Two forms of Ca2+-dependent cyclic 3':5'-nucleotide phosphodiesterase from human aorta and effect of free fatty acids.

Investigations were carried out on two DEAE-cellulose columnresolvable Ca²⁺-dependent nucleotide 3′:5′-phosphodiesterases from human aorta. An extract from human aorta when chromatographed on DEAE-cellulose yielded five active cyclic nucleotide 3′:5′-phosphodiesterase fractions designated as F I, F II, F III, F IV, and F V and these fractions eluted at about 0.02, 0.08, 0.18, 0.28, and 0.38 M sodium acetate. F 111 and F IV were found to be activated by a protein modulator and free fatty acids. The two Ca²⁺-dependent cyclic nucleotide phosphodiesterases (F III and FIV) were clearly separated by rechromatography on DEAE-cellulose column and were found to hydrolyze guanosine 3′:5′-monophosphate (cyclic GMP) preferentially. Fatty acids as well as a protein modulator increased the maximum velocity of one form (F III) without affecting the K_m values and decreased the K_m values of the other (F IV) without changing maximum velocity. The extent of maximum stimulation by behenic acid (C₂₂) and a protein modulator was similar at optimal conditions. Fatty acids did not require calcium for stimulation of the phosphodiesterases. Stimulating activity diminished as the hydrocarbon chain length of the fatty acid was shortened or when more than two unsaturated bonds were introduced. Behenic acid (C₂₂) and eruic acid (C_22:1) were the most potent stimulators among the saturated or unsaturated fatty acids tested. The other DEAE-cellulose-resolvable aortic phosphodiesterase forms (F I, FII, and F V) were neither activated by the protein modulator nor stimulated significantly by fatty acids.     

The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (M_r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K_0.5 for MnCl₂ of 2 mm. Enzyme II (M_r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn²⁺. This enzyme was strongly inhibited by 50 mm NaF or 1 mm ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn²⁺, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn²⁺. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.     

Two forms of O-methyltransferase in tobacco cell suspension culture

An S-adenosyl-l-methionine: o-dihydric phenol O-methyltransferase was isolated from tobacco cell suspension culture and was partially purified by (NH₄)₂SO₄ precipitation and successive chromatography on DEAE-Sepharose, Sephacryl S-200 and hydroxyapatite columns. It catalysed the O-methylation of 3 cinnamic acids, two coumarins and two flavonoids, but to different extents. Results obtained from polyacrylamide gel electrophoresis, m-/p-methylation ratios and mixed substrate experiments indicated the existence of two forms of the enzyme which were resolved by chromatography on DEAE-cellulose. One form (MW 74000, pI 6.1, opt. pH 7.3) catalysed the meta-methylation of caffeic acid, while the other (MW 70000, pI 6.3, opt. pH 8.3) mediated the para-methylation of quercetin, though each form exhibited some activity against other substrates.     

Metabolism of trans-Aconitic Acid in Maize : I. PURIFICATION OF TWO MOLECULAR FORMS OF CITRATE DEHYDRASE

Trans-aconitate synthesis via citrate dehydrase was determined in crude extracts of maize (Zea mays L.) coleoptiles. Two molecular forms of this enzyme were purified by substrate-specific elution from DEAE-cellulose, ammonium sulfate precipitation, and gel filtration. Each molecular form migrates as a single band in isoelectric focusing. Gel filtration and sodium dodecyl sulfate electrophoresis provided evidence that one enzyme form is composed of four 80,000-dalton subunits while the other is composed of two 60,000-dalton subunits. There was no evidence of proteolytic conversion of the large to the small molecular weight form when the former was incubated with either the 15,000_g supernatant or with proteases. The data indicate that the two molecular forms of citrate dehydrase are isozymes.     

Multiple forms of porcine pancreatic α-amylase

Porcine pancreatic α-amylase can be fractionated into two components by DEAE-cellulose chromatography and by disc electrophoresis. The basis for fractionation is tentatively ascribed to a charge difference. The two components displayed the same specific activity and their thermal and pH stability, as well as the variation of V_max and K_m with pH, were identical within experimental error. It is concluded that the multiple forms of the amylase are physically distinct, but structurally related, with a common active site.     

Metabolism of purine nucleotides in the tomato plant

The enzymes 5′-nucleotidase (EC 3.1.3.5) and adenine phosphoribosyltransferase (EC 2.4.2.7) from the roots and leaves of tomato (Lycopersicon esculentum) have been purified and characterized. Two forms (root 1 and root 2) of 5′-nucleotidase from tomato roots were separated by chromatography on DEAE-cellulose. These were further purified by affinity chromatography on Blue Sepharose CL-6B. The enzyme from leaves appeared in only one form (leaf) when purified by similar methods. Root 2 and leaf enzymes were very similar in all respects including M_r (ca 68 000) whilst root 1 appeared distinct with a M_r close to 18 000. Tomato 5′-nucleotidase catalysed hydrolysis of isopentenylAMP and its action on AMP was inhibited in the presence of nucleoside monophosphates including isopentenylAMP. Adenine phosphoribosyltransferase existed in one form in roots and leaves and these differed from one another in several respects, e.g. pH optimum, M_r. Both enzymes catalysed phosphoribosylation of benzyladenine and the conversion of adenine to AMP was inhibited by the presence of cytokinin bases. The enzymes from the two sources differed in their patterns of inhibition by cytokinin bases.     

Alkaline phosphatase from pig kidney. Microheterogeneity and the role of neuraminic acid

Several alkaline phosphatases (EC 3.1.3.1) could be obtained from pig kidney brush-border membrane on extraction with butan-1-ol. Three of the multiple forms were separated by DEAE-cellulose chromatography and further purified. They form a regular series with different degrees of glycosylation (mainly owing to N-acetylneuraminic acid), of charge, of molecular weight, of stability to temperature, to pH and to urea, of minimal requirement for Mg²⁺ and of extractability by butan-1-ol. In contrast, the detectable antigenic sites, the inhibition by amino acids and the pH-dependency of K_m and V_max. were identical for these multiple forms. On treatment with neuraminidase, the multiple forms became identical in all their properties. It was therefore concluded that the microheterogeneity of alkaline phosphatase is due to different degrees of glycosylation at polypeptide chains which appear to be otherwise identical.     

Homogeneous cytochrome b5 from human erythrocytes

Homogeneous cytochrome b₅ has been isolated from large volumes of human erythrocytes by sequential chromatography on DEAE-cellulose, Amberlite CG-50, Bio-Gel P-60, and DEAE-Sephadex A-50. A molecular weight of 15,300 was determined by SDS disc gel electrophoresis. Trypsin converted the protein to a smaller hemepeptide which was indistinguishable from trypsin-cytochrome b₅ of human liver microsomes by disc gel electrophoresis. The data suggest that erythrocyte cytochrome b₅ has the same structure as a segment of liver microsomal cytochrome b₅ and is intermediate in size between the trypsin- and detergent-solubilized forms of the liver protein.     

Isolation and characterization of multiple forms of malt deoxyribonuclease

A deoxyribonuclease (DNase), similar to bovine pancreatic DNase, has been isolated from germinating barley. Commerically available malt was used as source of the enzyme. The purification procedure involves (a) ammonium sulfate fractionation (45–65% saturation), (b) CM-cellulose chromatography at pH 4.7 and (c) DEAE-cellulose chromatography at pH 8. DEAE-cellulose separates the enzyme into 4 distinct forms, designed as DNases A, B, C, and D. DNase A and B may be rechromatographed on DEAE-cellulose employing a CaCl₂ instead of Tris-HCl gradient. Both forms appear homogeneous on regular and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. In addition, both forms have a sp. act. of ca 700 units per A unit at 280 nm, similar to the potency of the pancreatic enzyme. DNase C and D, which are present in relatively small quantities in malt, were not characterized. The MWs of DNases A and B, as estimated by the SDS gel electrophoresis techniques, are near 32 000, slightly larger than that of the pancreatic enzyme. In the presence of either Mn²⁺ or Mg²⁺, the pH-activity profile of the barley enzyme is similar to that obtained with the pancreatic enzyme. Like the pancreatic enzyme, barley DNase is protected by Ca²⁺ from inactivation. The amino acid compositions of the A and B forms are about the same; a comparison of the malt and pancreatic enzymes shows many similarities but major differences in the amounts of glutamic acid, proline and glycine. The hydrolysis products of DNA by malt DNase are indistinguishable from those obtained with pancreatic DNase. Further hydrolysis of these products by snake venom phosphodiesterase shows malt DNase to be a 5′-phosphate producer. Deoxythymidine 3′,5′-di-p-nitrophenyl phosphate, one of the synthetic substrates of pancreatic DNase, is also hydrolysed by malt DNase.     

Isolation of an inhibitor of ß-glucuronidase from porcine sublingual gland

An inhibitor of ß-glucuronidase was isolated from porcine sublingual gland by successive fractionation of trypsin extracts of the latter on Sephadex G-100, DEAE-cellulose, Sephadex G-200, and DEAE-cellulose. Its purity and homogeneity were established by DEAE-cellulose column chromatography, ultracentrifugation, and electrophoresis on cellulose-acetate membrane. The sedimentation coefficient of the purified ß-glucuronidase inhibitor was 3.75 S (S₂₀⁰, w), and the molecular weight was determined to be 340 000 from Sephadex G-200 column chromatography. The inhibitor contained 17.5% protein, 20.8% total hexoses, 19.9% hexosamine, 21.8% N-acetylneuraminic acid, and 9.6% fucose. The inhibition was non-competitive, and it was completely suppressed by the addition of NaCl, KCl, Na₂SO₄, or CaCl₂, respectively.     

Extraction,regeneration after bleaching,and ion-exchange chromatography of rhodopsin in Tween 80

Rhodopsin can be readily and somewhat, selectively extracted into Tween 80 solutions from the isolated photoreceptor particulate fraction of bovine retinal tissue. Approximately 80% of the rhodopsin is recovered from the particulate fraction with A₄₉₈ values of approximately 6 and spectral ratios (A₂₇₈:A₄₉₈) of 1.8-1.9. The solutions are estimated to be approximately 97% pure based upon assay of protein and rhodopsin content and 98% pure based upon chromatography on DEAE-cellulose. The bulk of the rhodopsin can be regenerated after bleaching in Tween 80. Partial regenerability is retained when solutions of unbleached or bleached rhodopsin in Tween 80 are further purified by DEAE-cellulose chromatography.     

β-Galactosidases of Lilium pollen

Lily (Lilium auratum) pollen contains very high levels of β-galactosidase. There are three forms: β-galactosidase I and II differ in M_r, while β-galactosidase III is firmly bound in the pollen wall. The two cytoplasmic forms were separated and partially purified using a combination of chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose 6B. Forms I and II appear to be glycoprotein in nature as shown by binding to Con A-Sepharose. The three enzymes were optimally active near pH 4, and all were inhibited by galactose and galactonolactone. The wall-bound enzyme, β-galactosidase III effectively hydrolysed nitrophenyl β-galactosidase but not lactose, and could not be released from the wall polysaccharide matrix by high salt concentrations or detergents. The total β-galactosidase activity of lily pollen remained constant during in vitro germination. A possible role for this enzyme may be in degradation of stylar arabinogalactans providing a carbon source for pollen tube nutrition.     

Multiple forms of (1 → 4)-α-d-glucan, (1 → 4)-α-d-glucan-6- glycosyl transferase from developing zea mays L. Kernels

Two major forms of branching enzyme from developing kernels of maize have been detected after DEAE-cellulose chromatography. Branching-enzyme I, which contained 24% of the activity based on a phosphorylase-stimulation assay, but 74% of the activity based on the branching of amylose as monitored by change in spectra of the iodine-glucan complex, eluted with the column wash and was unassociated with starch-synthase activity. Branching-enzyme II was bound to DEAE-cellulose and was coeluted with both primed and unprimed starch-synthase activities. Both fractions were further purified by chromatography on aminoalkyl-Sepharose columns. Single peaks were observed for both fractions by gel filtration on BioGel A_1.5m columns and native molecular weights were estimated at 70,000–90,000 for both enzymes. Subunit molecular weights of branching-enzymes I and II were estimated by dodecyl sodium sulfate-gel electrophoresis at 89,000 and 80,000, respectively. Thus both enzymes are primarily monomeric. Branching-enzymes I and II could be distinguished by chromatography on DEAE-cellulose or 4-aminobutyl-Sepharose, and by disc-gel electrophoresis with activity staining. Branching-enyme I had a lower ratio of activity (phosphorylase stimulation-amylose branching; based on enzyme units). The ratio varied from 30–60 as compared to about 300–500 for branching-enzyme II. Likewise, branching-enzyme I had a lower K_m value for amylose than branching- enzyme II, the values being 160 and 500 μg/ml, respectively. Both enzymes could introduce further branches into amylopectin, as decreases in the overall absorption and wavelength maxima of the iodine complexes were observed. Combined action of the branching enzymes and rabbit-muscle phosphorylase a (12:1 ratio based on enzyme units) resulted in similar patterns of incorporation of d-glucose into the growing α-d-glucan and the synthesis of high molecular-weight polymers. However, the α-d-glucans differed, as shown by spectra of iodine complexes and average unit-chain length. Branching-enzyine II was separated into two fractions (IIa and IIb) by chromatography on 4-aminobutyl-Sepharose. These Fractions differed only in the branching of amylopectin, fractional IIb being more active than IIa.     

Author index

The membrane-bound ATPase activity of Bacillus subtilis was inhibited by dicyclohexylcarbodiimide (DCCD). The DCCD-reactive proteolipid of B. subtilis was extracted, from labelled or untreated membranes containing F₁ or depleted of F₁, with neutral or acidic chloroform/methanol. Purification of the [₁₄C]DCCD-binding proteolipid was attempted by column chromatography on methylated Sephadex G-50 and on DEAE-cellulose. The maximal amount of DCCD which could be bound to the purified proteolipid was found to exceed the amount bound by the purified proteolipid extracted from membranes labelled with the lowest [₁₄C]DCCD concentration required for maximal inhibition of the membrane-bound ATPase activity. The radioactive protein peaks eluted by gel filtration and ion-exchange chromatography were analysed by urea-SDS polyacrylamide slab gel electrophoresis and autoradiography. Radioactivity was incorporated into two components of M_r 18 000 and 6000 when proteolipid was purified by methylated Sephadex. The 6000 polypeptide was always present, whatever the extraction and purification procedures. However, the 18 000 polypeptide was present in largest quantity only when proteolipid was extracted from membranes containing F₁ and purified by methylated Sephadex. When proteolipid was purified on DEAE-cellulose this [₁₄C]DCCD binding component of M_r 18 000 was absent.     

Malate dehydrogenase isoforms from ribbed mussel (Modiolus demissus) gill tissue

• 1.1. The malate dehydrogenase (MHD) activity from the ribbed mussel gill is polymorphic with two distinct mitochondrial forms (M1 and M2) and five forms that could be resolved from cytosolic extracts (C1 to C5) by DEAE-cellulose chromatography and starch gel electrophoresis.
• 2.2. Two of the cytosolic forms (C3 and C4) may represent interchangeable conformational states.
• 3.3. With kinetic analysis there appear to be three distinct cytosolic forms (C1, C2 and C3–C4), with C2 possibly behaving as a heterodimer.
• 4.4. The identity of C5 is uncertain.
• 5.5. The forms isolated from the mitochondria (M1 and M2) exhibited lower apparent K_ms for oxaloacetate (OAA) than the cytosolic forms.
• 6.6. For all isozymic forms, the apparent K_ms for OAA increased as the pH increased between pH 6 and 9
• 7.7. Increasing the salt concentration raised the K_m for OAA for all forms.
• 8.8. The mMDHs were more sensitive to inhibition by NaCl than the cMDHs.
• 9.9. Representative cMDH (C1) and mMDH (M2) isozymes exhibited substrate inhibition by high concentrations of OAA with the mMDH possessing lower K_is for substrate inhibition than the cMDH at each pH tested.
• 10.10. Differences and similarities in K_m app. for OAA at the different pHs and salt concentrations indicated that C1, C2 and C3–C4 and C5 were distinct forms, that M1 and M2 were distinct but very similar to each other, and that C1, C2, C3–C4 and C5 were distinct from M1 and M2.

     

Purification and properties of two different α1-protease inhibitors from mouse plasma

Two similar but distinct forms of α1-protease inhibitor (α1-PI) have been isolated and purified 120-fold to homogeneity from the plasma of female, white Swiss (Ha/ICR) mice. The two inhibitors can be separated by chromatography on DEAE-cellulose using a shallow NaCl gradient at pH 8.9 for elution. Because of their differing specificities for elastase and trypsin we have labeled the two inhibitors α1-PI(E) and α1-PI(T), respectively. The apparent M_r for both proteins, as estimated by gel exclusion chromatography, is approximately 53,000 daltons. However by polyacrylamide gel electrophoresis in the presence of SDS, α1-PI(T) has an apparent m_r of 65,000 while the apparent m_r of α1-PI(E) is 55,000. These results suggest differences in charge and carbohydrate composition. The two mouse inhibitors also have different AT-terminal amino acids. Like human α1-PI the mouse inhibitors form stable complexes with proteases. However they differed from human α1-PI in that they were not found to neutralize either human thrombin or plasmin. While α1-PI(E) inhibits bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase, α1-PI(T) is an effective inhibitor only of trypsin. Plasma levels of α1-PI(E) increase significantly 24 h after stimulation of the acute phase reaction while those of α1-PI(T) do not. Our data suggest that α1-PI(E) and α1-PI(T) are products of different genes.     

Differential regulation by triiodothyronine of substrate-specific uridinediphosphoglucuronate glucuronosyl transferases in rat liver

Hepatic uridinediphosphoglucroonate glucuronosyl transferase (UDPglucuronyltransferase, EC 2.4.1.17) functionally heterogeneus; 4-nitrophenol and bilirubin are representative subtrates for two separated from of the enzyme. UDPglucuronyltransferase activity for bilirubin and 4-nitrophenol was separated from solubilized rat liver microsomes by DEAE-cellulose chromatography and corresponding enzymes were purified. A radioimmunoassay was developed using a rabbit antiserum against purified rat 4-nitrophenol-specific UDPglucuronyltransferase, which precipitated enzyme activities toward both 4-nitrophenol and bilirubin. After treatment with triiodothyronine(T₃) (0.55 mg/kg body weight), hepatic microsomal UDPglucuronyltransferase activity for 4-nitropheelos was increased 400% as compared to controls; the enzyme activity for bilirubin was decreased by 80%; the changes in the substrate-specific enzyme activities were reflected in the enzymatically active fractions separated after DEAE-cellulose chromatography. The changes in enzyme activities paralleled changes in the concentrations of the two corresponing UDP glucuronyltransferase proteins in the chromatographic fractions, as measured by radioimmunoassay. The results indicate that the opposite effects of T₃ on the two forms of UDPglucuronyltransferase activity is due to its differential effect on corresponding enzyme proteins.     

Two forms of glutamine synthetase in leaves of Cucurbita pepo

It has been shown that the leaves of pumpkin (Cucurbita pepo) contain two molecular forms of glutamine synthetase (GS), one occurring in the cytosol (GS₁)and the other in the chloroplasts (GS₂). The activities of both forms were greater when ammonium ion was infiltrated into the leaves and this was shown to be due to de novo synthesis. The two synthetases were purified by ammonium sulphate fractionation, ion exchange chromatography on DEAE-cellulose, selective adsorption on calcium phosphate gel, and preparative polyacrylamide gel electrophoresis. The MWs of GS₁ and GS₂, estimated by gel filtration on Sephacryl S-200, were 480 000 and 370 000 respectively. During polyacrylamide gel electrophoresis in the presence of SDS both GS₁ and GS₂ were dissociated into polypeptide chains with MWs of 58 000 and 50 000 respectively, suggesting that GS, ₁ and GS₂ are octamers consisting of identical monomers. The synthetases showed noticeable differences in their amino acid composition. In GS₁ and GS₂ the proportions of α- helical segments were 34 and 17 % respectively. In the presence of Mg²⁺, the pH optima for GS₁ and GS₂ were 7.25 and 7.75 respectively, and K_m values toward l-glutamate were 13 and 46 mM respectively. From the experimental data it is inferred that GS₁ and GS₂ are isoenzymes.