Resolution of myocardial phospholipase C into several forms with distinct properties.

Phospholipase C activity capable of hydrolysing phosphatidylinositol in bovine heart was resolved into four forms (I-IV) by ion-exchange chromatography. Some of these forms could only be detected if the assay was performed at acidic pH (I and IV) or in the presence of deoxycholate (II). Gel-filtration chromatography indicated that the four forms had different molecular weights in the range 40000-120000. I, II and III all had pH optima in the range 4.5-5.5. However, the major form (III) also had substantial activity at pH 7.0 and above. The activities of I, II and III at pH 7.0 were stimulated by deoxycholate; this effect was most marked with I and II, which had very low activity at this pH. All forms of the enzyme were inhibited by EGTA and required 2-5 mM-CaCl2 for maximal activity. When the fractions eluted from the ion-exchange and gel-filtration columns were assayed with polyphosphoinositides as substrates there was a close correspondence to the elution profile obtained with phosphatidylinositol as substrate; there was no evidence for the existence in heart of phospholipase C activities specific for individual phosphoinositides.     

Multiple forms of myeloperoxidase from human neutrophilic granulocytes: evidence for differences in compartmentalization, enzymatic activity, and subunit structure

Multiple forms of myeloperoxidase from normal human neutrophilic granulocytes obtained from a single donor can be resolved by carboxymethyl (CM)-cellulose ion-exchange column chromatography into three forms (I, II, and III) designated in order of elution of adsorbed enzyme using a linear salt gradient. Selective solubilization of individual forms of the enzyme by detergent (form I) or high-ionic-strength procedures (forms II and III) suggested that these forms of the enzyme were compartmentalized differently. All three forms were purified by a combination of preferential extraction, manipulation of ionic strength, and ion-exchange and molecular sieve chromatography. Purified forms II and III had similar specific activities for a variety of substrates. Form I was less active toward several of these same substrates, most notably iodide, with a specific activity about one-half that of forms II and III. All forms had similar spectral properties characteristic of a type alpha heme. The amino acid compositions of the three forms were similar, yet significant differences were found in selected residues such as the charged amino acids. Native polyacrylamide gel electrophoresis resolved small differences in mobility between the forms which were consistent with the charge heterogeneity observed on CM-cellulose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis data were consistent with the generally accepted subunit structure of two heavy chains and two light chains. All three forms contained a small-molecular-weight subunit of Mr 11,500. Form I contained a large subunit of Mr 63,000, while forms II and III contained a corresponding subunit of Mr approximately 57,500. We conclude that heterogeneity of human myeloperoxidase is accompanied by differences in cellular compartmentalization, enzymatic activity, and subunit structure.     

Purification and characterization of a phosphoinositide-specific phospholipase C from guinea pig uterus. Phosphorylation by protein kinase C in vivo

Two peaks of phosphoinositide-specific phospholipase C (PI-PLC) activity were resolved when guinea pig uterus cytosolic proteins were chromatographed on a DEAE-Sepharose column. The first peak of enzyme activity eluting from the DEAE-Sepharose column (PI-PLC I) was further purified to homogeneity, whereas the second peak of enzyme activity was enriched 300-fold. PI-PLC I migrated as a 62-kDa protein on sodium dodecyl sulfate-polyacrylamide gels. Antibodies prepared against PI-PLC I failed to react with PI-PLC II. PI-PLC I hydrolyzed all three phosphoinositides, exhibiting a greater Vmax for phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. Hydrolysis of phosphatidylinositol was calcium-dependent, whereas significant hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate occurred in the presence of 2.5 mM EGTA. At physiological concentrations of calcium, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were the preferred substrates. Antibodies specific for PI-PLC I reacted with a 62-kDa protein in both the cytosol and membrane fractions from guinea pig uterus. Quantitation of the immunoblots revealed that 25% of the 62-kDa protein was membrane-associated, whereas only 5% of the total enzyme activity was membrane-associated. Approximately 20% of the membrane-bound phospholipase C activity and immunoreactive material were loosely bound, whereas the remainder required detergent extraction for complete solubilization. The 62-kDa protein associated with the membrane fractions did not bind lectin affinity columns, suggesting that it was not glycosylated. PI-PLC I was identified as a phosphoprotein in [32P]orthophosphate-labeled rat basophilic leukemia (RBL-1) cells by two-dimensional gel electrophoresis followed by immunoblotting. In untreated cells, 32P-labeled PI-PLC I was found in the cytosolic fraction. Treatment of RBL-1 cells with those phorbol esters which are known to activate the Ca2+/phospholipid-dependent enzyme protein kinase C, resulted in a time-dependent increase in the phosphorylation of both membrane-bound and cytosolic PI-PLC I. Thus, in RBL-1 cells, protein kinase C may play an important role in the regulation of phospholipase C through protein phosphorylation.     

Phosphorylation of aortic plasma membranes by protein kinase C

A calcium-sensitive, phospholipid-dependent protein kinase (protein kinase C) and its three isozymes were purified from rat heart cytosolic fractions utilizing a rapid purification method. The purified protein kinase C enzyme showed a single polypeptide band of 80 KDa on SDS-polyacrylamide gel electrophoresis, and was totally dependent on the presence of Ca²⁺ and phospholipid for activity. Diacylglycerol was also found to stimulate enzymatic activity. Autophosphorylation of the purified PKC showed an 80 KDa polypeptide. The identity of the purified protein was also verified with monoclonal antibodies specific for PKC. Further fractionation of the purified PKC on a hydroxylapatite column yielded three distinct peaks of enzyme activity, corresponding to type I, II and III based on similar chromatographic behaviour as the rat brain enzyme. All three forms were entirely Ca^2– and phosphatidylserine dependent. Type II was found to be the most abundant. Type I was found to be highly unstable. PKC activity studies demonstrate that types II and III isozymic forms are different with respect to their sensitivity to Ca²⁺.Abbreviations PKC Protein Kinase C - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - K_m Michaelis constant - NBT Nitro-Blue Tetrazolium - BCIP 5-Bromo-4-Chloro-3-Indolyl Phosphate     

Secretion of myeloperoxidase isoforms by human neutrophils.

The human neutrophil lysosomal enzyme, myeloperoxidase (MPO), exists in three major and chromatographically distinct forms, MPO I, MPO II, and MPO III. We used cation-exchange medium-pressure liquid chromatography and kinetic microenzyme assay (or spectrophotometric monitoring) to analyze the secretion of MPO isoforms by neutrophils exposed to N-formylmethionylleucylphenylalanine (FMLP), digitonin, the ionophore A23187, and serum-opsonized zymosan A (SOZ). All three MPO isomers were released into the fluid phase after neutrophils were exposed to these secretagogues. A significant proportional increase in MPO I was released when neutrophils were stimulated with SOZ. MPO I was released in higher proportions than found in the whole cell constituency when neutrophils were stimulated with FMLP + cytochalasin B, A23187, and digitonin, but this was not statistically significant.     

Three distinct forms of rat brain protein kinase C: differential response to unsaturated fatty acids

Although the three distinct forms of protein kinase C isolated from rat brain soluble fraction are structurally very similar, they respond differently to free unsaturated fatty acids such as arachidonic acid to exhibit their catalytic activity. Type I enzyme encoded by gamma-sequence, as predicted by cDNA clone analysis, responds to these fatty acids only slightly, whereas Type III enzyme determined by alpha-sequence is activated by free unsaturated fatty acids in the presence of Ca2+ in a comparable manner to phosphatidylserine plus diacylglycerol. Type II, a mixture of two enzymes encoded by beta I- and beta II-sequence, resulting from alternative splicing, shows properties in between those of Type I and Type III. Some of these forms of protein kinase C may function at a relatively later phase of cellular responses when large amounts of unsaturated fatty acids and Ca2+ are mobilized.     

Heterogeneity of Rice Glutelin

Three forms of endopolygalacturonase from Saccharomyces fragilis (Kluyveromyces fragilis) were separated by a procedure including adsorption on Amberlite IRC-50, CM Sephadex C-50 column chromatography and repeated preparative disc electrophoresis. Each endo-PG was almost homogenoeus as judged by polyacrylamide gel electrofocusing and disc electrophoresis. The three enzyme were designated as enzymes I, II and III. Enzymes I and II were similar but enzyme HI different from I and II in isoelectric point. The three enzymes resembled one another in eznyme action on pectic acid and other properties. All the three enzymes showed macerating activity toward the potato and carrot tissues.     

Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points

Purified rat-liver dihydropteridine reductase is homogeneous by gel filtration (Mr approximately 51,000), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 25,500), and native polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. However, analysis by isoelectric focusing has revealed three enzyme forms with approximate isoelectric points of 6.5, 5.9, and 5.7 (designated forms, I, II, and III, respectively). The three forms, isolated in 65% yield by preparative chromatofocusing, are stable in 0.05 M phosphate buffer, pH 6.8, containing 1 mM beta-mercaptoethanol and exhibit similar kinetic constants when the catalytic activities of the isolated forms are compared with quinonoid dihydrobiopterin as substrate. All forms generate complexes with the enzymatic cofactor NADH which are also detectable by IEF. When examined further by IEF under denaturing conditions in 6 M urea the enzyme demonstrates a differing subunit composition for its three forms. Two distinct subunits, designated alpha and beta, can be identified, and additional evidence suggests that the native enzyme forms I, II, and III represent the three differing dimeric combinations alpha alpha (form I), alpha beta (form II), and beta beta (form III).     

Purification and characterization of the multiple forms of 3 alpha-hydroxysteroid dehydrogenase in rat liver cytosol

The presence of multiple forms of 3 alpha-hydroxysteroid dehydrogenase in the cytosol of male rat livers was demonstrated. The enzyme activity was separated into two fractions (F3 and F4) by DEAE-cellulose chromatography, and further fractionation of F3 into four (I-IV) and F4 into three (I-III) fractions was achieved by subsequent CM-Sephadex chromatography. Six forms (F3-II-IV and F4-I-III) were further purified by chromatofocusing and Red-Sepharose 4B chromatography. Two (F4-II and III) of the isolated enzymes were homogeneous, based on polyacrylamide gel electrophoresis. No shift of pI values was observed, when isoelectric focusing was performed with the F4 enzyme species in the presence of NAD(P)+ or NAD(P)H. All six enzyme species migrated closely with each other on dodecyl sulfate-polyacrylamide gel electrophoresis, from which the molecular masses were estimated to be 32 500 Da. Gel filtration gave similar values for the F4 enzyme species, indicating that each enzyme is a monomeric peptide. All enzyme species were able to catalyse the dehydrogenation of 3 alpha-hydroxysteroids (C19 to C26), and not C27 compound having a 1,5-dimethylhexyl side chain. The catalytic properties with steroids were very similar for five of the enzyme species, but F3-IV especially preferred androsterone. When male rat livers were used for isolation, the enzyme activity ratio of F3 to F4 for androsterone was about 1 to 8, whereas the ratio was about 1 to 20 for female rat livers. Considering the biosynthetic pathway of bile acids, the enzymes isolated here might play a specific role in the conversion of a 3 beta-hydroxy group to a 3 alpha-hydroxy group via a 3-oxo group of an intermediate in the synthesis of bile acids.     

Three forms of DNA polymerase from Drosophila melanogaster embryos. Purification and properties.

DNA polymerase was purified from Drosophila melanogaster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, II, and III, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)-16 and poly(rA) . (dT)-16. At the final step of purification, enzymes I, II, and III were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)-16. The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and II eluted as distinct peaks and enzyme III eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5-7.3 S, enzyme II sedimented at 7.3-8.3 S, and enzyme III sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme III, which was inactive with poly(rA) . (dT)-16. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)-16 activity from poly(rA) . (dT)-16 activity. The DNA polymerase preferred poly(dA) . (dT)-16 (with Mg2+) as a primer-template, although it was also active with poly(rA) . (dT)-16 (with Mn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)-16 was inhibited by K+ and activity with poly(rA) . (dT)-16 was stimulated by K+ and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophila embryos have physical properties similar to those of DNA polymerase-alpha and enzymatic properties similar to those of all three vertebrate DNA polymerases.     

Propionyl-CoA condensing enzyme from Ascaris muscle mitochondria. II. Coenzyme A modulation.

The propionyl-CoA condensing enzyme which catalyzes the first step in the biosynthesis of 2-methylbutyrate and 2-methylvalerate by Ascaris muscle appears to exist in at least three forms in the mitochondria of this parasitic nematode. Two forms, A and B, were separated by ion exchange chromatography on CM-Sephadex. Chromatography on phosphocellulose resulted in the recovery of one minor peak (I) and two major peaks with activity (II and III). A and B as well as I, II, and III differed in their specific activities. Forms B and III were the most retained by their resins, and were the most active forms of the enzyme in each case. Inhibition studies with metabolites from Ascaris mitochondria indicate that CoASH, a product of the condensation reaction, and acetyl-CoA are effective inhibitors of the condensing and thiolytic activities of the Ascaris enzyme, respectively. Incubation of the active enzyme form B for 2 h with 0.1 mM CoASH at room temperature under nitrogen caused the loss of 92% of the propionyl-CoA condensing activity and 67% of the thiolase activity when assayed in standard mixtures. The propionyl-CoA condensing enzyme exhibited a hyperbolic dependence of the condensation velocity to changes in substrate concentration. However, in the presence of CoASH the Michaelis-Menten kinetics was transformed into a sigmoidal kinetics indicating a deviation from a simple product inhibition. Inactivation of the most active forms of the enzyme with CoASH was accompanied by (a) a change in the chemical reactivity of the protein toward p-chloromurcuribenzoate, (b) a change in the uv-visible spectrum of the protein, and (c) a change in the elution patterns from both CM-Sephadex and phosphocellulose column chromatography, where-upon one, two, or more protein peaks were obtained. The several protein peaks resolved by rechromatography of the [14C]CoASH-inactivated enzyme III on phosphocellulose had different CoASH contents. The elution positions were correlated with the less active forms (I and II) having increased [14C]CoASH activities. Similarly, the two peaks isolated upon rechromatography of the CoASH-partially inactivated enzyme B on CM-Sephadex had different isotope contents and the elution position of enzyme A corresponded to the less active form. The results described indicate that the CoASH modification of Ascaris propionyl-CoA condensing enzyme may be responsible for the existence of several forms of the enzyme and might represent a mode of control by chemically modulating the amount of the active forms of the enzyme.     

Partial purification of phosphoinositide phospholipase C from human platelet cytosol; characterization of its three forms

Three forms (I, IIa and IIb) of phospholipase C, hydrolyzing specifically inositol phospholipids, were resolved from human platelet cytosol and partially purified by DEAE-cellulose, phenyl-Sepharose, Ultrogel ACA-44 and hydroxylapatite column chromatographies. All three forms exhibited pH optimum at 6.5 - 7.0 in the presence of deoxycholate and their molecular weights were 67,000 (form I), 120,000 (IIa) and 70,000 (IIb). These enzymes hydrolyzed both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate in Ca2+-dependent manner; their maximal activities for phosphatidylinositol hydrolysis were obtained at 10(-4) to 10(-3) M Ca2+, whereas phosphatidylinositol 4,5-bisphosphate was preferentially hydrolyzed at lower concentration of Ca2+.     

Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells.

Corticosteroid-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 X g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I leads to II leads to III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cell subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.     

Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine spermatozoa

1. The distribution of phosphatidylinositol3, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis or phosphatidylinositol-specific phospholipase C (PI-PLC), activity in the bull reproductive system showed the highest specific activity in the isolated spermatozoa (SZ) followed by testis and different epididymal segments. Both the head and tail fractions of SZ were active. 2. The optimal solubilization of the enzyme from SZ was obtained with 0.2% Triton X-100 or at 0.05% detergent concentration when combined with a 60 sec sonication. The sucrose gradient centrifugation showed that PI-PLC was enriched in membrane fraction distinct from mitochondria and acrosomes. 3. The enzyme was purified by ammonium sulphate precipitation and fractionations by hydrophobic interaction chromatography, gel filtration, Con A-Sepharose affinity and chromatofocusing columns. The purified enzyme was able to hydrolyse all phosphatidylinositol substrates with optimum at pH 7.0 and activation by Ca2+, Cd2+ and Mn2+ but not phospholipids lacking the inositol residue. 4. In PAGE (8-25% gradient) the purified (aggregated) enzyme did not enter the gel. In SDS-PAGE two closely located bands were found with Mr-values of 15,000 and 18,000. Isoelectric focusing showed a wide band at pl 4.5-5.1. 5. Gel filtration resulted in a broad elution peak indicating multiple molecular forms (aggregates); the basic form had an apparent molecular weight of 100,000. The binding of the enzyme to Con A-Sepharose indicated that the enzyme is a glycoprotein.     

Specific membrane binding of the carboxypeptidase Y inhibitor I(C), a phosphatidylethanolamine-binding protein family member

I(C), an endogenous cytoplasmic inhibitor of vacuolar carboxypeptidase Y in the yeast Saccharomyces cerevisiae, is classified as a member of the phosphatidylethanolamine-binding protein family. The binding of I(C) to phospholipid membranes was first analyzed using a liposome-binding assay and by surface plasmon resonance measurements, which revealed that the affinity of this inhibitor was not for phosphatidylethanolamine but for anionic phospholipids, such as phosphatidylserine, phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate, with K(D) values below 100 nm. The liposome-binding assay and surface plasmon resonance analyses of I(C), when complexed with carboxypeptidase Y, and the mutant forms of I(C) further suggest that the N-terminal segment (Met1-His18) in its carboxypeptidase Y-binding sites is involved in the specific and efficient binding to anionic phospholipid membranes. The binding of I(C) to cellular membranes was subsequently analyzed by fluorescence microscopy of yeast cells producing the green fluorescent protein-tagged I(C), suggesting that I(C) is specifically targeted to vacuolar membranes rather than cytoplasmic membranes, during the stationary growth phase. The present findings provide novel insights into the membrane-targeting and biological functions of I(C) and phosphatidylethanolamine-binding proteins.     

Partial purification and kinetic properties of three different d-glucosamine 6-P:N-acetyltransferase forms from human placenta

Three distinct forms of -glucosamine 6-P (Gm 6-P):N-acetyltransferases (EC 2.3.1.4) were partially purified from human placental homogenates by carboxy methyl-Sephadex chromatography. Purification of forms I and II were 13.5-fold, while that of form III was 114-fold. All three forms had a pH optimum value of 9.7 in glycine–NaOH buffer. Enzymes II and III had a K_m value for Gm 6-P of 3.0 mM, which was less than half of that observed for form I (7.1 mM). The corresponding K_m values for acetyl CoA were 0.157 (form I), 0.187 (form II) and 0.280 mM (form III), respectively. Activities of all three forms were inhibited at high concentrations of either substrate. These enzymes were inhibited from 82 to 92% by 2.5 mM p-chloromercuribenzoate. The inhibition was largely reversible by inclusion of 2.5 mM dithiothreitol in the incubation mixtures. There was no requirement for divalent cations, as demonstrated by lack of inhibition of enzyme activity by ethylene diamine tetraacetate. The results are discussed in terms of differences among the enzyme properties of human placental, rodent and porcine liver forms.     

Interconversion of multiple forms of tyrosine aminotransferase in vitro and in vivo in cultured hepatoma cells

Corticosteroi-induced tyrosine aminotransferase (EC 2.6.1.5) from cultured hepatoma cells was separated by carboxymethyl-Sephadex chromatography into three molecular forms resembling those described previously in the rat liver. Enzyme forms were isolated and used as purified substrates to examine their in vitro interconversion by various subcellular fractions. Isolated form III was converted to forms II and I, and isolated form II was converted to form I by the coarse particulate fraction sedimenting at 1000 × g. This activity was inhibited by the serine enzyme inhibitor phenylmethane sulfonyl fluoride or by raising the pH to 8.7. Conversion of enzyme forms in vitro in the opposite direction (I → II → III) could not be detected. The distribution of enzyme forms in vivo was examined by the use of experimental conditions that prevent their in vitro interconversion during cell extraction. Tyrosine aminotransferase extracted from cells subjected to various treatments that affect the rates of enzyme synthesis or degradation existed always predominantly as form III. It appears, therefore, that multiple forms of tyrosine aminotransferase are not related to the turnover of this enzyme in vivo.     

Endogenous factor in rat liver that converts tyrosine aminotransferase form III to form I: a rapid assay of this converting factor.

After induction by cortisol, tyrosine aminotransferase (l-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5) present in rat liver homogenates can be resolved into three peaks of activity by CM-Sephadex chromatography. Based on differential elution of these forms by a linear KCl gradient, a three-tube assay was developed that quantitates the amount of form III relative to total enzyme. The assay was used to determine the presence of a factor in the liver that converts tyrosine aminotransferase form III to form I. Definitive evidence for the liberation of such a factor is presented.     

Characterization of arylsulfatase activity in brine shrimp, Artemia salina

Arylsulfatase from Artemia salina exists in at least two forms (AS I and AS II). The paper presents characterization of the AS II form of the arylsulfatase. The enzyme was able to hydrolyze p-nitrocatechol sulfate (pNCS) as well as ascorbate sulfate. It exhibited maximum activity at temperature of 50 °C and was stable for 2 h at 4-10 °C. Optimum pH shifted from 6.2 at 4 mM pNCS (substrate) to 4.8 at 20 mM pNCS. The enzyme displayed linear kinetics. AS II arylsulfatase exists in two molecular forms (349 and 460 kDa) composed of identical subunits with molecular mass of 53 kDa. Sulfite and phosphate ions were the most potent inhibitors of the enzyme. Cyanide proved to be a weak inhibitor. Sulfate and low concentrations of silver ions had no effect on the enzyme activity. Based on the above results, modifications in the assay for determination of enzyme activity are proposed.     

alpha-Amylase from developing embryos of the camel tick Hyalomma dromedarii

alpha-Amylase activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of alpha-amylase III to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). alpha-Amylase III with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of alpha-amylase III was 106 kDa for the native enzyme, composed of two subunits of 43 and 66 kDa, respectively. alpha-Amylase had a value of 10 mg starch/ml. Varying alpha-amylase activity was detected when supplied with various substrates. alpha-Amylase III had a temperature optimum at 40 degrees C with heat stability up to 50 degrees C, and a pH optimum of 7.0. The enzyme activity was activated by CaCl2, MgCl2 and NaNO3, but not activated by NaCl, p-CMB, N-ethylmaleimide and iodoacetamide. EDTA and beta-mercaptoethanol strongly inhibited activity.