Soluble and membrane-bound neutral alpha-glucosidase from the human kidney

In human kidney cortex neutral alpha-glucosidases 1 and 2 are represented by two forms, soluble (cytosolic) and membrane-bound (brush border) ones. It has been shown that the soluble enzyme preexists in human kidney but does not derive from the membrane-bound form. Similar to the membrane-bound enzyme the soluble form is a glycoprotein. Both enzyme forms possess identical electrophoretic mobility, pH-optimum, heat sensibility and Km values for maltose (0.7 mM) and 4-methylumbelliferyl-alpha-D-glucopyranoside (0.57 mM), but differ by molecular weights as determined by gel filtration chromatography. The molecular weights of the soluble neutral alpha-glucosidases 1 and 2 are lower than those of the comparable brush border enzymes (470 000, 360 000, 520 000 and 440 000, correspondingly). Neutral membrane-bound alpha-glucosidase 1 is a sialylated enzyme with a pI of 4.10 +/- 0.02. The soluble enzyme contains no or only traces of neuraminic acid and has a pI 4.40 +/- 0.03. The soluble and membrane-bound neutral alpha-glucosidases are apparently independent forms of the enzyme, differing by the degree of sialylation and by the presence of an "anchor" in the membrane-bound enzyme. The synthesis of both forms is presumably coded by the same structural gene.     

Human chitotriosidase-catalyzed hydrolysis of chitosan

Chitotriosidase (HCHT) is one of two family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the human defense mechanism against fungal parasites, but its precise role and the details of its enzymatic properties have not yet been fully unraveled. We have studied the properties of HCHT by analyzing how the enzyme acts on high-molecular weight chitosans, soluble copolymers of β-1,4-linked N-acetylglucosamine (GlcNAc, A), and glucosamine (GlcN, D). Using methods for in-depth studies of the chitinolytic machinery of bacterial family 18 enzymes, we show that HCHT degrades chitosan primarily via an endoprocessive mechanism, as would be expected on the basis of the structural features of its substrate-binding cleft. The preferences of HCHT subsites for acetylated versus nonacetylated sugars were assessed by sequence analysis of obtained oligomeric products showing a very strong, absolute, and a relative weak preference for an acetylated unit in the -2, -1, and +1 subsites, respectively. The latter information is important for the design of inhibitors that are specific for the human chitinases and also provides insight into what kind of products may be formed in vivo upon administration of chitosan-containing medicines or food products.     

A comparative study of ribonuclease hydrolysis of rat brain-cortex and liver membrane-bound ribosomes

Because it has been proposed that the ribosome–membrane interaction is different in endoplasmic reticulum derived from a non-secretory and secretory cell we undertook a study to determine whether attachment of the ribosome to the membrane involved ribosomal RNA and if the rRNA in ribosomes derived from the two classes of cell possessed an altered susceptibility to RNAase (ribonuclease) hydrolysis. We found that brain ribosomes appeared to possess more regions accessible to nuclease attack, independent of whether a sequence-dependent RNAase (T₁) or a sterically hindered RNAase bound to Enzite polymer was employed. These results were independent of whether the ribosomes were membrane-bound or detached from the endoplasmic reticulum membranes, but at high RNAase concentration these differences became negligible. No conclusions, however, could be drawn as to whether ribosomal RNA is involved in the attachment of the ribosome to the endoplasmic reticulum membrane, because of the presence of endogeneous membrane-associated RNAases. Analysis of the rRNA fragments by polyacrylamide-gel electrophoresis suggests that the sites available for attack by low concentrations of nuclease in bound-ribosomes derived from brain cortex are different from those of liver.     

Phosphoinositide hydrolysis in Ki-ras-transformed fibroblasts stimulated by platelet-derived growth factor and bradykinin

• 1.1. Signal transduction in response to platelet-derived growth factor (PDGF)-BB and bradykinin (BK) have been examined by measuring inositol polyphosphate formation in NIH3T3 fibrobalst and v-Ki-ras -transformed NIH3T3 fibroblast (DT).
• 2.2. The PDGF-induced inositol polyphosphate formation in NIH3T3 was greater than that in DT cells, in which autophosphorylation of PDGF receptor and tyrosine phosphorylation of phospholipase C (PLC)-γ 1 were suppressed when examined by immunoblotting with anti-phosphotyrosine antibody.
• 3.3. On the other hand, BK-stimulation produced a much higher level of inositol polyphosphate in DT cells which have a greater number of BK receptors.
• 4.4. These results indicate that in Ki-ras transformed cells the decrease (caused by PDGF) and the increase (caused by BK) in phosphoinositide hydrolysis are due to the defective autophosphorylation of PDGF receptors leading to a reduction in PLC-γ 1 tyrosine phosphorylation and the overexpression of BK receptors, respectively.

     

Pulmonary phosphatidic acid phosphohydrolase: further studies on the activities in rat lung responsible for the hydrolysis of membrane-bound and aqueously dispersed phosphatidate

Rat lung cytosol and microsomal fractions both contain phosphohydrolase activity towards membrane-bound phosphatidic acid (PAmb) and aqueously dispersed phosphatidic acid (PAaq) which cannot be explained through contamination with the other fraction. The phosphohydrolase activities with PAaq demonstrated Km and Vmax values which were more than an order of magnitude greater than those observed with PAmb and with vesicles prepared from the lipids extracted from [32P]PA-labelled microsomes. The PAaq-dependent activities in both fractions were stimulated by preparing mixed liposomes with phosphatidylcholine. The PAmb-dependent activities in rat lung microsomes and cytosol were markedly stimulated by high concentrations of Triton X-100 and Nonidet P-40. The PAmb- and PAaq-dependent activities in the microsomes were stimulated by deoxycholate. Although no difference was observed in the inhibition profiles of the PAmb- and PAaq-dependent activities of the cytosol in the presence of various mercurials, the PAmb-dependent activity in the microsomes was somewhat more susceptible than the PAaq-dependent activity. The PAmb-dependent activities in both fractions were more susceptible to inhibition by iodoacetamide. These results support the view that separate rat lung enzymes were involved in the hydrolysis of PAmb and PAaq. The relative abilities of rat lung cytosol and microsomes to hydrolyse PA endogenously generated on the microsomes were compared using relative concentrations of cytosol corresponding to the levels in intact rat lung. During the initial period (5-10 min) the cytosol phosphohydrolase activity was more effective than the microsomal activity. At later stages (10-20 min), the rates were comparable.     

Purification of a membrane-bound neutral endopeptidase from rat kidney and its activation by polyamines

An endopeptidase which cleaves succinyl trialanine p-nitroanilide (Suc(Ala)3-pNA) into succinyl dialanine and alanine p-nitroanilide (Ala-pNA) was solubilized from a microsomal membrane fraction of rat kidney with Nonidet P-40 following treatment with 1 M KCl and Brij 35. The solubilized enzyme was purified to homogeneity by DEAE-Sephadex chromatography, Sepharose CL-6B gel filtration and sucrose gradient centrifugation. The final enzyme preparation had a specific activity of 1.69 mumol/min/mg protein, representing about 140-fold purification over the starting membrane. The enzyme hydrolyzes Suc(Ala)3-pNA with a Km value of 0.28 mM and a Vmax value of 1.3 mumol/min. The molecular weight of the undenatured enzyme was estimated to be 360,000 by gel filtration on a Sepharose CL-6B column and that of the denatured enzyme to be 92,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, revealing the presence of a single polypeptide chain. The enzyme was markedly activated by polyamines, producing increases in the values of both Km and Vmax. Comparatively less activation was found in the presence of some monovalent cations and Ca2+. The activation by polyamines was inversely proportional to the concentration of monovalent cations, but Ca2+ and polyamines seemed to stimulate additively.     

A neutral pH thermal hydrolysis method for quantification of structured RNAs

Riboswitch aptamers adopt diverse and complex tertiary structural folds that contain both single-stranded and double-stranded regions. We observe that this high degree of secondary structure leads to an appreciable hypochromicity that is not accounted for in the standard method to calculate extinction coefficients using nearest-neighbor effects, which results in a systematic underestimation of RNA concentrations. Here we present a practical method for quantifying riboswitch RNAs using thermal hydrolysis to generate the corresponding pool of mononucleotides, for which precise extinction coefficients have been measured. Thermal hydrolysis can be performed at neutral pH without reaction quenching, avoids the use of nucleases or expensive fluorescent dyes, and does not require generation of calibration curves. The accuracy of this method for determining RNA concentrations has been validated using quantitative ³¹P-NMR calibrated to an external standard. We expect that this simple procedure will be generally useful for the accurate quantification of any sequence-defined RNA sample, which is often a critical parameter for in vitro binding and kinetic assays.     

ATP hydrolysis and synthesis by the membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum.

The membrane-bound ATP synthetase complex of Methanobacterium thermoautotrophicum showed maximum activity for ATP hydrolysis at pH 8, at temperatures between 65 and 70 degrees C, and at an ATP-Mg2+ ratio of 0.5. Anaerobic conditions were not prerequisite for enzyme activity. The enzyme showed a Km value for ATP of 2 mM, and activity was Mg2+ dependent; Mn2+, Co2+, Ca2+, and Zn2+ could replace Mg2+ to some extent. Other nucleoside triphosphates could be hydrolyzed. N,N'-dicyclohexylcarbodiimide inhibited ATP hydrolysis. A proton-motive force, artificially imposed by a pH shift or valinomycin, resulted in ATP synthesis in whole cells. The ATP synthetase complex of the thermophilic methanogenic bacterium is similar to those described in aerobic and anaerobic microorganisms.     

The hydrolysis of endothelins by neutral endopeptidase 24.11 (enkephalinase)

Endothelins 1-3 are a family of 21-amino acid peptides whose structure consists of two rings formed by intra-chain disulfide bonds and a linear "COOH-terminal tail." These peptides were originally described on the basis of their potent vasoconstrictor activity. The hydrolytic inactivation of endothelin action has recently been implicated to be attributed, at least in part, to the enzyme neutral endopeptidase 24.11 (Scicli, A. G., Vijayaraghavan, J., Hersh, L., and Carretero, O. (1989) Hypertension 14, 353). The kinetic properties and mode of hydrolysis of the endothelins by this enzyme are reported in this study. The Km for endothelins 1 and 3 hydrolysis is approximately 2 microM while endothelin2 exhibits a 5-fold higher Km. Endothelins 1 and 2 exhibit similar Vmax values while endothelin3 is hydrolyzed considerably more slowly. The initial cleavage site in endothelin1 is at the Ser5-Leu6 bond located within one of the cyclic structures. Thermolysin, a bacterial neutral endopeptidase with a similar substrate specificity to neutral endopeptidase 24.11 initially cleaves endothelin1 between His16-Leu17 which lies within the COOH-terminal linear "tail" portion of the molecule. The cleavage of endothelins 2 and 3 by neutral endopeptidase 24.11 differs from that observed with endothelin1 in that cleavage of these endothelins occurs at Asp18-Ile19 within the linear COOH-terminal tail structure. These results demonstrate that the endothelins are good substrates for neutral endopeptidase 24.11 and suggest that their mode of cleavage is dependent upon both amino acid sequence as well as peptide conformation.     

Chloramine-T-mediated chemoselective hydrolysis of thioglycosides into glycosyl hemiacetals under neutral conditions

A metal-free, mild, efficient and chemoselective hydrolysis of several thioalkylglycosides (1) into their corresponding 1-hydroxy sugars (2) using sodium N-chloro-p-toluenesulfonamide trihydrate (chloramine-T) without affecting other functional groups is reported.     

Chloride-insensitive, glycine-phenylalanine-naphthylamide hydrolysis at neutral pH in human skin fibroblasts

Crude lysosomal preparations from a cultured human skin fibroblast line were found to contain significant levels of a neutral pH hydrolase activity towards glycine--phenylalanine--beta-naphthylamide (NA), a substrate normally used for the assay of lysosomal dipeptidyl aminopeptidase I. However, the activity was chloride ion insensitive, nonlatent, and inhibitable by cationic detergents and amino acids. Assays of substrate selectivity, relative substrate affinity, pH and anion and cation sensitivity indicated the activity to be distinct from dipeptidyl aminopeptidases I (chloride-dependent hydrolysis of Pro-Phe-, Gly-Phe-, Gly-Arg-, and Pro-Arg-NA's at acid pH), II (Lys-Ala-NA hydrolysis), III (Arg-Arg-NA hydrolysis), and IV (Gly-Pro-NA hydrolysis). The lysosomal preparations also contained significant activity towards several amino acid--naphthylamides, notably Arg-NA. Only dipepidyl aminopeptidase I activity showed sensitivity to chloride anions, both dipeptidyl aminopeptidases I and II showed substantial latency, and none of the activities displayed a significant metal cation dependent.     

Soluble and membrane-bound forms of lipoprotein lipase in mouse lung tissue

Homogenates of mouse lungs were separated by differential centrifugation into two fractions containing lipoprotein lipase, namely, a soluble and a membrane-bound fraction. Lipoprotein lipase was specifically identified by its inhibition by both protamine sulfate (3 mg/ml) and sodium chloride (0.9 mol/l). The enzymatic activity of each fraction was enhanced when serum was preincubated with the enzyme. Both enzyme fractions showed optimum activity at alkaline pH, but the membrane-bound enzyme showed a higher pH optimum. In addition, the apparent Km of the soluble enzyme was lower than that of the membrane-bound enzyme. It is concluded that there are two different forms of lipoprotein lipase in mouse lung tissue that differ in a number of aspects.