Studies on starch-degrading enzymes Part XIV. The multiple forms of porcine, pancreatic alpha-amylase

Crystalline, porcine, pancreatic alpha-amylase has been fractionated into four distinct fractions by ion-exchange chromatography on DEAE-cellulose. Each fraction hydrolyses amylose in a manner identical to that of the parent enzyme, i.e., at optimal pH, the reaction pattern corresponds to multiple attack, whereas in the presence of glycerol, or at high pH, it changes to multichain attack. Ultracentrifugation and gel exclusion-chromatography showed that the molecular weights of the fractions are similar to one another and to the parent enzyme, suggesting that the fractions are isoenzymes. However, determination of the amino-acid content of the multiple forms failed to reveal any reason for their different migratory rates through DEAE-cellulose. It is suggested that the multiple forms are artefacts, arising during the isolation of the enzyme.     

Structural biology of starch-degrading enzymes and their regulation

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Identification and genetic control of starch-degrading enzymes in maize endosperm

Three starch-degrading enzymes from liquid endosperm of maize have been separated by means of horizontal acrylamide gel electrophoresis. The three enzymes are tentatively identified as -amylase (zone 1), -amylase (zone 2), and -glucan phosphorylase (zone 3). Electrophoretic variants of these enzymes were found among ten inbred strains examined. Results of genetic crosses with respect to zone 2 amylase show that it is controlled by a pair of alleles (Amy-2 ^A and Amy-2 ^B) acting without dominance. It further appears that Amy-2 and Ct (catalase) are linked with 5% recombination frequency.This work was supported by the U.S. Atomic Energy Commission, under contract No. AT(11-1)-1338.     

Chloroplast and extrachloroplastic starch-degrading enzymes in Pisum sativum L.

The organelles of soybean (Glycine max (L.) Merr.) protoplasts were separated using a recently developed procedure which allows rapid (3-h) recovery of a fraction enriched for coated vesicles (CVs). As determined by marker-enzyme enrichment and ultrastructural analysis of isolated membrane fractions, endoplasmic reticulum, Golgi membranes, glucan-synthase-II (EC 2.4.1.34)-containing membranes (putative plasma membrane), mitochondria, and CVs were enriched in separate fractions in a sucrose density gradient. Glucan synthase I (EC 2.4.1.12) had the highest specific activity in the Golgi-enriched and CV-enriched fractions and was found to comigrate with CVs upon rate-zonal centrifugation of a CV-enriched fraction. For further elucidation of the role of these latter organelles in cell-wall regeneration, freshly isolated protoplasts were pulsed with [³H]glucose for 20 min, and the disappearance of label from the organelles was followed for the ensuing 1 h. Although a CV-enriched fraction contained glucan synthase I, it contained very small amounts of labelled polysaccharide during the period of study. Pulse-chase experiments with [³H]glucose helped to confirm the role of the Golgi apparatus in secretion of matrix polysaccharides by protoplasts.Abbreviations CV(s) coated vesicle(s) - Da dalton - ER endoplasmic reticulum - GSI,II glucan synthase I and II, respecitively Two whom correspondence should be directed. Address after February 1986:Department of Biology, Texas A&M University. College Station, TX 77843-3258, USA     

Studies on bovine serum amylase; evidence for two loci

Two genetically controlled polymorphic systems of amylase enzymes are described in sera from Friesian, Jersey, Guernsey, Hereford, Ayrshire and Charolais cattle. One of these Ami has been described previously and is controlled by two codominant alleles in British cattle, AmI ^B and AmI ^C. Evidence presented here suggests that it could be a gamma.amylase. The other enzyme which is described here for the first time has many of the properties of an alpha amylase. It is also polymorphic and controlled by two codominant alleles AmII ^A and AmII ^B. AmII ^A is only present in low frequency. The inheritance of both enzyme systems has been studied in 419 families.     

Studies on the interaction between tetraphenylporphyrin compounds and bovine serum albumin.

The interaction of three porphyrin compounds with bovine serum albumin (BSA) was examined by fluorescence emission spectra at the excitation wavelength 280 nm and in UV-Vis absorption spectra. Through fluorescence quenching experiments, it was confirmed that the combination of three porphyrin compounds with BSA was a single static quenching process. The binding constant K(A), the thermodynamic parameters enthalpy change (DeltaH(0)), Gibbs free energy change (DeltaG(0)) and entropy change (DeltaS(0)) were obtained. It was found that hydrophobic interaction played a main role in tetraphenylporphyrin (TPP) or tetraparacholophenylporphyrin (TClPP) binding to BSA, while tetraparamethoxyphenylporphyrin (TMEOPP) mainly based on van der Waals' force. According to F?ster energy transfer, the separate distance r, the energy transfer efficiency E and F?ster radium R(0) were calculated. The results obtained from the above experiments showed that three porphyrin compounds were tightly bound to BSA.     

Studies on the transverse localization of lysophospholipase in bovine liver microsomes using proteolytic enzymes.

1. Sonication of bovine liver microsomes completely solubilized the membrane-bound lysophospholipase II (EC 3.1.1.5). Co-chromatography with purified 125I-labelled lysophospholipase indicated that the enzyme was solubilized from microsomes in a lipid-free state. 2. In the presence of residual microsomal membranes, the solubilized lysophospholipase could only be partly degraded by trypsin (EC 3.4.21.4). Therefore, trypsin could not be used to study the transmembrane disposition of lysophospholipase in intact microsomes. 3. Chymotrypsin (EC 3.4.21.1) destroyed the solubilized lysophospholipase activity, even in the presence of residual microsomal membranes. 4. Lysophospholipase in intact microsomal vesicles was resistant to chymotrypsin digestion. 5. When microsomal vesicles were made leaky with lysophosphatidylcholine, chymotrypsin destroyed more than 95% of the lysophospholipase activity. 6. It is concluded from these experiments that at least the active center of lysophospholipase is located at the luminal side of the bovine liver microsomal membrane.     

Studies on the interaction between benzophenone and bovine serum albumin by spectroscopic methods

The interaction between benzophenone (BP) and bovine serum albumin (BSA) was investigated by the methods of fluorescence spectroscopy combined with UV–Vis absorption and circular dichroism (CD) measurements under simulative physiological conditions. The experiment results showed that the fluorescence quenching of BSA by BP was resulted from the formation of a BP–BSA complex and the corresponding association constants (K _a) between BP and BSA at four different temperatures had been determined using the modified Stern–Volmer equation. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be –43.73 kJ mol⁻¹ and −53.05 J mol⁻¹ K⁻¹, respectively, which suggested that hydrogen bond and van der Waals force played major roles in stabilizing the BP–BSA complex. Site marker competitive experiments indicated that the binding of BP to BSA primarily took place in site I (sub-domain IIA). The conformational investigation showed that the presence of BP decreased the α-helical content of BSA and induced the slight unfolding of the polypeptides of protein, which confirmed some micro-environmental and conformational changes of BSA molecules.     

Studies on the interaction between Oxaprozin-E and bovine serum albumin by spectroscopic methods

The interaction between Oxaprozin-E and bovine serum albumin (BSA) was studied by spectroscopic methods including fluorescence and UV–vis absorption spectroscopy. The quenching mechanism of fluorescence of BSA by Oxaprozin-E was discussed to be a dynamic quenching procedure. The number of binding sites n and apparent binding constant K was measured by fluorescence quenching method. The thermodynamics parameter ΔH, ΔG, ΔS were calculated. The results indicate the binding reaction was mainly entropy-driven and hydrophobic forces played major role in the binding reaction. The distance r between donor (BSA) and acceptor (Oxaprozin-E) was obtained according to Förster theory of non-radioactive energy transfer.     

Studies on the interaction between benzidine and bovine serum albumin by spectroscopic methods

The interaction between bovine serum albumin (BSA) and benzidine (BD) in aqueous solution was investigated by fluorescence spectroscopy, circular dichroism (CD) spectra and UV–Vis spectroscopy, as well as resonance light scattering spectroscopy (RLS). It was proved from fluorescence spectra that the fluorescence quenching of BSA by BD was a result of the formation of BD–BSA complex, and the binding constants (K _a) were determined according to the modified Stern–Volmer equation. The enthalpy change (ΔH) and entropy change (ΔS) were calculated to be ?34.11 kJ mol^?1 and ?25.89 J mol^?1 K^?1, respectively, which implied that van der Waals force and hydrogen bond played predominant roles in the binding process. The addition of increasing BD to BSA solution caused the gradual enhancement in RLS intensity, exhibiting the forming of the aggregate. Moreover, the competitive experiments of site markers suggested that the binding site of BD to BSA was located in the region of subdomain IIA (sudlow site I). The distance (r) between the donor (BSA) and the acceptor (BD) was 4.44 nm based on the Förster theory of non–radioactive energy transfer. The results of synchronous fluorescence and CD spectra demonstrated the microenvironment and the secondary conformation of BSA were changed.     

Studies on the biological activity of an insulin-stimulating peptide from a tryptic digest of bovine serum albumin

Summary A two-chain polypeptide, which corresponds to amino acid residues 115–143 and 144–184(185) of bovine serum albumin, connected to each other by a disulfide bridge, potentiated the effects of insulin on glucose transport and glucose metabolism in isolated rat adipocytes. Although the peptide alone had little activity, it shifted the concentration-response curves of insulin-stimulated D-[I-¹⁴C]glucose oxidation, 2-deoxyglucose transport, and lipid synthesis from D-[U-¹⁴C]glucose to lower insulin concentrations. It also increased the maximal responses of these parameters to insulin. However, it did not affect insulin binding to adipocytes. The peptide protected insulin considerably from degradation, but this effect alone cannot account for its effect in increasing the maximal responses to the hormone, and even when degradation of a submaximal concentration of insulin was suppressed by bacitracin, the peptide still had an enhancing effect. These results suggest not only that the peptide influences a step distal to receptor-mediated insulin binding but also that inhibition of insulin degradation alone cannot explain its total effect.The peptide lost its insulin-stimulating activity completely when it was further digested with V8 or lysinespecific endopeptidase, or when it was reduced and then carboxamidomethylated or oxidized with performic acid. Similar active tryptic fragments were obtained from human and rat albumins.Insulin-stimulating peptides should be useful in studies on the mechanisms of insulin action including both the sensitivities and responsiveness of target cells to the hormone.Abbreviations ISP insulin-stimulating peptide - HEPES N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid - HPLC high-performance liquid chromatography - SDS sodium dodecyl sulfate     

Characterization of the starch-degrading enzyme ofCorynebacterium diphtheriae typegravis

An inducible cell-surface-bound starch-degrading enzyme produced byCorynebacterium diphtheriae typegravis in tryptose-sodium chloride-maltose broth was partially purified by diethylaminoethylcellulose (DEAE) column chromatography. The partially purified enzyme had approximately six times the specific activity of the crude enzyme. Forty-five percent of the enzyme was recovered in the partially purified form after DEAE chromatography. A molecular weight for the enzyme of approximately 61 200 was obtained by glycerol density gradient centrifugation. The enzyme was inhibited by mercuric ions,p-chloromercuribenzoate, and iodoacetic acid. It was inhibited slightly by EDTA and 1,10-phenanthroline. The enzyme was not inhibited by mercaptoethanol, dithiothreitol, fluoride, bromide, or chloride ions. Activity was lost after precipitation with ethanol, and after concentration with Lyphogel, Sephadex G-15, or lyophilization, but no activity was lost after precipitation with ammonium sulfate. Calcium ions did not activate the enzyme. Maltose was the best inducer of the enzyme. All the saccharides tested induced the enzyme slightly. No phosphorylase was found associated with the organism. However, a phosphatase and possibly an α-glucosidase were produced by the organism. The properties of the starch-degrading enzyme(s) ofCorynebacterium diphtheriae typegravis resemble those of either an amylase, an α-glucosidase, or both. This investigation was supported by Public Health Service grant A102924-10 from the National Institute of Allergies and Infectious Diseases.     

Studies on the thermal inactivation of immobilized enzymes

The thermal inactivation of a great number of immobilized enzymes shows a biphasic kinetics, which distinctly differs from the first-order inactivation kinetics of the corresponding soluble enzymes. As shown for alpha-amylase, chymotrypsin, and trypsin covalently bound to silica, polystyrene, or polyacrylamide, the dependence of the remaining activities on the heating time can be well described by the sum of two exponential terms. To interpret this mathematical model function, the catalytic properties of immobilized enzymes (number of active sites in silica-bound trypsin, K(M) and E(a) values in silica-bound alpha-amylase and chymotrypsin) at different stages of inactivation and the influence of various factors (coupling conditions, addition of denaturants or stabilizers, etc.) on the thermal inactivation of silica-bound alpha-amylase were studied. Furthermore, conformational alterations in the thermal denaturation of spin-labeled soluble and silica-bound beta-amylase were compared by electron spin resonance (ESR) studies. The results suggest that the biphasic inactivation kinetics reflects two different pathways according to which catalytically identical enzyme molecules are predominantly inactivated.