical M_r 18,000 subunits) and cellobiase (a trimer of three identical M_r 27,000 subunits) occur mainly in the crop and midgut contents. Subcellular fractions of midgut cells were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation. The results suggest that part of the aminopeptidase and carboxypeptidase A activity is bound to microvilli, that major amounts of trypsin and maltase are trapped in the cell glycocalyx and finally that soluble aminopeptidase, amylase and cellobiase occur in intracellular vesicles. The data support the hypothesis that most protein and carbohydrate digestion takes place in the crop under the action of enzymes passed forward from the midgut, after being secreted by exocytosis. Nevertheless, part of the intermediate and final digestion occurs at the surface of the midgut cells. The peculiar features of the digestion of P. aequinoctialis beetles, including their partly fluid peritrophic membranes, are thought to be derived from putative Coleoptera ancestors.     

Acid carboxypeptidase from a wood-deteriorating basidiomycete, Pycnoporus Sanguineus

An acid carboxypeptidase (EC 3.4.16.1) has been isolated from the culture filtrate of a wood-degrading Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme were determined. The extracellular acid carboxypeptidase was homogeneous on polyacrylamide gel electrophoresis at pH 9.4 and SDS-disc gel electrophoresis. The MWs as determined by gel filtration and SDS-gel electrophoresis were 50 000 and 54 000, respectively. The isoelectric point was pH 4.78 using electrofocusing. The purified enzyme had a pH optimum of 3.4, a K_m of 0.74 mM and a k_cat of 16/sec with benzyloxycarbonyl-l-glutamyl-l-tyrosine. The K_m and k_cat values for bradykinin at pH 3.4 and 30° were 2.0 mM and 25/sec. Values for angiotensin at pH 3.4 and 30° were 0.76 mM and 2.4/sec, respectively.     

Proteins in the nervous system — structure and function : Progress in clinical and biological research; volume 79

We have purified a unique enzyme, α-amino-?-caprolactam racemase 945-fold from an extract of Achromobacter obae by Octyl—Sepharose CL-4B and Thiopropyl—Sepharose 6B and some other chromatographies. The purified enzyme was found homogeneous by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a monomeric structure with M_r ～ 50 000 and a sedimentation coefficient (s_20,w) of 4.28 S. The enzyme contains pyridoxal 5'-phosphate as a coenzyme. The pH optimum for the enzyme activity is ～9.0. D- and L-α-amino-?-caprolactams are the only substrates. The K_m values for the D- and L-isomers are, 8 and 6 mM, respectively.     

Purification and properties of glutamine synthetase from Bacillus polymyxa

Glutamine synthetase (EC 6.3.1.2) was purified to homogeneity from a free-living nitrogen fixing bacteria, Bacillus polymyxa. The holoenzyme, relative molecular mass (M_r) of 600 000 is composed of monomeric sub-units of 60 000 (M_r). The isoelectric point of the sub-units was 5.2. The pH optimum for the biosynthetic and transferase enzyme activity was 8.2 and 7.8, respectively. The apparent K _m values (K _m ^app ) in the biosynthetic reaction for glutamate, NH₄Cl and ATP were 3.2, 0.22 and 1 mM, respectively. In the transferase reaction the K _m values for glutamine, hydroxylamine and ADP were 6.5, 3.5 and 8×10^-4 mM respectively. L-Methionine-D-L-sulfoximine was a very potent inhibitor in both biosynthetic and transferase reactions. Similar to most Gram positive bacteria there was no evidence of in vivo adenylylation and the enzyme seemed to be mainly regulated by feed-back mechanism.Abbreviations PMSF phenylmethylsulfonylfluoride - TCA trichloroacetic acid - GS glutamine synthetase - MSO L-Methionine-D-L-sulfoximine - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis - SVPDE snake venum phosphodiesterase     

Comparative study of digestive enzymes of squid (Todarodes pacificus) viscera after supercritical carbon dioxide and organic solvent extraction

Three major classes of digestive enzymes of squid viscera were characterized following extraction of oil by supercritical carbon dioxide (SCO₂) and organic solvent, n-hexane. Squid viscera were extracted at temperature, 35∼45°C and pressure, 15∼25 MPa for 2.5 h by SCO₂ with a constant flow rate of 22 g/min. Oil extraction yield increased with the increasing of extraction pressure and temperature. The highest oil extracted residues of squid viscera were used for characterization of digestive enzymes. The activities of protease, lipase, and amylase were highest in n-hexane treated squid viscera samples and lowest in SCO₂ treated samples. The crude extracts of SCO₂ and n-hexane treated squid viscera samples showed almost same optimum pH and pH stability for each of the digestive enzymes. The optimum temperature of protease, lipase, and amylase were found to almost similar in SCO₂ and n-hexane treated samples. But the thermal stability for each digestive enzyme in SCO₂ treated squid viscera were slightly higher than that of n-hexane treated squid viscera. Studies using SDS-PAGE showed no significant differences in protein patterns of the crude extracts of untreated and SCO₂ and n-hexane treated squid viscera indicating no denaturation of proteins.     

A hysteretic invertase from Equisetum giganteum L

The invertase from Equisetum giganteum L., a lower vascular sporophytic plant, was purified to chromatographic and electrophoretic homogeneity. The enzyme appears to be a pentamer, M_r 91,000, formed by identical subunits (M_r 18,000). An isoelectric point of 4.5 was found for the protein. The optimum pH was about 4.5 and the preferred substrate is sucrose, K_m=10.4 mM. Glucose and fructose are classical non-competitive (K_i=120 mM) and competitive (K_i=96 mM) inhibitors, respectively. Proteins which behave as activators of the enzyme suppress the inhibitory action of the reaction products. The activation energy of the hydrolytic reaction is 18,000 cal/mol. The outstanding property of the invertase is a hysteretic behavior when the pH changes from 3.05 to 4.5. The lag time is independent of the enzyme concentration suggesting that slow conformational changes are induced by pH variation and not by different polymerization states.     

Purification of ctp: cholinephosphate cytidylyl-transferase from pea stems

CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) was purified from pea (Pisum sativum) stems. The purification involved ammonium sulphate fractionation, ion exchange chromatography, removal of proteases with α₂-macroglobulin and gel filtration. The purified enzyme had K_m values for phosphorylcholine and CTP of 2.1 mM and 0.55 mM respectively. It was found to have a pH optimum of 7.5, a requirement for Mg²⁺ and an M_r of 56000. It could not utilize phosphorylethanolamine and its activity was not stimulated by added phospholipids.