Characterization and kinetics of beta-D-gluco/fuco/galactosidase from sheep liver

This enzyme shows beta-D-glucosidase, beta-D-fucosidase and beta-D-galactosidase activities, all associated in a single peak in Sephadex G-200, DEAE-cellulose, concanavalin A-Sepharose chromatographies, and in high resolution isoelectric focusing (pI 4.56), having the optimal pH in the range 4.5-5.5. The enzyme is very stable under different conditions: (i) at pH in the range 5.5-7.0; (ii) in successive freezing-thawing cycles; (iii) at 4 degrees C; (iv) after exhaustive ultrasonic treatment. It is not stable beyond 40 degrees C, and in the presence of urea, Triton X-100, SDS or mercaptoethanol. HgCl2, KCN, Tris, maltose and the lactones were inhibitors of the enzyme. With glucose, fucose and galactose the inhibition is competitive. In addition, a transglycosylation mechanism seems to occur. The kinetic studies suggest a substrate-activation model and the presence of two primary active sites: fuco-gluco and galacto.     

Studies on the hydrophobic properties of sphingomyelinase.

Crude liver lysosomal sphingomyelinase (EC 3.1.4.12) displays a heterogeneous electrofocusing profile. The majority of the enzyme resolves into two major components with acidic pI values near pH 4.6 and 4.8. Several additional minor peaks of activity are seen at more basic pH values (up to pH 8.0). In the presence of 0.1% Triton X-100 (or Cutscum), the location of sphingomyelinase is shifted by about 1 pH unit to more basic pH values. Triton X-100 also increases the apparent heterogeneity of sphingomyelinase. Removal of detergent by treatment with Bio Beads SM-2 restores the acidic pI profile. This behaviour appears to be specific, since it was not shared by six glycosidases several of which hydrolyse sphingolipids. The electrofocusing profile of 3H-labelled Triton X-100 was distinct and separate from sphingomyelinase, suggesting that only a small fraction of detergent interacted directly with the enzyme. To study this behaviour in more detail we examined the effect of detergents on elution of sphingomyelinase from sphingosylphosphocholine-Sepharose. Sphingosylphosphocholine is a competitive inhibitor of sphingomyelinase (Ki 0.5 mM). Binding of enzyme was pH-dependent. Triton X-100, Cutscum and Tween 20 eluted significant amounts of enzyme at 0.01-0.02%. Total elution was achieved with up to 0.1% detergent. These data suggest that sphingomyelinase binds to neutral detergent monomers with a high degree of affinity. In excess detergent (5-7 times the critical micellar concentration) the surface charge on the protein is changed, leading to a pI shift. This behaviour probably does not occur at the active site of the enzyme, since there is no appreciable effect on substrate hydrolysis and substrate analogues were ineffective in eluting the enzyme.     

Proteins of the kidney microvillar membrane. Purification and properties of carboxypeptidase P from pig kidneys.

Carboxypeptidase P has been purified by immunoaffinity chromatography from pig kidneys. A single-step assay with Z-Pro-Met (where Z represents benzyloxycarbonyl) as substrate was used, methionine being determined by using L-amino acid oxidase and horseradish peroxidase. The enzyme constitutes about 1.5% of the kidney microvillar proteins. Triton X-100-solubilized and papain-released forms of the enzyme were isolated. The former had an apparent subunit Mr of 135 000, and the latter form contained two polypeptide chains of Mr 128 000 and 95 000. The undenatured forms were dimeric proteins. In common with other microvillar hydrolases, carboxypeptidase P was a glycoprotein and each subunit contained one Zn atom. MnCl2 (1 mM) in the assay was necessary for maximum activity; in its absence, 0.5 mM-ZnSO4 produced a limited activation, but was inhibitory at higher concentrations. The Km for Z-Pro-Met, in the presence of MnCl2, was 4.1 mM, and the kcat. for freshly prepared enzyme was 1230 min-1. The enzyme lost activity during storage at -20 degrees C. In a limited survey of peptides, hydrolysis was observed only with substrates containing a proline, alanine or glycine residue in the P1 position, and these included angiotensins II and III. The best substrate in this series was Val-Ala-Ala-Phe.     

Properties of acid ceramidase from human spleen

We have characterised ceramidase activity in extracts of human spleen from control subjects and from patients with Gaucher disease. In Triton X-100 extracts of control spleens, a broad pH optimum of pH 3.5-5.0 was found; no ceramidase activity was detectable at neutral or alkaline pH. About 45-60% of acid ceramidase could be extracted from spleen without detergents, but for complete extraction, Triton X-100 was required. For the radiolabelled substrate oleoylsphingosine, a Km of 0.22 +/- 0.09 mM and a Vmax of 57 +/- 11 nmol/h per mg protein was calculated in spleen from a control subject. Flat-bed isoelectric focussing in the presence of Triton X-100 revealed a pI of 6.0-7.0 for acid ceramidase; similar values were found for sphingomyelinase and glucerebrosidase. HPLC-gel filtration indicated that in the presence of Triton X-100, acid ceramidase has an Mr of about 100 kDa. In the absence of detergents, the enzyme forms high-molecular-weight aggregates. Similar aggregation behaviour was observed for sphingomyelinase, while the elution of beta-hexosaminidase was not affected by detergents. The elution profile of glucocerebrosidase was only slightly altered by Triton X-100. There was no difference in the properties of acid ceramidase present in spleen from control subjects and from patients with type I Gaucher disease.     

Purification, characterisation and reconstitution of glutaconyl-CoA decarboxylase, a biotin-dependent sodium pump from anaerobic bacteria

Glutaconyl-CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X-100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate-binding site are located on the same side of the membrane. The decarboxylase was solubilized with Triton X-100 and purified by affinity chromatography on monomeric avidin-Sepharose. The enzyme is composed of three types of polypeptides: the group of alpha chains (Mr 120000-140000) containing the biotin, the beta chain (60000) and an apparently hydrophobic gamma chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na+ channel. The beta and gamma chains but not the alpha chain could be labelled by N-ethyl-[14C]maleimide. Similar decarboxylases but with much smaller biotin peptides (Mr 15000-20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum. The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na+ uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K+ indicating an electrogenic Na+ pump. The coupling between Na+ transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2. The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl-CoA decarboxylase are able to ferment glutamate and require 10 mM Na+ for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.     

Purification, characterization, and kinetic analysis of a 55-kDa form of phosphatidylinositol 4-kinase from Saccharomyces cerevisiae.

A 55-kDa form of membrane-associated phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 10,166-fold from Saccharomyces cerevisiae. The purification procedure included solubilization of microsome membranes with 1% Triton X-100 followed by chromatography with DE52, hydroxylapatite I, Q-Sepharose, Mono Q, and hydroxylapatite II. The procedure resulted in a nearly homogeneous 55-kDa phosphatidylinositol 4-kinase preparation. The 55-kDa phosphatidylinositol 4-kinase and the previously purified 45-kDa phosphatidylinositol 4-kinase differed with respect to their amino acid composition, isoelectric points, and peptide maps. Furthermore, the two forms of phosphatidylinositol 4-kinase did not show an immunological relationship. Maximum 55-kDa phosphatidylinositol 4-kinase activity was dependent on magnesium (10 mM) or manganese (0.5 mM) ions and Triton X-100 at the pH optimum of 7.0. The activation energy for the reaction was 12 kcal/mol, and the enzyme was labile above 30 degrees C. The enzyme was inhibited by thioreactive agents, MgADP, and calcium ions. A detailed kinetic analysis of the purified enzyme was performed using Triton X-100/phosphatidylinositol-mixed micelles. 55-kDa phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol and followed surface dilution kinetics. The interfacial Michaelis constant (Km) and the dissociation constant (Ks) for phosphatidylinositol in the Triton X-100 micelle surface were 1.3 mol % and 0.035 mM, respectively. The Km for MgATP was 0.36 mM. 55-kDa phosphatidylinositol 4-kinase catalyzed a sequential reaction mechanism as indicated by the results of kinetic and isotopic exchange reactions. The enzyme bound to phosphatidylinositol before ATP and released phosphatidylinositol 4-phosphate before ADP. The enzymological and kinetic properties of the 55-kDa phosphatidylinositol 4-kinase differed significantly from those of the 45-kDa phosphatidylinositol 4-kinase. This may suggest that the two forms of phosphatidylinositol 4-kinase from S. cerevisiae are regulated differentially in vivo.     

Substrate specificity and binding loci for inhibitors in an aminopeptidase purified from the plasma membrane of midgut cells of an insect (Rhynchosciara americana) larva

Plasma membrane-bound aminopeptidases (EC 3.4.11.2) are found in the midgut cells from Rhynchosciara americana larvae, and are recovered in soluble form after papain treatment. The major papain-released aminopeptidase (Mr 207,000 and pI 7.8) was shown to be a true aminopeptidase with a broad specificity toward aminoacyl-beta-naphthylamides and to be more active on tetra and tripeptides than on dipeptides. The purified aminopeptidase is inactivated by EDTA according to a kinetics which is half order in relation to EDTA. Leucine hydroxamate (Ki 27 microM) and hydroxylamine (Ki 5.4 mM) completely protect the enzyme from inactivation by EDTA, whereas isoamyl alcohol (Ki 62 mM) increases the inactivation rate. There are 2.3 binding sites in the enzyme for phenanthroline, which makes the binding of the substrate in the enzyme difficult, changes the enzyme-substrate into a more productive complex, and increases the inactivation rate of the enzyme by EDTA by 87-fold. The data support the proposal that the enzyme has a metal ion which is catalytically active and that the enzyme displays two subsites in its active center: a hydrophobic subsite, to which isoamyl alcohol binds exposing the metal ion, and a polar subsite, to which hydroxylamine binds.     

Characterization of Fe2+-activated acid phosphatase in rat epidermis

A particulate acid phosphatase (EC 3.1.3.2, orthophosphoric monoester phosphohydrolase (acid optimum)) was extracted in 1 M KCl, from 2-day rat epidermis. The enzyme has a Mr of 32,000, but two forms, F1 and F2 with pI values of 8.6 and 8.3, respectively, were identified while the pI values of other acid phosphatases soluble in sucrose and Triton X-100 were all acidic. F1 and F2 also differed from other epidermal acid phosphatases because they were (a) activated by Fe2+ and reducing agents, (b) showed immunological cross-reactivity with purple acid phosphatase of rat spleen and (c) dephosphorylated phosvitin and alpha-casein even though they had rather high Km values.     

Purification and properties of L-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus.

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a 'wall + membrane' fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.     

Comparison of the size and physical properties of gamma-glutamyltranspeptidase purified from rat kidney following solubilization with papain or with Triton X-100.

gamma-Glutamyltranspeptidase is associated with the brush border membrane of kidney proximal straight tubule cells. It can be solubilized qualitatively by treatment with papain or Triton X-100. Neither procedure affects its catalytic activity but the two resulting forms of the enzyme differ considerably in their physical properties. The papain-solubilized transpeptidase is soluble in aqueous buffers and was purified 430-fold. It has an s20,w of 4.9 S, a Stokes radius of 36 A, and a calculated molecular weight of 69,000. It appears homogeneous by sedimentation equilibrium centrifugation (Mr=66,700). In contrast, the Triton-solubilized transpeptidase is soluble only in the presence of detergents and was purifed 300-fold. This form of the enzyme has a Stokes radius of 70 A but an s20,w of only 4.15 S. Aggregation of the enzyme just below the critical micelle concentration of Triton X-100 and its ability to bind 1.16 mg of Triton X-100-protein complex was calculated to be 169,000, but the glycoprotein portion of the complex is 52% of the total mass (87,000). The mass of Triton X-100 (82,000) is consistent with its reported micelle molecular weight. Treatment of the Triton-purified transpeptidase with papain or bromelain results in a form of the enzyme identical in all respects with the papain-purified enzyme. Both the Triton- and papain-purified transpeptidase exhibit two protein bands on sodium lauryl sulfate-polyacrylamide gel electrophoresis. The smaller subunits of the two forms appear identical (Mr=27,000), while the larger subunits of the Triton- and papain-purified enzyme have apparent molecular weights of 54,000 and 51,000, respectively. These data suggest that a peptide (3,000 to 19,000) in the larger subunit of gamma-glutamyltranspeptidase is responsible for its binding to Triton micelles and probably for holding the enzyme in the brush border membrane.     

Identification of the membrane form of phenylalanine hydroxylase from the human liver and characteristics of its subunit composition

Phenylalanine hydroxylase was detected among human liver bioptats and autoptats extracted with 0.4% Triton X-100 from the 105,000 g homogenate fraction. In contrast to the membrane form of the rat liver enzyme, human liver phenylalanine hydroxylase is detected both by its enzymatic activity and immunochemically under non-denaturating conditions. The enzymatic activity of phenylalanine hydroxylase makes 5-15% of that of the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction and 20-30% of the amount of antigen in the cytoplasmic fraction as can be evidenced from rocket immunoelectrophoresis data. Immunoblotting of proteins performed after denaturating electrophoresis of the membrane and cytoplasmic fractions revealed an antigen band with a similar electrophoretic mobility. The subunit composition of the enzyme in both fractions was characterized by two-dimensional electrophoresis with subsequent immunoblotting. It was found that the membrane fraction of the enzyme is represented only by the L-subunit with Mr of 55 kD, whereas the cytoplasmic fraction, besides the predominant L-subunit, also contains 2H-subunits of the enzyme with Mr = 57 kD. These 2H-subunits differ between themselves as well as from the L-subunit by the pI value.     

Purification and characterization of phosphatidylinositol kinase from Saccharomyces cerevisiae

The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified 8,000-fold from Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of microsomal membranes, DE-52 chromatography, hydroxylapatite chromatography, octyl-Sepharose chromatography, and two consecutive Mono Q chromatographies. The procedure resulted in the isolation of a protein with a subunit molecular weight of 35,000 that was 96% of homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidylinositol kinase activity was associated with the purified Mr 35,000 subunit. Maximum phosphatidylinositol kinase activity was dependent on magnesium ions and Triton X-100 at pH 8. The true Km values for phosphatidylinositol and MgATP were 70 microM and 0.3 mM, and the true Vmax was 4,750 nmol/min/mg. The turnover number for the enzyme was 166 min-1. Results of kinetic and isotopic exchange reactions indicated that phosphatidylinositol kinase catalyzed a sequential Bi Bi reaction mechanism. The enzyme bound to phosphatidylinositol prior to ATP and phosphatidylinositol 4-phosphate was the first product released in the reaction. The equilibrium constant for the reaction indicated that the reverse reaction was favored in vitro. The activation energy for the reaction was 31.5 kcal/mol, and the enzyme was thermally labile above 30 degrees C. Phosphatidylinositol kinase activity was inhibited by calcium ions and thioreactive agents. Various nucleotides including adenosine and S-adenosylhomocysteine did not affect phosphatidylinositol kinase activity.     

An isoelectric focusing study of acid phosphohydrolases in rat liver lysosomes.

The differentiation of rat liver lysosomal acid phosphatase, acid ATPase, acid phosphodiesterase, acid ribonuclease, and acid deoxyribonuclease was studied by isoelectric focusing. To prevent autolytic digestion, inhibitors of cathepsins and neuraminidase were used. The proportion of acidic forms of acid phosphatase, acid ATPase and acid phosphodiesterase was increased by the use of extraction medium containing 0.05% Triton X-100. To investigate the identity of acid ATPase and acid phosphodiesterase, the relative activities among the multiple forms of these enzymes, the acid phosphodiesterase/acid ATPase ratio at each activity peak, and the degree of enzyme inhibition by p-chloromercuriphenyl sulfonic acid were estimated. The results suggest that acid ATPase is not identical with acid phosphodiesterase. With extraction medium free of Triton X-100, acid ribonuclease appeared in two forms. However, in addition to these forms, a new form of this enzyme with a more acidic pI (4.22) emerged when extraction medium containing 0.05% Triton X-100 was used. The major peak of acid deoxyribonuclease with pI=8.40-9.39 was obtained regardless of the extracting method.     

Blood digestion in the mosquito, Anopheles stephensi Liston (Diptera: Culicidae): partial characterization and post-feeding activity of midgut aminopeptidases

Aminopeptidase activity was partially characterized from midguts of Anopheles stephensi Liston which had been dissected 30 h after blood feeding. In crude midgut homogenate supernatants the aminopeptidases showed optimum activity at pH 8.0 and preferentially hydrolyzed alanine- and leucine-terminal amino acid substrates. Methionine, proline, lysine, and arginine terminal substrates were hydrolysed, but not glutamic acid. Activity was stimulated by Mg2+, EDTA, and low Ca2+ concentrations, while Mn2+, Tris, 1,10 phenanthroline, and higher Ca2+ concentrations were inhibitory. Supernatants from midguts homogenized in 1% Triton X-100 showed a two-fold increase in activity. Differential centrifugation of midgut homogenates demonstrated 45% of the total activity in a putative microvillar pellet and 32% in a soluble fraction. More than 92% of the total activity was solubilized after homogenization in Triton X-100. Activity in homogenate supernatants was restricted to one major peak (Mr = 552,000) with a higher molecular weight shoulder. Three distinct peaks of aminopeptidase activity were observed following Triton X-100 treatment: a minor high molecular weight peak (Mr = 552,000), and two major peaks at Mr = 123,000 and Mr = 32,000 respectively. The activity of aminopeptidase increased after a blood meal, in parallel to the post-feeding changes in trypsin activity, indicating its important role in secondary digestion of blood meal proteins.     

Identification and extraction of proteins that compose the triad junction of skeletal muscle

Treatment of both transverse tubules and terminal cisternae with a combination of Triton X-100 and hypertonic K cacodylate causes dissolution of nonjunctional proteins and selective retention of membrane fragments which are capable of junction formation. Treatment of vesicles with Triton X-100 and either KCl or K gluconate causes complete dissolution of all components. Therefore K cacodylate exerts a specific preservative action on the junctional material. The membrane fragment from treatment of transverse tubules with Triton X-100 + cacodylate contains a protein of Mr = 80,000 in SDS gel electrophoresis as the predominant protein while lipid composition is enriched in cholesterol. The membrane fragment retains in electron microscopy the trilaminar appearance of the intact vesicles. Freeze fracture of transverse tubule fragments reveals a high density of low-profile, intercalated particles, which frequently form strings or occasional small arrays. The fragments from Triton X-100 plus cacodylate treatment of terminal cisternae include the protein of Mr = 80,000 as well as the spanning protein of the triad, calsequestrin, and some minor proteins. The fragments are almost devoid of lipid and display an amorphous morphology suggesting membrane disruption. The ability of the transverse tubular fragment, which contains predominantly the Mr = 80,000 protein, to form junctions with terminal cisternae fragments suggests that it plays a role in anchoring the membrane to the junctional processes of the triad. The junctional proteins may be solubilized in a combination of nonionic detergent and hypertonic NaCl. Subsequent molecular sieve chromatography gives an enriched preparation of the spanning protein. This protein has subunits of Mr = 300,000, 270,000 and 140,000 and migrates in the gel as a protein of Mr = 1.2 X 10(6) indicating a polymeric structure.     

Partial purification and characterization of phosphatidylinositol kinases from human platelets

Most of human platelet phosphatidylinositol (PI) kinase activity (approx. 80%) was associated with the membrane fraction and its majority was released by the extraction with Triton X-100 after KCl treatment. Two major activity peaks (mPIK-I and mPIK-III) were obtained by Mono Q column chromatography. They were distinct from each other with regard to Mr (76,000 and 80,000 as determined by gel-filtration chromatography), apparent Km values for ATP, effect of arachidonic acid and phosphatidylserine and detergent requirement. Triton X-100 inhibited the activity of mPIK-I but rather weakly enhanced the mPIK-III activity, and sodium cholate remarkably inhibited both mPIK-I and mPIK-III activities. Their products were identified to be phosphatidylinositol 4-phosphate. On the other hand, about 20% of PI kinase activity was recovered from the cytosolic fraction and two activity peaks (cPIK-I and cPIK-II) were resolved on Mono Q column chromatography. There were no significant differences in biochemical properties between cPIK-I and cPIK-II. Both of them had Mr approx. 550,000 as determined by gel-filtration chromatography and were activated by sodium cholate to a greater extent than by Triton X-100. The results suggest that the major PI kinases (mPIK-I and mPIK-III) are PI 4-kinase and mPIK-I is distinct from PI 4-kinases in other sources especially with regard to the effect of Triton X-100.     

NAD(P)(+)-glycohydrolase from human spleen: a multicatalytic enzyme

NAD(P)(+)-glycohydrolase (NADase, EC 3.2.2.6) was partially purified from microsomal membranes of human spleen after solubilization with Triton X-100. In addition to NAD+ and NADP+, the enzyme catalyzed the hydrolysis of several NAD+ analogues and the pyridine base exchange reaction with conversion of NAD+ into 3-acetylpyridine adenine dinucleotide. The enzyme also catalyzed the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and the hydrolysis of cADPR to adenosine diphosphoribose (ADPR). Therefore, this enzyme is a new member of multicatalytic NADases recently identified from mammals, involved in the regulation of intracellular cADPR concentration. Human spleen NADase showed a subunit molecular mass of 45 kDa, a pI of 4.9 and a Km value for NAD+ of 26 microM. High activation of ADPR cyclase activity was observed in the presence of Ag+ ions, corresponding to NADase inhibition.     

Characterization of NAD-dependent malate dehydrogenases from spinach leaves

Summary. Spinach leaves were used to extract isoforms of NAD-dependent malate dehydrogenase (NAD-MDH) (EC 1.1.1.37), either soluble or bound to microsomal, plasma, or chloroplast envelope membranes. All fractions were subjected to isoelectric focusing analysis, which showed that purified chloroplast envelopes contain an NAD-MDH isoform tightly bound to the membranes, since treatment with 0.5 or 1% Triton X-100 was not able to release the enzyme from the envelopes. In contrast, plasma membranes released an isoform with a pI of 3.5 following treatment with 0.5% Triton X-100. The most abundant soluble leaf isoform had a pI of 9, while the chloroplast stroma contained an isoform with a pI of 5.3. Kinetic analysis of oxaloacetate (OAA)-dependent NADH oxidation in different fractions gave different K _m values for both substrates, the envelope- and plasma membrane-bound NAD-MDH exhibiting the highest affinities for OAA. Leaf plasma membrane-bound MDH exhibited a high capacity for both reaction directions (malate oxidation and OAA reduction), while the two chloroplast isoforms (stromal and envelope-bound) preferentially reduced OAA. Our results indicate that the chloroplast envelope contains a specifically attached NAD-MDH isoform that could provide direct coupling between chloroplast and cytosol adenylate pools. Correspondence: T. Cvetić, Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia.     

Demonstration of two molecular forms of dipeptidyl peptidase IV in normal human serum

The heterogeneity of dipeptidyl peptidase IV (EC 3.4.14.5) was investigated in normal human serum. Thin-layer analytical isoelectric focusing revealed the presence of multiple molecular forms of the enzyme, their isoelectric points being in the pH range of 3.30-4.25. The maximum of enzyme activity appeared around pH 3.50. After treatment with neuraminidase the pI shifted to 4.70-5.40 with two maxima at pH 5.00 and 5.15. The Triton X-100 solubilized as well as the papain-treated-Triton X-100 solubilized enzyme from the whole human adult jejunal biopsy were also found to be heterogeneous. They focused--both before and after neuraminidase treatment--at pH values different from those of the enzyme of normal human serum. There was almost no pI shift after neuraminidase treatment of the intestinal enzyme from adult enterobiopsy. Electrophoresis in continuous polyacrylamide gradient gels as well as gel chromatography on Bio-Gel A-1.5m revealed two molecular forms of dipeptidyl peptidase IV in normal human serum. The estimated relative molecular mass of the major enzyme form was 250 000 in both the separation techniques used. On the other hand, the apparent relative molecular mass of the minor enzyme form was 450 000 as assessed by gradient gel electrophoresis, and 550 000, when estimated by gel chromatography. The Km values for glycyl-L-proline-4-nitroanilide as substrate with the major and minor forms of the serum enzyme were 1.60 +/- 0.39 X 10(-4) mol/l and 1.60 +/- 0.13 X 10(-4) mol/l, respectively. Our results indicate that the dipeptidyl peptidase IV in normal human serum is a heterogeneous enzyme as far as its charge and molecular size are concerned.     

The cAMP-dependent protein kinase in sea urchin sperm tails: association of the enzyme with the flagellar axonemes

When sea urchin spermatozoa were treated with a Triton X-100 solution, cAMP-dependent protein kinase (cA-kinase) activity was extracted. Further extraction with Triton X-100 of axonemes isolated from the Triton-extracted sperm again released a considerable amount of the cA-kinase activity. The activity which remained after extraction three times with Triton X-100 was released by treatment with a low salt solution. These activities found in the various extracts were likely to be due to the same cA-kinase, which was a mammalian type II-like enzyme. The cA-kinase activity that remained in the axonemes after the first Triton X-100 extraction may be involved in the regulation of flagellar movement in the Triton-extracted sperm.