Guy Marchis-Mouren

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic α-amylase

Abstract Porcine pancreatic α-amylase (1,4-α- d -glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary α-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian α-amylases.

Barley malt-α-amylase. Purication, action pattern, and subsite mapping of isozyme 1 and two members of the isozyme 2 subfamily using p-nitrophenylated maltooligosaccharide substrates

Isoforms AMY1, AMY2-1 and AMY2-2 of barley alpha-amylase were purified from malt. AMY2-1 and AMY2-2 are both susceptible to barley alpha-amylase/subtilisin inhibitor. The action of these isoforms is compared using substrates ranging from p-nitrophenylmaltoside through p-nitrophenylmaltoheptaoside. The kcat/Km values are calculated from the substrate consumption. The relative cleavage frequency of different substrate bonds is given by the product distribution. AMY2-1 is 3-8-fold more active than AMY1 toward p-nitrophenylmaltotrioside through p-nitrophenylmaltopentaoside. AMY2-2 is 10-50% more active than AMY2-1. The individual subsite affinities are obtained from these data. The resulting subsite maps of the isoforms are quite similar. They comprise four and six glucosyl-binding subsites towards the reducing and the non-reducing end, respectively. Towards the non-reducing end, the sixth and second subsites have a high affinity, the third has very low or even lack of affinity and the first (catalytic subsite) has a large negative affinity. The affinity declines from moderate to low for subsites 1 through 4 toward the reducing end. AMY1 has clearly a more negative affinity at the catalytic subsite, but larger affinities at both the fourth subsites, compared to AMY2. AMY2-1 has lower affinity than AMY2-2 at subsites adjacent to the catalytic site, and otherwise mostly higher affinities than AMY2-2. Theoretical kcat/Km values show excellent agreement with experimental values.

The determination of subsite binding energies of porcine pancreatic α-amylase by comparing hydrolytic activity towards substrates

The active centre of porcine pancreatic alpha-amylase contains five subsites. Their occupancy has been studied using as a substrate maltooligosaccharide of various chain lengths (maltose up to maltoheptaose), some of their p- and o-nitrophenylated derivatives, and 412-residue amylose. Quantitative analysis of the digestion products allowed the determination of the subsite occupancy for the various productive complexes, the bond cleavage frequency and respective kcati (where i is the binding mode). The catalytic efficiency (kcat/Km) increases with chain length from maltose (2 M-1 X S-1) up to amylose (1.06 X 10(7) M-1 X S-1). The kinetic parameters of p-nitrophenylmaltoside hydrolysis are quite close to those of maltose, and the ortho compound behaves as maltotriose. Determination of binding energy of glucose residue at the various subsites calculated according to the method of Hiromi et al. (Hiromi, K., Nitta, Y., Numata, C. and Ono, S. (1973) Biochim. Biophys. Acta 302, 362-375) did not give consistent results. A method is proposed based on certain properties of porcine pancreatic alpha-amylase, especially the non-interaction of the p-nitrophenyl moiety of the maltose derivative with subsites 1 and 2, and the o-nitrophenyl group which interacts in a similar way to a glucose residue at the reducing end, and on the grounds that the amylase-amylose complexes are of the productive type. In addition, binding energy differences were calculated from substrates with the same chain length. The subsite energy profile is characterized by a low value at subsite 3 which confirms this subsite as the catalytic one. Another consequence is that the hydrolysis rate constant of productive complexes (kintn) (where n is the number of glucose or glucose equivalent residues for a given substrate) varies with chain length which is in conflict with the hypothesis of Hiromi et al.

The human pancreatic α-amylase isoforms: isolation, structural studies and kinetics of inhibition by acarbose

A rapid method is proposed for isolating the two main components of human pancreatic alpha-amylase (HPA I and HPA II). The isoelectric point of HPA I (7.2), the main component, was determined using an isoelectrofocusing method and found to differ from that of HPA II (6. 6). The molecular mass of HPA I (55862+/-5 Da) and that of HPA II (55786+/-5 Da) were determined by performing mass spectrometry and found to be quite similar to that of the protein moiety calculated from the amino acid sequence (55788 Da), which indicates that the human amylase is not glycosylated. The structure of both HPA I and HPA II was further investigated by performing limited proteolysis. Two fragments with an apparent molecular mass of 41 kDa and 14 kDa were obtained by digesting the isoforms with proteinase K and subtilisin, whereas digestion with papain yielded two cleaved fragments with molecular masses of 38 kDa and 17 kDa. Proteinase K and subtilisin susceptible bonds are located in the L8 loop (A domain), while the papain cut which occurs in the presence of the calcium chelator EDTA is in the L3 loop (B domain). The kinetics of the inhibition of HPA I and HPA II by acarbose, a drug used to treat diabetes and obesity, were studied using an amylose substrate. The Lineweaver-Burk primary plots of HPA I and HPA II, which did not differ significantly, indicated that the inhibition was of the mixed non-competitive type. The secondary plots gave parabolic curves. All in all, these data provide evidence that two acarbose molecules bind to HPA. In conclusion, apart from the pI, no significant differences were observed between HPA I and HPA II as regards either their molecular mass and limited proteolysis or their kinetic behavior. As was to be expected in view of the high degree of structural identity previously found to exist between human and porcine pancreatic amylases, the present data show that the inhibitory effects of acarbose on the kinetic behavior of these two amylases are quite comparable. In particular, the process of amylose hydrolysis catalyzed by HPA as well as by PPA in both cases requires two carbohydrate binding sites in addition to the catalytic site.

Solubility, phase transition, kinetic ripening and growth rates of porcine pancreatic α-amylase isoenzymes

Abstract Two polymorphic modifications, A and B, of porcine pancreatic α-amylase were grown between 4 and 30°C. A and B crystals are made up by two isoenzymes so that four crystal varieties (AI, AII, BI, BII) exist. A and B are easily distinguished due to their typical crystal habits but there is no difference between AI and AII or BI and BII respectively at least as concerns their unit cells, crystal habits and solubilities for instance. On the other hand, the growth rates are somewhat different, even if the overall rate determining step is volume diffusion. The transition temperature between A and B polymorphs is 18°C, A being stable above this temperature. A and B can undergo a phase transition by slightly changing the temperature around the transition point. Kinetic ripening experiments show that ripening can be used for growing larger crystals at the expenses of smaller ones.

Subsite profile of the active center of porcine pancreatic α-amylase. Kinetic studies using maltooligosaccharides as substrates

The hydrolysis of several maltooligosaccharides catalysed by porcine pancreatic alpha-amylase was performed in order to determine their kinetic parameters. Maltose behaves as a substrate. Molecular activity (Ko) increases with chain length up to maltopentaose, remaining practically unchanged from maltopentaose to maltoheptaose. Maltose shows the highest Km value while the one for maltotriose is the lowest. Only maltose and maltotriose were directly cleaved to glucose. From Km and Ko values, the binding energy of the total complexes and of the productive ones respectively were calculated. The binding energy at each subsite was determined assuming that each substrate forms a single productive complex and that maltose and maltotriose differ in their binding mode from higher oligosaccharides. The model was checked by calculating theorical Km and Ko. Theorical values agree reasonably well with experimental ones.

Comparison of barley malt α-amylase isozymes 1 and 2: construction of cDNA hybrids by in vivo recombination and their expression in yeast

Germinating barley produces two alpha-amylase isozymes, AMY1 and AMY2, having 80% amino acid (aa) sequence identity and differing with respect to a number of functional properties. Recombinant AMY1 (re-AMY1) and AMY2 (re-AMY2) are produced in yeast, but whereas all re-AMY1 is secreted, re-AMY2 accumulates within the cell and only traces are secreted. Expression of AMY1::AMY2 hybrid cDNAs may provide a means of understanding the difference in secretion efficiency between the two isozymes. Here, the efficient homologous recombination system of the yeast, Saccharomyces cerevisiae, was used to generate hybrids of barley AMY with the N-terminal portion derived from AMY1, including the signal peptide (SP), and the C-terminal portion from AMY2. Hybrid cDNAs were thus generated that encode either the SP alone, or the SP followed by the N-terminal 21, 26, 53, 67 or 90 aa from AMY1 and the complementary C-terminal sequences from AMY2. Larger amounts of re-AMY are secreted by hybrids containing, in addition to the SP, 53 or more aa of AMY1. In contrast, only traces of re-AMY are secreted for hybrids having 26 or fewer aa of AMY1. In this case, re-AMY hybrid accumulates intracellularly. Transformants secreting hybrid enzymes also accumulated some re-AMY within the cell. The AMY1 SP, therefore, does not ensure re-AMY2 secretion and a certain portion of the N-terminal sequence of AMY1 is required for secretion of a re-AMY1::AMY2 hybrid.

Subsite mapping of porcine pancreatic alpha-amylase I and II using 4-nitrophenyl-α-maltooligosaccharides

The catalytic efficiency (kcat/Km) and the cleaved bond distribution for the nitrophenylated maltooligosaccharides, p-NPGlcn (2 < or = n < or = 7) hydrolysed by porcine pancreatic alpha-amylase isozymes I and II were determined. The subsite affinities (Ai) were calculated from the p-NPGlcn (4 < or = n < or = 7) hydrolysis data. Five subsites (-3 to 2) bind glucosidic residues with a positive affinity. No additional subsites could be detected both at the reducing end (3, 4, 5) and at the nonreducing end (-4, -5, -6). The energetic profiles of both isozymes are similar. The energetic profile of PPA differs from other alpha-amylases by having both a small number of subsites, and a catalytic subsite with a high positive affinity. Excellent agreement was found between observed catalytic efficiency values and those calculated from the subsite affinities.

Activation of cyclic AMP-dependent protein kinases in human gut adenocarcinoma (HT 29) cells in culture.

Vasoactive intestinal peptide, secretin, catecholamines and prostaglandin E1 stimulate the accumulation of cyclic AMP in HT 29 cells (see Laburthe, M. et al. (1978) Proc. Natl. Acad. Sci. U.S. 75, 2772-2775). In the present work maximal activation of protein kinases has been obtained at similar or even lower concentrations of the effectors. Maximal stimulation also requires a phosphodiesterase inhibitor. Type I and type II cyclic AMP-dependent protein kinases from basal and stimulated cells have been characterized by DEAE-Sepharose chromatography. Further identidication of the kinase has been carried out by gel electrophoresis and assay of the enzymes in the gel slabs. Comparison of the radioautography patterns of high speed supernatant lysate from basal and stimulated cells shows: First, that one type I and two type II cyclic AMP-dependent protein kinases plus one or two major and two minor cyclic AMP-independent protein kinases are present in HT 29 cells. Second, that all three holoenzymes are fully dissociated upon maximal stimulation, while the activity of the independent kinases appears unchanged.

Parallel activation of cyclic AMP phosphodiesterase and cyclic AMP-dependent protein kinase in two human gut adenocarcinoma cells (HT 29 and HRT 18) in culture, by vasoactive intestinal peptide (VIP) and other effectors activating the cyclic AMP system

Vasoactive intestinal peptide (VIP), secretin, catecholamines and prostaglandin E1 (PGE1) in the presence of a cyclic nucleotide phosphodiesterase inhibitor stimulate the accumulation of cyclic AMP in two colorectal carcinoma cell lines (HT 29 and HRT 18) with subsequent activation of the cyclic AMP-dependent protein kinases. In HT 29 cells incubated without phosphodiesterase inhibitor, 10(-9) M VIP promotes a rapid and specific activation of the lower Km cyclic AMP phosphodiesterase (1.7-fold); at 25 degrees C the effect is maintained for more than 15 min, while at 37 degrees C the activity returns to basal value within 15 min. As shown by dose-response studies, VIP is by far the most effective inducer (Ka equals 4 x 10(-10) M) of the cyclic AMP phosphodiesterase activity; partial activation of the enzyme is obtained by 3 x 10(-7) M secretin, 10(-5) M isoproterenol and 10(-5) M PGE1; PGE2 and epinephrine are without effect. In HRT 18 cells VIP is less active (Ka equals 2 x 10(-9) M) whereas 10(-6) M PGE1, 10(-6) M PGE2 and 10(-5) M epinephrine are potent inducers of th phosphodiesterase activity. The positive cell response to dibutyryl-cyclic AMP further indicates that cyclic AMP is a mediator in the phosphodiesterase activation process. The incubation kinetics and dose response effects of the various agonists on the cyclic AMP-dependent protein kinase activity determined for both cell types in the same conditions show a striking similarity to those of phosphodiesterase. Thus coordinate regulation of both enzymes by cyclic AMP was observed in all incubation conditions.