Small Molecular Substrates Research Articles

Fluorescence‐activated screening of polyester‐depolymerizing enzymes based on pseudo‐PET polythioester plastics

AbstractWhile fluorescence‐based high‐throughput screening (HTS) has been valuable for identifying enzymes and microorganisms for polyethylene terephthalate (PET) depolymerization, the traditional approach relies on cleaving ester bonds in small‐molecule probes, limiting its ability to directly detect activity towards polymeric PET substrates. This study proposes a novel fluorescence‐based screening strategy for detecting the release of sulfhydryl groups during the depolymerization of pseudo‐PET polythioesters by polyester hydrolases. The strategy successfully identifies the polyester‐depolymerizing activity of leaf‐branch compost cutinase (LCCICCG), whereas porcine liver esterase (PLE) only hydrolyses small molecular substrates. Furthermore, the strategy can be integrated with a droplet microfluidic system for high‐throughput screening of LCCICCG. This work further demonstrates the applicability of the microplate reader platform for screening polyester‐depolymerizing bacteria. This novel fluorescence‐based approach offers an efficient strategy to identify enzymes and microbial resources for depolymerizing polyester‐like plastics.

Mitochondrial uncoupling proteins regulate the metabolic function of human Sertoli cells.

Mitochondrial uncoupling proteins (UCPs) regulate mitochondrial activity and reactive oxygen species production through the transport of protons and metabolites. This study identified the expression of UCPs in human Sertoli cells, which proved to be modulators of their mitochondrial activity. Mitochondrial uncoupling proteins (UCPs) are mitochondrial channels responsible for the transport of protons and small molecular substrates across the inner mitochondrial membrane. Altered UCP expression or function is commonly associated with mitochondrial dysfunction and increased oxidative stress, which are both known causes of male infertility. However, UCP expression and function in the human testis remain to be characterized. This study aimed to assess the UCP homologs (UCP1-6) expression and function in primary cultures of human Sertoli cells (hSCs). We identified the mRNA expression of all UCP homologs (UCP1-6) and protein expression of UCP1, UCP2, and UCP3 in hSCs. UCP inhibition by genipin for 24 h decreased hSCs proliferation without causing cytotoxicity (n = 6). Surprisingly, the prolonged UCP inhibition for 24 h decreased mitochondrial membrane potential, oxygen consumption rate (OCR), and endogenous reactive oxygen species (ROS) production. The metabolism of hSCs was also affected as UCP inhibition shifted their metabolism toward an increased pyruvate consumption. Taken together, these findings demonstrate that UCPs play a role as regulators of the mitochondrial function in hSCs, emphasizing their potential as targets in the study of male (in)fertility.

A Thermococci-to-Clostridia Pathway for the Evolution of the Bacteria Domain

With the identification of an archaeal Last Universal Common Ancestor (LUCA) related to the archaeon Methanopyrus, the origin of Bacteria became a choice between autonomic development versus descent from Archaea. The similarity bitscores between paralogous valyl-tRNA synthetase (VARS) and isoleucyl-tRNA synthetase (IARS) suggest that the five oldest bacteria were Mahella australiensis, Thermincola potens, Halobacteroides halobius, Desulfosporosinus orientis and Caldicellulosiruptor lactoaceticus, which were all Clostridia species and hydrogen producers. A search for archaea that could be a candidate Progenitor of Bacteria endowed with an Emden-Myerhof-Parnas type glycolytic pathway and a clostridial-like dark-fermentation mechanism for generating hydrogen pointed to such a role for the anaerobic chemoorganotroph Thermococci, which were known to engage in rapid evolutionary changes at high-biodiversity sites abundant in sugars and other small molecular substrates, and constituted together with Clostridia the two most powerful microbial generators of hydrogen. Moreover, two-domain maximum-likelihood and maximum- parsimony phylogenetic trees for VARS showed that Thermococci and Clostridia formed sister clades on both trees, and close similarity was evident between their VARS sequences, which were consistent with kinship between them. On this basis, it was proposed that the Bacteria domain emerged from a thermocococal or thermococcal-like Progenitor of Bacteria possibly at some high-biodiversity site, through the formation of a Thermincola-proximal Last Bacterial Common Ancestor (LBCA).

Rapid regulations of metabolic reactions in Escherichia coli via light-responsive enzyme redistribution.

Protein-based condensates have been proposed to accelerate biochemical reactions by enriching reactants and enzymes simultaneously. Here, we engineered those condensates into a photo-activated switch in Escherichia coli (PhASE) to regulate enzymatic reactions via tuning the spatial correlation of enzymes and substrates. In this system, scaffold proteins undergo liquid-liquid phase separation (LLPS) to form light-responsive compartments. Tethered with a light-responsive protein, enzymes of interest (EOIs) can be recruited by those compartments from cytosol within only a few seconds after a pulse of light induction and fully released in 15min. Furthermore, we managed to enrich small molecular substrates simultaneously with enzymes in the compartments and achieved the acceleration of luciferin and catechol oxidation by 2.3- and 1.6-folds, respectively. We also developed a quantitative model to guide the further optimization of this demixed regulatory system. Our tool can thus be used to study the rapid redistribution of proteins, and reversibly regulate enzymatic reactions in E. coli.

EuPIP1;2, a Plasma Membrane Aquaporin Gene from Eucommia ulmoides, Enhances Drought and Salt Tolerance in Transgenic Tobacco

Aquaporins are a specific type of membrane channel proteins that efficiently transport water molecules and other small molecular substrates in plants. In this study, we isolated the plasma membrane aquaporin gene EuPIP1;2 from Eucommia ulmoides, an important medicinal plant in China. The EuPIP1;2 protein was localized on the plasma membrane. Quantitative RT-PCR analysis revealed that EuPIP1;2 was constitutively expressed in all analyzed tissues, with the highest expression levels detected in the fruit and root. Overexpression of EuPIP1;2 in transgenic tobacco enhanced plant tolerance of drought and salinity. Under drought and salt stress, the transgenic lines exhibited higher percentage germination, longer roots, and enhanced percentage survival compared with wild-type plants. The contents of malonaldehyde and proline suggested that EuPIP1;2 improved drought and salt tolerance in transgenic lines by reducing damage to membranes and improving osmotic adjustment. We predict that EuPIP1;2 could be applied to improve drought and salt tolerance in transgenic plants.

Iocasia fonsfrigidae NS-1 gen. nov., sp. nov., a Novel Deep-Sea Bacterium Possessing Diverse Carbohydrate Metabolic Pathways.

Resolving metabolisms of deep-sea microorganisms is crucial for understanding ocean energy cycling. Here, a strictly anaerobic, Gram-negative strain NS-1 was isolated from the deep-sea cold seep in the South China Sea. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain NS-1 was most closely related to the type strain Halocella cellulosilytica DSM 7362T (with 92.52% similarity). A combination of phylogenetic, genomic, and physiological traits with strain NS-1, was proposed to be representative of a novel genus in the family Halanaerobiaceae, for which Iocasia fonsfrigidae NS-1 was named. It is noteworthy that I. fonsfrigidae NS-1 could metabolize multiple carbohydrates including xylan, alginate, starch, and lignin, and thereby produce diverse fermentation products such as hydrogen, lactate, butyrate, and ethanol. The expressions of the key genes responsible for carbohydrate degradation as well as the production of the above small molecular substrates when strain NS-1 cultured under different conditions, were further analyzed by transcriptomic methods. We thus predicted that part of the ecological role of Iocasia sp. is likely in the fermentation of products from the degradation of diverse carbohydrates to produce hydrogen as well as other small molecules, which are in turn utilized by other members of cold seep microbes.

Polymer Coatings on Virus-like Particle Nanoreactors at Low Ionic Strength-Charge Reversal and Substrate Access.

Virus-like particles (VLPs) are a class of biomaterials which serve as platforms for achieving the desired functionality through interior and exterior modifications. Through ionic strength-mediated electrostatic interactions, VLPs have been assembled into hierarchically ordered materials. This work builds on predictive models to prepare polymer-coated VLP clusters at very low ionic strength. Zeta potential measurements showed that the clusters carried a strongly positive charge, a complete charge reversal from the VLP building block. SAXS analysis confirmed polymer adsorption onto the VLP exterior. We then studied the activity of an encapsulated enzyme toward small molecular and macromolecular substrates to determine the effect of each component of the hierarchically assembled material. We found that while encapsulation and polymer coating did not have a large effect on access to the enzyme by its native, small molecular substrate, substrate modification with a macromolecule caused the polymer coating and encapsulation to affect the access to the enzyme.

Visible Light-Activatable Oxidase Mimic of 9-Mesityl-10-Methylacridinium Ion for Colorimetric Detection of Biothiols and Logic Operations.

In this work, 9-mesityl-10-methylacridinium ion (Acr+-Mes) is found to act as an effective photocatalyst mimicking the function of oxidase. Upon visible light illumination, the excited Acr+-Mes is able to exhibit superior enzymatic catalytic activity for small molecular substrates as well as protein biomacromolecule (cytochrome c). The experiment results demonstrate that the Acr+-Mes oxidase mimic shows higher affinity to 3,3',5,5'-tetramethylbenzidine (TMB) than natural horseradish peroxidase or the reported molecule oxidase mimic. The reaction mechanism is ascribed to the strong oxidation property of the long-lived electron-transfer state (Acr•-Mes•+) and the electron transfer from Acr•-Mes radical to dissolved oxygen to generate superoxide radicals, which can easily oxidize various substrates. On the basis of these observations, the light-activatable Acr+-Mes with an oxidase-like activity as the probe is utilized for cost-effective, sensitive, and highly selective colorimetric detection of two biothiols (L-cysteine and L-glutathione). The lowest detectable concentrations of L-Cys and L-GSH is 100 nM, which is lower than that of most of the reported methods for biothiols. Beyond this, we construct a series of visual molecular logic gates (AND, INH, and NOR) using the oxidase mimic-involved reaction systems.

Focused library with a core structure extracted from natural products and modified: application to phosphatase inhibitors and several biochemical findings.

Synthesis of a focused library is an important strategy to create novel modulators of specific classes of proteins. Compounds in a focused library are composed of a common core structure and different diversity structures. In this Account, we describe our design and synthesis of libraries focused on selective inhibitors of protein phosphatases (PPases). We considered that core structures having structural and electronic features similar to those of PPase substrates, phosphate esters, would be a reasonable choice. Therefore, we extracted core structures from natural products already identified as PPase inhibitors. Since many PPases share similar active-site structures, such phosphate-mimicking core structures should interact with many enzymes in the same family, and therefore the choice of diversity structures is pivotal both to increase the binding affinity and to achieve specificity for individual enzymes. Here we present case studies of application of focused libraries to obtain PPase inhibitors, covering the overall process from selection of core structures to identification and evaluation of candidates in the focused libraries. To synthesize a library focused on protein serine-threonine phosphatases (PPs), we chose norcantharidin as a core structure, because norcantharidin dicarboxylate shows a broad inhibition profile toward several PPs. From the resulting focused library, we identified a highly selective PP2B inhibitor, NCA-01. On the other hand, to find inhibitors of dual-specificity protein phosphatases (DSPs), we chose 3-acyltetronic acid extracted from natural product RK-682 as a core structure, because its structure resembles the transition state in the dephosphorylation reaction of DSPs. However, a highly selective inhibitor was not found in the resulting focused library. Furthermore, an inherent drawback of compounds having the highly acidic 3-acyltetronic acid as a core structure is very weak potency in cellulo, probably due to poor cell membrane permeability. Therefore, we next modified the core structure from acidic to neutral by transformation to the enamine derivative and constructed a second-generation focused library (RE derivatives). The resulting compounds showed dramatically improved cell membrane permeability and inhibitory selectivity and included VHR (vaccinia VH1-related)-selective RE12 and CDC25A/B (cell division cycle 25A/B)-selective RE44. These inhibitors act on target enzymes in cellulo and do not generate reactive oxygen species, which is a potential problem with quinoid-type inhibitors of CDC25s. The cellular activity of RE12 was further improved by replacement of the side chain to afford RE176, which showed more potent antiproliferative activity than RE12 against HeLa cells. The dramatic change of inhibitory selectivity obtained by core structure modification from 3-acyltetronic acid to its enamine derivative was associated with a change in the mode of action. Namely, RE derivatives were found to be noncompetitive inhibitors with respect to a small-molecular substrate of CDC25A/B, whereas RK-682 was a competitive inhibitor of VHR. We identified the binding site of RE derivatives on the CDC25A as a pocket adjacent to the active site; this appears to be a promising target site for development of further novel inhibitors of CDC25s.

Processive catalysis.

Nature's enzymes are an ongoing source of inspiration for scientists. The complex processes behind their selectivity and efficiency is slowly being unraveled, and these findings have spawned many biomimetic catalysts. However, nearly all focus on the conversion of small molecular substrates. Nature itself is replete with inventive catalytic systems which modify, replicate, or decompose entire polymers, often in a processive fashion. Such processivity can, for example, enhance the rate of catalysis by clamping to the polymer substrate, which imparts a large effective molarity. Reviewed herein are the various strategies for processivity in nature's arsenal and their properties. An overview of what has been achieved by chemists aiming to mimic one of nature's greatest tricks is also included.

Functional transformations of bile acid transporters induced by high-affinity macromolecules

Apical sodium-dependent bile acid transporters (ASBT) are the intestinal transporters that form intermediate complexes with substrates and its conformational change drives the movement of substrates across the cell membrane. However, membrane-based intestinal transporters are confined to the transport of only small molecular substrates. Here, we propose a new strategy that uses high-affinity binding macromolecular substrates to functionally transform the membrane transporters so that they behave like receptors, ultimately allowing the apical-basal transport of bound macromolecules. Bile acid based macromolecular substrates were synthesized and allowed to interact with ASBT. ASBT/macromolecular substrate complexes were rapidly internalized in vesicles, localized in early endosomes, dissociated and escaped the vesicular transport while binding of cytoplasmic ileal bile acid binding proteins cause exocytosis of macromolecules and prevented entry into lysosomes. This newly found transformation process of ASBT suggests a new transport mechanism that could aid in further utilization of ASBT to mediate oral macromolecular drug delivery.

Oligomeric bile acid-mediated oral delivery of low molecular weight heparin

Intestinal transporters are limited to the transport of small molecular substrates. Here, we describe the development of apical sodium-dependent bile acid transporter (ASBT)-targeted high-affinity oligomeric bile acid substrates that mediate the transmembrane transport of low molecular weight heparin (LMWH). Several oligomers of deoxycholic acid (oligoDOCA) were synthesized to investigate the substrate specificity of ASBT. To see the binding of oligoDOCA on the substrate-binding pocket of ASBT, molecular docking was used and the dissociation rate constants (KD) were measured using surface plasmon resonance. The KD for tetrameric DOCA (tetraDOCA) was 50-fold lower than that for monomeric DOCA, because tetraDOCA interacted with several hydrophobic grooves in the substrate-binding pocket of ASBT. The synthesized oligoDOCA compounds were subsequently chemically conjugated to macromolecular LMWH. In vitro, tetraDOCA-conjugated LMWH (LHe-tetraD) had highest selectivity for ASBT during its transport. Orally administered LHe-tetraD showed remarkable systemic anticoagulation activity and high oral bioavailability of 33.5±3.2% and 19.9±2.5% in rats and monkeys, respectively. Notably, LHe-tetraD successfully prevented thrombosis in a rat model of deep vein thrombosis. These results represent a major advancement in ASBT-mediated LMWH delivery and may facilitate administration of many important therapeutic macromolecules through a non-invasive oral route.

Two Structures of a Thiazolinyl Imine Reductase from Yersinia enterocolitica Provide Insight into Catalysis and Binding to the Nonribosomal Peptide Synthetase Module of HMWP1

The thiazolinyl imine reductase from Yersinia enterocolitica (Irp3) catalyzes the NADPH-dependent reduction of a thiazoline ring in an intermediate for the formation of the siderophore yersiniabactin. Two structures of Irp3 were determined in the apo (1.85 Å) and NADP(+)-bound (2.31 Å) forms. Irp3 is structurally homologous to sugar oxidoreductases such as glucose-fructose oxidoreductase and 1,5-anhydro-d-fructose reductase, as well as to biliverdin reductase. A homology model of the thiazolinyl imine reductase from Pseudomonas aeruginosa (PchG) was generated. Extensive loop insertions are observed in the C-terminal domain that are unique to Irp3 and PchG and not found in the structural homologues that recognize small molecular substrates. These loops are hypothesized to be important for binding of the nonribosomal peptide synthetase modules (found in HMWP1 and PchF, respectively) to which the substrate of the reductase is covalently attached. A catalytic mechanism for the donation of a proton from a general acid (either histidine 101 or tyrosine 128) and the donation of a hydride from C4 of nicotinamide of the NADPH cofactor is proposed for reduction of the carbon-nitrogen double bond of the thiazoline.

Highly Potent and Selective Histone Deacetylase 6 Inhibitors Designed Based on a Small-Molecular Substrate

To find novel histone deacetylase 6 (HDAC6)-selective inhibitors and clarify the structural requirements for HDAC6-selective inhibition, we prepared thiolate analogues designed based on the structure of an HDAC6-selective substrate and evaluated the histone/alpha-tubulin acetylation selectivity by Western blot analysis. Aliphatic compounds 17b-20b selectively caused alpha-tubulin acetylation over histone H4 acetylation. In enzyme assays using HDAC1, HDAC4, and HDAC6, compounds 17a-19a exhibited HDAC6-selective inhibition over HDAC1 and HDAC4.

A phage antibody to the active site of human placental alkaline phosphatase with higher affinity to the enzyme–substrate complex

Selection of specific antibodies from large repertoires is of importance in generating antibodies to specific structural determinants and in studying structure–function relationships. Alkaline phosphatase (AP) has several isozymes with various degrees of homology and a range of common synthetic substrates. We have previously reported the generation of isozyme specific anti-enzyme antibodies to an oncofetal antigen, placental alkaline phosphatase (PLAP) by using a specific uncompetitive inhibitor, l-Phe-Gly-Gly along with the substrate para-nitrophenyl phosphate ( pNPP), to elute scFvs from a phage-displayed immunoglobulin library. These antibodies were directed to the active site and inhibited enzyme activity. An uncompetitive inhibitor acts by stabilizing the enzyme–substrate (ES) complex. In the present work, we report the characteristics of a clone VE5, selected by the same method. This clone has a higher binding affinity for ES complex than for enzyme alone. This is true for all the three isozymes (placental, bone and intestinal) tested. However, the other synthetic small molecular substrate, disodium phenyl phosphate inhibits phage binding. The clone possibly binds to the conserved structures of the active site of the AP isozymes and the higher affinity binding to AP– pNPP complex reflects the method of selection. Such anti-enzyme antibodies have a possible potential role in dissecting structure–function relationship of enzymatic antigens.

The lipoprotein-associated coagulation inhibitor that inhibits the factor VII-tissue factor complex also inhibits factor Xa: insight into its possible mechanism of action

Blood coagulation is initiated when plasma factor VII(a) binds to its essential cofactor tissue factor (TF) and proteolytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of VII(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipoprotein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing VIIa, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin III alpha alone had no effect, but antithrombin III alpha with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2 of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phospholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondenaturing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its gamma-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with alpha-chymotrypsin, was used to further investigate the Xa requirement for VIIa/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of VIIa/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a VIIa-TF-XA-LACI complex that requires the GD of XA.(ABSTRACT TRUNCATED AT 400 WORDS).

The Lipoprotein-Associated Coagulation Inhibitor That Inhibits the Factor VII-Tissue Factor Complex Also Inhibits Factor Xa: Insight Into Its Possible Mechanism of Action

AbstractBlood coagulation is initiated when plasma factor Vll(a) binds to its essential cofactor tissue factor (TF) and proteo-lytically activates factors X and IX. Progressive inhibition of TF activity occurs upon its addition to plasma. This process is reversible and requires the presence of Vll(a), catalytically active Xa, Ca2+, and another component that appears to be associated with the lipoproteins in plasma, a lipopro-tein-associated coagulation inhibitor (LACI). A protein, LACI(HG2), possessing the same inhibitory properties as LACI, has recently been isolated from the conditioned media of cultured human liver cells (HepG2). Rabbit antisera raised against a synthetic peptide based on the N-terminal sequence of LACI(HG2) and purified IgG from a rabbit immunized with intact LACI(HG2) inhibit the LACI activity in human serum. In a reaction mixture containing Vila, Xa, Ca2+, and purified LACI(HG2), the apparent half-life (t1/2) for TF activity was 20 seconds. The presence of heparin accelerated the initial rate of inhibition threefold. Antithrombin IIIα alone had no effect, but antithrombin IIIα with heparin abrogated the TF inhibition. LACI(HG2) also inhibited Xa with an apparent t1/2of 50 seconds. Heparin enhanced the rate of Xa inhibition 2.5-fold, whereas phos-pholipids and Ca2+ slowed the reaction 2.5-fold. Xa inhibition was demonstrable with both chromogenic substrate (S-2222) and bioassays, but no complex between Xa and LACI(HG2) could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Nondena-turing PAGE, however, showed that LACI(HG2) bound to Xa but not to X or Xa inactivated by diisopropyl fluorophosphate. Thus, LACI(HG2) appears to bind to Xa at or near its active site. Bovine factor Xa lacking its γ-carboxyglutamic acid-containing domain, BXa(-GD), through treatment with a-chymotrypsin, was used to further investigate the Xa requirement for Vlla/TF inhibition by LACI(HG2). LACI(HG2) bound to BXa(-GD) and inhibited its catalytic activity against a small molecular substrate (Spectrozyme Xa), though at a rate approximately sevenfold slower than native BXa. Preincubation of LACI(HG2) with saturating concentrations of BXa(-GD) markedly retarded the subsequent inhibition of BXa. The VII(a)/TF complex was not inhibited by LACI(HG2) in the presence of BXa(-GD), and further, preincubation of LACI(HG2) with BXa(-GD) slowed the inhibition of Vlla/TF after the addition of native Xa. The results are consistent with the hypothesis that inhibition of VII(a)/TF involves the formation of a Vlla-TF-Xa-LACI complex that requires the GD of Xa. Because the GD contains the a-carboxyglutamic acids required for the Ca2+-dependent binding of factor Xa to phospholipid surfaces, the results also suggest that Ca2+ may be required for the native Xa-LACI complex to bind to and inhibit VII(a)/TF. LACI is a novel inhibitor that can rapidly affect feedback inhibition of the Vlla-Ca2+-TF enzymatic complex after the generation of small amounts of Xa and probably plays an important role in the regulation of in vivo coagulation.1988 by Grune & Stratton, Inc. 0006-4971/88/7102-0004$3.00/0

Rodent and human acid -glucosidase. Purification, properties and inhibition by antibodies. Investigation in type II glycogenosis.

The lysosomal acid α‐glucosidase has been purified more than 10000‐fold from rat and mice liver and from human placenta. The kinetic properties of the human enzyme are similar to those previously described for the rodent enzyme, with the main exception of the absence of inhibition by an excess of maltose. Antibodies were obtained by injection of the pure rat liver, mouse liver or human placental α‐glucosidase to rabbits. The property of the enzyme of hydrolysing glycogen or to catalyse transglucosylation from maltose to glycogen could be nearly completely inhibited by the antibodies whereas the hydrolysis of small molecular substrates was much less inhibited. Within the same species, the same amount of enzyme was inhibited by a given amount of antibodies, whatever the tissue used as a source of enzyme. A partial cross‐reactivity was observed from species to species. The presence of an immunologically reacting protein in tissues from patients with type‐II‐glycogenosis could not be demonstrated either by double‐diffusion test or by antibodies consumption. Similar negative results were also obtained with normal human liver in which acid α‐glucosidase has been inactivated at alkaline pH.

SEMIPERMEABLE MICROCAPSULES.

Simple methods have been developed for encapsulating aqueous solutions of protein within polymer membranes. Stable microcapsules 1 to 100 micro in diameter, with semipermeable membranes, can be made by depositing polymer around emulsified aqueous droplets, either by interfacial coacervation or by interfacial polycondensation. Aqueous suspensions of enzyme-loaded microcapsules act well on small-molecular substrates both in vitro and in vivo.