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).     

Isolation and properties of multiple forms of histidine decarboxylase from rat gastric mucosa.

The heterogeneity of histidine decarboxylase from rat gastric mucosa was studied. The partially purified enzyme was fractionated by preparative isoelectric focusing on a flat-gel bed by using narrow pH-range carrier ampholytes and a short focusing time. The activity was resolved, with about 95% recovery, into three forms, designated I, II and III, with pI values of 5.90, 5.60 and 5.35 respectively. These three forms exhibited similar molecular weights, indicating that the forms were not the result of different degrees of polymerization. By preparative refocusing each form refocused as a single peak of enzyme activity with reproducible pI, but a high loss of activity occurred with repeated focusing. Forms I, II and III were purified by the combined use of preparative isoelectric focusing and gel chromatography and other fractionation methods. The active forms could be distinguished by electrophoresis and isoelectric focusing on polyacrylamide gels and displayed protein heterogeneity. These forms were found in the crude extract and in the partially purified preparations in the presence or absence of proteinase inhibitors. Form II had the highest specific activity, but all three forms had the same optimum pH and Km value for histidine.     

Molecular forms of myeloperoxidase in human plasma.

A radioimmunoassay for myeloperoxidase was established with the use of affinity-purified anti-(human myeloperoxidase) immunoglobulins. By the use of ion-exchange followed by immunoaffinity chromatography a preparation of immunoreactive, catalytically active myeloperoxidase was obtained from fresh human plasma. In non-denaturing gel electrophoresis, the plasma preparation showed about four catalytically active components of mobility very similar to that of the granulocyte enzyme. SDS/polyacrylamide-gel electrophoresis combined with protein blotting showed that the two polypeptides of strongest antigenicity in the plasma preparation corresponded in Mr to the large and the small subunits of the granulocyte enzyme. In addition, the plasma preparation contained a higher-Mr immunoreactive polypeptide, possibly a precursor form of the enzyme, together with another of Mr similar to that of the large subunit of eosinophil peroxidase.     

Differential distribution of distinct forms of myeloperoxidase in different azurophilic granule subpopulations from human neutrophils

Myeloperoxidase (MPO), a characteristic enzyme of human polymorphonuclear neutrophils (PMN), is localized in specialized lysosomal or azurophilic granules, and can be resolved into three distinct forms (I, II, III) by ion-exchange chromatography. Granules were isolated from single donor PMN and fractionated with centrifugation into two different azurophilic subpopulations (high and low density) by banding in a continuous sucrose density gradient. Ion-exchange chromatography of granule extracts indicated that the lower density granules contained mainly MPO forms II and III while the higher density granules appeared to contain all three forms, but in much reduced amounts. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed that, the mobilities of the heavy subunits of MPO appeared to be inversely related to the density of the granule population from which they were extracted. These observations suggest that the different forms of MPO may have distinct functional roles and/or are a possible reflection of maturational differences among the granule subpopulations.     

Purification and characterization of, and preparation of an antibody to, transketolase from human red blood cells

Transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase, EC 2.2.1.1) was purified 16 000-fold from human red blood cells, using DEAE-Sephadex A-50, Sephadex G-150, FPLC on Mono P, and Sephadex G-100. The purified enzyme migrated as a single protein band on SDS-polyacrylamide gel electrophoresis. The FPLC step resolved transketolase into three peaks, designated I, II and III. From results of re-FPLC on Mono P, SDS-polyacrylamide gel electrophoresis, gel filtration, catalytic studies, amino acid analysis and immunological studies, it was concluded that I, II and III were originally the same protein, modified during storage and purification. Transketolase had a subunit (Mr 70 000) and appeared to be composed of two identical subunits. 1 mol of subunit contained 0.9 mol of thiamine pyrophosphate. The pH optimum of the reaction lay within the range 7.6-8.0, and the Km values were determined to be 1.5 X 10(-4) M for xylulose 5-phosphate and 4.0 X 10(-4) M for ribose 5-phosphate. Hg2+ and p-chloromercuribenzoate inhibited the enzyme reaction, and the inhibition of the latter disappeared upon the addition of cysteine. Thiamine and its phosphate esters did not, but cysteine (1 X 10(-2) M) and ethanol (10% and 1% v/v) did activate the enzyme reaction. Antibody prepared to II bound all forms of transketolase in the hemolysate, but inhibited the reaction only about 20%.     

Distinct chromatographic forms of human hemi-myeloperoxidase obtained by reductive cleavage of the dimeric enzyme. Evidence for subunit heterogeneity

The enzyme myeloperoxidase (MPO) is a functionally important glycoprotein of neutrophilic granulocytes and occurs in three major isoforms (forms 1, 2, and 3) that are dimeric structures composed of two heavy subunit-light subunit protomers, each of which is associated with a chlorine-like prosthetic group. In the present study, highly purified MPO isoforms were obtained from the cells of a single normal donor, and each protein was subjected to reductive alkylation under nondenaturing conditions. The resulting enzymatically active protomers were separated from unreacted dimer using gel filtration chromatography. Use of a fast protein liquid chromatography cation exchange system with a Mono S matrix revealed heterogeneity of the protomers, and allowed essentially complete resolution of the protomers of MPO form 2. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two resolved protomeric species under reducing conditions revealed small but reproducible differences in the Mr of their heavy subunits (59,000 and 57,000). Treatment with either endo-beta-N-acetylglucosaminidase or peptide N-glycohydrolase F reduced the Mr of each heavy subunit by approximately 3000 but did not change their relative electrophoretic mobilities. Heavy and light subunits were prepared from each of the MPO isoforms by reductive alkylation under conditions that allowed full retention of the prosthetic group with the heavy subunit. Reverse-phase chromatography and amino-terminal sequencing showed that each MPO isoform contained one major species of light subunit and several minor species. No differences in peroxidatic activity or inhibition by salicylhydroxamic acid were observed among any of the MPO isoforms or resolved protomers, but the latter were considerably more heat labile than dimeric forms of the enzyme and a monomeric form isolated from HL-60 cells. This is the first report of the isolation and partial characterization of distinct protomers from a single isoform of human MPO and suggests that the structure of MPO is more complex than considered previously.     

Comparison between the monomeric and binary-complex forms of procarboxypeptidase A from whole pig pancreas.

Two different forms of procarboxypeptidase A (I and II) were obtained from pig pancreas extracts. The Mr values, the pattern found on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, and the sedimentation coefficients indicate that form I is a binary complex formed by two different subunits, whereas form II is a monomer. The carboxypeptidase A-precursor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx. 12500) and, in the case of the binary complex, the activation with trypsin follows a rather complex pattern, suggesting that the accompanying subunit of form I might play a modulating role in the activation process. Although the appearance of enzymic activity is rather slow, a protein with an Mr equivalent to that of active carboxypeptidase A is found very early in the activation process. Both zymogens are glycoproteins (so far no carbohydrate has been reported in any procarboxypeptidase A) and both contain two strongly bound Zn2+ ions/molecule. Other chemical and physical properties were also determined.     

Purification and some properties of the extracellular alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum.

The novel alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum E 101-69 was purified as two forms (I and II) from culture medium, by using gel filtration in 6 M-guanidine hydrochloride as the final step. Renatured alpha-amylase-pullulanase I and II had apparent Mr values of 370,000 +/- 85,000 and 330,000 +/- 85,000 respectively, as determined by native polyacrylamide-gradient-gel electrophoresis. Both forms appear to be dimers of two similar subunits, with Mr values of 190,000 +/- 30,000 for enzyme I and 180,000 +/- 30,000 for enzyme II according to SDS/polyacrylamide-gradient-gel electrophoresis. The two forms had similar amino acid compositions, the same N-terminal sequence (Glu-Ile-Asp-Thr-Ala-Pro-Ala-Ile) and the same pI of 4.25. Both forms contained sugars having mobilities identical with those of rhamnose, glucose, galactose and mannose. The amount of neutral hexoses relative to protein was 11-12% (w/w) for both forms.     

Evidence for and partial characterization of three major and three minor chromatographic forms of human neutrophil myeloperoxidase

Myeloperoxidase (MPO) is an enzyme found in the azurophil granules of neutrophils. Cation-exchange chromatography on carboxymethyl-cellulose previously has been used to demonstrate the heterogeneity of the peroxidase enzymes isolated from human neutrophils. In this study, fast protein liquid chromatography (FPLC) was used to separate and purify three major (I, II, and III) and three minor (IIa, IIIa, IIIb) forms of MPO from isolated neutrophil granules. Purity was confirmed by polyacrylamide gel electrophoresis in the presence of cetyltrimethylammonium bromide (CETAB-PAGE), by crossed immunoelectrophoresis, and by spectral characteristics. All three major forms were indistinguishable by immunodiffusion against rabbit antiserum, scanning spectrophotometry, and amino acid composition. They differed in their elution from a cation-exchange resin, inhibition by 3-amino-1,2,4-triazole, migration rate in CETAB-PAGE, and subunit molecular weight. Subunit molecular weight was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All three major forms appeared to consist of heavy (H), intermediate (M), and light (L) peptides. The M peptide appeared to be derived from the H subunit. All L subunits exhibited a molecular weight of 14,500. The molecular weights for the H subunits varied, and were 60,000, 59,000, and 57,000 for MPO I, II, and III, respectively. The molecular weights for the M peptides were 44,100, 43,000, and 42,000 for MPO I, II, and III, respectively. The treatment of neutrophils, granules, and extracts with protease inhibitors and sodium azide did not block the appearance of three major forms of MPO. Thus, neither protease activity nor MPO autooxidation during extraction and purification procedures is responsible for the appearance of multiple chromatographic forms of MPO derived from human neutrophils.     

Purification and characterization of multiple S6 phosphatases from the rat parotid gland

S6 phosphatase activities, which dephosphorylate the phosphorylated S6 synthetic peptide, RRLSSLRASTSKSESSQK, were purified to near homogeneity from the membrane and cytosolic fractions of the rat parotid gland. Multiple S6 phosphatases were fractionated on Mono Q and gel filtration columns. In the cytosolic fraction, at least three forms of S6 phosphatase, termed peaks I, II, and III, were differentially resolved. The three forms had different sizes and protein compositions. The peak I enzyme, which had an approximately Mr of 68 kDa on gel filtration, appears to represent a dimeric form of the 39 kDa protein. This S6 phosphatase showed the high activity in the presence of EGTA and was completely inhibited by nanomolar concentrations of either okadaic acid or inhibitor 2. The peak II S6 phosphatase enzyme, with an Mr of 35 kDa, was activated by Mn²⁺. This form could be a proteolytic product of the catalytic subunit of type 1 phosphatase, due to its sensitivities to okadaic acid and inhibitor 2. The peak III enzyme, with an Mr of 55 kDa, is a Mn²⁺-dependent S6 phosphatase. This S6 phosphatase can be classified as a type 1 phosphatase, due to its sensitivity to okadaic acid, since the IC₅₀ of okadaic acid is 4 nM. However, the molecular mass of this S6 phosphatase differs from that of the type 1 catalytic subunit (37 kDa) and showed less sensitivity to inhibitor 2. On the other hand, the membrane fraction contained one form of the S6 phosphatases, termed peak V (Mr 34 and 28 kDa), which could be classified as a type 1 phosphatase. This S6 phosphatase activity was greatly stimulated by Mn²⁺.Abbreviations PP1-C catalytic subunit of type 1 protein phosphatase - SDS sodium dodecyl sulfate - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - Mops 4-morpholine propanesulfonic acid - EDTA ethylenediaminetetraacetate - EGTA [ethylenbis (oxyethylenenitrilo)]-tetra acetic acid     

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.     

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.     

Bovine liver formaldehyde dehydrogenase. Kinetic and molecular properties

The dimeric formaldehyde dehydrogenase from bovine liver has been resolved into three nearly homogeneous enzyme forms by the successive use of ion-exchange, affinity, and ampholine (chromatofocusing) chromatography. The different enzyme species were isolated in the approximate proportions 3:2:1, having pI values of 6.5, 6.2, and 6.0, respectively. The subunit molecular weights of the three forms are all similar (Mr congruent to 41,000), on the basis of sodium dodecyl sulfategel electrophoresis. The enzyme species appear to arise from covalent differences unrelated either to partial proteolysis during isolation or to differential sialization of homodimeric protein. Human liver contains a single major form and two minor forms of formaldehyde dehydrogenase having pI values very similar to those found for the bovine liver enzyme. The macroscopic kinetic constants (V, V/K) for the three forms of the dehydrogenase from bovine liver are all similar in magnitude, using NADH and S-hydroxymethylglutathione as substrates. The isotope-sensitive hydride transfer step is not significantly rate-limiting during catalysis by any of the forms, as evidenced by the near-unity primary deuterium isotope effects on both V and V/KS (for S-hydroxymethylglutathione); catalysis may be limited by the rate of dissociation of at least one (and possibly both) of the product molecules. In support of rate-limiting dissociation of NAD+ in the normal reaction, V increases by approximately 22-fold and isotope effects of approximately 1.4 are observed on both V and V/KS, using the coenzyme analog 3-acetylpyridine adenine dinucleotide. Product dissociation from the active site appears to be accelerated by the presence of dilute denaturing agents, perhaps indicative of a rate-limiting conformational transition associated with product release.     

Purification and characterization of human salivary carbonic anhydrase

A novel carbonic anhydrase was purified from human saliva with inhibitor affinity chromatography followed by ion-exchange chromatography. The molecular weight was determined to be 42,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the human salivary enzyme is larger than the cytosolic isoenzymes CA I, CA II, and CA III (Mr 29,000) from human tissue sources. Each molecule of the salivary enzyme had two N-linked oligosaccharide chains which were cleaved by endo-beta-N-acetylglucosaminidase F but not by endo-beta-N-acetylglucosaminidase H, indicating that the oligosaccharides are complex type. The isoelectric point was determined to be 6.4, but significant charge heterogeneity was found in different preparations. The human salivary isozyme has lower specific activity than the rat salivary isozyme and the human red blood cell isozyme II in the CO2 hydratase reaction. The inhibitory properties of the salivary isozyme resemble those of CA II with iodide, sulfanilamide, and bromopyruvic acid, but the salivary enzyme is less sensitive to acetazolamide and methazolamide than CA II. Antiserum raised in a rabbit against the salivary enzyme cross-reacted with CA II from human erythrocytes, indicating that human salivary carbonic anhydrase and CA II must share at least one antigenic site. CA I and CA III did not crossreact with this antiserum. The amount of salivary carbonic anhydrase in the saliva of the CA II-deficient patients was greatly reduced, indicating that the CA II deficiency mutation directly or indirectly affects the expression of the salivary carbonic anhydrase isozyme. From these results we conclude that the salivary carbonic anhydrase is immunologically and genetically related to CA II, but that it is a novel and distinct isozyme which we tentatively designate CA VI.     

Studies on the subunits of human myeloperoxidase.

The subunit composition of human myeloperoxidase was studied with the use of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration. The subunit pattern observed depended on the manner in which the enzyme was treated before analysis. Reduction before heat treatment in detergent led to two main protein species (Mr 57 000 and 10 500), whereas reduction during or after heat treatment yielded an additional species of Mr 39 000. Heating without any reductive pretreatment yielded the 39 000-Mr form as the major electrophoretic species. Carbohydrate staining showed large amounts of sugar on the 57 000-Mr species and little on the 10 500-Mr form. Significant amounts of haem were associated with this latter subunit. Haem also seemed to be associated with the 57 000-Mr form but not with the 39 000-Mr one. These three subunit forms were isolated and their amino acid composition analysed. The 57 000-Mr and 39 000-Mr forms had very similar amino acid composition and yielded an apparently identical collection of fragments on incubation with CNBr. Once separated, the subunits could not be interconverted. Generally, minor amounts of other molecular-mass forms were observed. The nature of the various molecular-mass forms originating from myeloperoxidase is discussed.     

Isolation and characterization of two 3-phosphatases that hydrolyze both phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate.

Inositol-polyphosphate 3-phosphatase catalyzes the hydrolysis of the 3-position phosphate bond of inositol 1,3-bisphosphate (Ins(1,3)P2) to form inositol 1-monophosphate and inorganic phosphate (Bansal, V.S., Inhorn, R.C., and Majerus, P.W. (1987) J. Biol. Chem. 262, 9444-9447). Phosphatidylinositol 3-phosphatase catalyzes the analogous reaction utilizing phosphatidylinositol 3-phosphate (PtdIns(3)P) as substrate to form phosphatidylinositol and inorganic phosphate (Lips, D.L., and Majerus, P.W. (1989) J. Biol. Chem. 264, 19911-19915). We now demonstrate that these enzyme activities are identical. Two forms of the enzyme, designated Type I and II 3-phosphatases, were isolated from rat brain. The Type I 3-phosphatase consisted of a protein doublet that migrated at a relative Mr of 65,000 upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The Mr of this isoform upon size-exclusion chromatography was 110,000, suggesting that the native enzyme is a dimer. The Type II enzyme consisted of equal amounts of an Mr = 65,000 doublet and an Mr = 78,000 band upon SDS-polyacrylamide gel electrophoresis. This isoform displayed an Mr upon size-exclusion chromatography of 147,000, indicating that it is a heterodimer. The Type II 3-phosphatase catalyzed the hydrolysis of Ins(1,3)P2 with a catalytic efficiency of one-nineteenth of that measured for the Type I enzyme, whereas PtdIns(3)P was hydrolyzed by the Type II 3-phosphatase at three times the rate measured for the Type I 3-phosphatase. The Mr = 65,000 subunits of the two forms of 3-phosphatase appear to be the same based on co-migration on SDS-polyacrylamide gels and peptide maps generated with Staphylococcus aureus protease V8 and trypsin. The peptide map of the Mr = 78,000 subunit was different from that of the Mr = 65,000 subunits. Thus, we propose that the differing relative specificities of the Type I and II 3-phosphatases for Ins(1,3)P2 and PtdIns(3)P are due to the presence of the Mr = 78,000 subunit of the Type II enzyme.     

Multiple forms of DNA-dependent RNA polymerases in Xenopus laevis. Rapid purification and structural and immunological properties

DNA-dependent RNA polymerases I, II, and III (EC 2.7.7.6) were isolated from Xenopus laevis ovaries. The soluble enzymes were precipitated with polyethyleneimine and subjected to chromatography on heparin-Sepharose, DEAE-Sephadex, and phosphocellulose. RNA polymerase I was subjected to an additional chromatographic step on CM-Sephadex. The procedure required 40 h and produced purified RNA polymerase forms IA, IIA, and III in yields of 5 to 40%. The specific activities of RNA polymerases IIA and III (on native DNA) were comparable to those reported from other eukaryotic sources, whereas that of form IA was severalfold greater than the specific activities reported for other purified class I RNA polymerases. The complex subunit compositions of chromatographically purified RNA polymerases IA, IIA, and III were distinct when analyzed by polyacrylamide gradient gel electrophoresis under denaturing conditions, although all three classes contained polypeptides with Mr = 29,000, 23,000, and 19,000. Antibodies prepared against RNA polymerase III showed common antigenic determinants within the class I, II, and III enzymes. The sites responsible for the cross-reaction are located, at least in part, on the common 29,000-dalton polypeptide.     

Purification and characterization of aspartate aminotransferase isoenzymes from carrot suspension cultures

Three aspartate aminotransferase isoenzymes were identified from extracts of carrot (Daucus carota L.) cell suspension cultures. These isoenzymes were separated by DEAE chromatography and were analyzed on native gradient polyacrylamide gels. The relative molecular weights of the isoenzymes were 111,000 ± 5000, 105,000 ± 5000, and 94,000 ± 4000 daltons; they were designated forms I, II, and III, respectively. Form I, the predominant form, has been purified to apparent homogeneity (>300-fold) using immunoaffinity chromatography with rabbit anti-pig AAT antibodies. Form I has a subunit size of 43,000 M_r, as determined on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Isoelectric focusing (IEF)-PAGE has resolved three bands at a pl of approximately 5.2. Form I may be composed of subunits of similar molecular weight and different charges, and the three bands with AAT activity on the IEF-PAGE gel are a combination of hetero- and homodimers. Form I has a broad pH optimum of 7.5 to 10.0. K_m values of 23.6, 2.8, 0.05, and 0.22 millimolar were obtained for glutamate, aspartate, oxaloacetate, and α-ketoglutarate, respectively. The mode of action is a ping-pong-bi-bi mechanism.     

Studies on Rice Glutelin

In order to obtain information of the gross-structure of glutelin, chemical and physicochemical properties of S-cyanoethyl glutelin were investigated. Glutelin remained at the origin in polyacrylamide gel electrophoresis, while S-cyanoethyl glutelin migrated in the gel and resolved into two components. The ion-exchange chromatography by carboxymethyl Sephadex C-50 gave further resolution of S-cyanoethyl glutelin into one neutral component corresponding to the anodic component and two basic components corresponding to the cathodic component in polyacrylamide gel electrophoresis at neutral pH. The amino-terminal residue of the neutral component (Component I) could not be detected by the fluorodinitrobenzene method, while both the basic components (Component II and III) had only glycine as the amino-termini. On the basis of dinitrophenyl-glycine found, the minimum molecular weights of Component II and III were calculated at about 35,000 and 43,000 respectively. The relative concentration of these three components was as follows; Component I: Component II: Component III=8:1:1. These facts obviously indicate that glutelin is a very large molecule composed from these three components polymerized by disulfide linkage, Component I being the major subunit.     

Purification and characterization of two porcine liver nuclear histone acetyltransferases

Two forms of porcine histone acetyltransferase (types I and II) have been purified to apparent homogeneity from liver nuclei. Both activities are extracted from nuclei by 0.5 M NaCl and display a native Mr of 110,000 as determined by gel filtration. Saline enzyme extracts were subject to ammonium sulfate precipitation and sequential chromatography on Q-Sepharose, Sephacryl S-200, hydroxylapatite, and Mono Q supports. The histone acetyltransferase type I fraction contains three polypeptide chains with apparent Mr values of 105,000, 62,000, and 45,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyanogen bromide peptide mapping and immunoblotting suggest that the Mr 62,000 and 45,000 polypeptides are derived by cleavage of the Mr 105,000 polypeptide. Histone acetyltransferase type II contains two different subunits with apparent Mr values of 50,000 and 40,000, respectively. The amino acid composition, heat inactivation profiles, and Michaelis constants with respect to both acetyl coenzyme A and histones were indistinguishable for types I and II. However, affinity-purified polyclonal antibodies to both forms of the enzyme do not cross-react; cyanogen bromide-derived in situ cleavage digest patterns show few similarities; and the turnover number for type I is approximately 15-fold lower than that for type II. We estimate that there is one enzyme molecule for every 500 nucleosomes. The existence of two distinct forms of nuclear histone acetyltransferase in pig liver suggests that they may have separate functions in vivo.