Catalase activity in developing seedling of opium poppyPapaver somniferum L

Catalase is an enzyme unique to glyoxysomes in developing poppy seedlings. Catalase activity is very low in endosperm and in embryo of germinating poppy seeds. During postgerminative growth and development the enzyme activity increases rapidly with maximum in endosperm on day 2 and in developing seedling on day 3. A rapid decline of enzyme activity parallells the extension growth of poppy seedlings. Three electrophoretic forms of catalase have been detected in isolated glyoxysomes and partially purified catalase preparation. Electron microscopic observation indicates the presence of catalase in glyoxysomes of parenchyma cells of poppy seedling cotyledons. Numerous lipid bodies and electron-dense deposits in vacuoles are the most characteristic feature of these cells.     

Heterogeneity of catalase in maturing and germinated cotton seeds

To investigate possible charge and size heterogeneity of catalase (EC 1.11.1.6) in cotton (Gossypium hirsutum L. cv Deltapine 62), extracts of cotyledons from different developmental ages were subjected to nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Special precautions (e.g. fresh homogenates, reducing media) were necessary to prevent artefacts due to enzyme modification during extraction and storage. When the gels were stained for enzyme activity, two distinct electrophoretic forms of catalase were resolved in extracts of maturing and mature cotton seeds. In germinated seeds, three additional cathodic forms were detected revealing a total of five electrophoretic variants. In green cotyledons, the two anodic forms characteristic of ungerminated seeds were less active; whereas, the most cathodic form was predominant. All forms of catalase were found in isolated glyoxysomes. Corresponding electrophoretic patterns were found on Western blots probed with anticatalase serum; no immunoreactive, catalytically inactive forms were detected. Western blots of sodium dodecyl sulfate-polyacrylamide gels revealed only one immunoreactive (55 kilodaltons) polypeptide in cotton extracts of all developmental ages. Results from isoelectric focusing and Ferguson plots indicate that the electrophoretic variants of catalase are charge isomers with a molecular weight of approximately 230,000.     

Isozyme differences between the developmental stages of the parthenogenetic gall wasp Diplolepis rosae

The value of electrophoretic analysis of enzymes as an aid in connecting a morphologically deviating form to one of several possible species is dependent on the proportion of shared enzyme loci between the forms. In order to determine this proportion for the different instars of the parthenogenetic gall wasp Diplolepis rosae, 16 different enzyme systems were analysed. Out of the 37 loci detected, 25 (68%) were active in all instars. The larvae, pupae, and imagines had two, one and five unique loci, respectively. Larvae and pupae shared four loci not expressed in the imagines. In the ten enzymes analysed for comparison of males and females a total of 22 loci were detected, 21 found in both sexes and one unique to the females. The difference in isozyme pattern was found to be much more pronounced between pupa and imago than between larva and pupa. The chance to find a locus in an instar when already detected in another was calculated to 87%. If this estimate is valid for other forms within species, differential activity should not present a problem when electrophoretic analysis is used in order to connect such forms.     

Erythrocyte glutathione S-transferase. Electrophoretic identification of two enzyme forms.

Starch-gel electrophoresis was used to demonstrate two forms of glutathione S-transferase in human erythrocytes. Whereas considerable inter-individual differences in enzyme activity and electrophoretic patterns were detected, intra-individual differences were small.     

Bacillopeptidase F: two forms of a glycoprotein serine protease from Bacillus subtilis 168

Bacillopeptidase F is a serine endopeptidase excreted by Bacillus subtilis 168 after the end of exponential growth. As a step toward discovering a physiological function for this protease, an enzymological and immunological study was undertaken. When bacillopeptidase F was purified at pH 10, a number of enzymically active, rapidly moving electrophoretic forms were observed, as had been previously reported. Rabbit antiserum was prepared against one form. When the enzyme was purified at pH 6.0 in the presence of the covalent inhibitor phenylmethylsulfonyl fluoride, using the rabbit antiserum to detect the bacillopeptidase F protein, no fast-moving electrophoretic forms were observed. Instead, only two forms of the enzyme were isolated. One form had a molecular weight of 33,000, and the other had a molecular weight of 50,000, as determined by equilibrium sedimentation methods. Both forms appeared to be glycoproteins, both contained compounds, released on acid hydrolysis, which cochromatographed with phosphoserine and galactosamine, and the two gave identical immunoprecipitin lines in Ouchterlony double-diffusion tests. The smaller form had a pI of 4.4, whereas the larger had a pI of 5.4. The data suggest that bacillopeptidase F is distinct from all other proteases of B. subtilis.     

Substrate-containing gel electrophoresis: sensitive detection of amylolytic, nucleolytic, and proteolytic enzymes

Electrophoresis of hydrolytic enzymes under nondenaturing conditions on acrylamide gels containing the appropriate high-molecular-weight substrates entrapped on the gel has been explored as a general method for sensitive enzyme resolution and detection. Under electrophoresis conditions of optimal enzyme activity, the enzymes may bind tightly to the fixed substrate and can only migrate in the electrophoretic field as the substrate is hydrolyzed. When the gels after electrophoresis in this “binding mode” are stained with substrate-detecting reagents, clear tracks of enzyme migration are observed, and the length of each track is a function of the amount of enzyme present in that track. Multiple forms of a given enzyme activity have not been and are not likely to be observed under these conditions. Under electrophoresis conditions of minimal (or suboptimal) enzyme activity, the enzymes do not bind to the fixed substrate and their mobility in the electrophoretic field does not appear to be significantly affected by the presence of substrate. After electrophoresis in this “nonbinding mode” the gels are incubated under conditions of optimal enzyme activity to allow substrate hydrolysis to take place before they are stained with substrate-detecting reagents, and active enzymes are detected as clear bands. Multiple forms of a given activity which were resolved during electrophoresis in the nonbinding mode are reflected by the presence of individual bands. The substrate-containing gel electrophoresis technique does not appear to be amenable to precise quantification of enzymes. By comparing the length of the clear tracks or the degree of staining of the activity bands for a range of enzyme concentrations, however, it is possible to establish the smallest amount of enzyme that can unequivocally be detected under a given set of conditions; from such studies we estimate that the sensitivity of detection with the substrate-containing gel electrophoresis technique can be orders of magnitude better than that obtained with other methods. The levels of detection observed in the work presented here were about 50 pg for α-amylase run on starch-containing gels, 1 pg to 1 ng for nucleases run on DNA- or RNA-containing gels, and 100 pg to 10 ng for 11 different pure and crude protease preparations run on gels containing heat-denatured bovine serum albumin.     

The presence of two forms of the phosphocarrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system in streptococci.

The protein, HPr, a necessary component of the phosphoenolpyruvate phosphotransferase system (PTS) in bacteria, was purified from Streptococcus salivarius by column chromatography. The purified preparation gave only one band when analyzed by sodium dodecylsulfate gel electrophoresis or by isoelectric focusing in polyacrylamide gel (pI = 4.85). However, electrophoresis in Tris-containing buffers under non-denaturing conditions revealed 2 bands that could be phosphorylated by PEP in the presence of enzyme I of the PTS or by ATP with the HPr kinase. Homogeneous preparations of these 2 forms could be obtained by preparative electrophoresis. Each preparation exhibited only 1 band when analyzed by electrophoresis under non-denaturing conditions, indicating that the doublet observed before preparative electrophoresis was not an electrophoretic artefact. The electrophoretic mobility of each protein was not modified following heat-treatment at 100 degrees C for 20 min or storage at -40 degrees C for several months. Both HPr proteins catalyzed in vitro the PEP-dependent phosphorylation of glucose, but at a rate slightly lower than that observed with a preparation of HPr containing both forms of the protein. Both forms were also able to transfer the phosphate group from PEP to the other specific PTS proteins known in S salivarius. Rabbit polyclonal antibodies directed against each form reacted with both proteins. The presence of the 2 forms of HPr was detected in fresh cellular extracts of S salivarius; however, their intracellular ratio varied according to growth conditions. A doublet was also found in many other streptococcal species tested (S mutans, S sobrinus, S sanguis, S thermophilus, S bovis, S rattus) and also in L lactis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on enzyme variation in the murine malaria parasites Plasmodium berghei, P. yoelii, P. vinckei and P. chabaudi by starch gel electrophoresis.

Electrophoretic variation of the enzymes glucose phosphate isomerase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase and glutamate dehydrogenase (NADP-dependent) has been studied in the African murine malaria parasites Plasmodium berghei, P. yoelii, P. vinckei and P. chabaudi and their subspecies. Horizontal starch gel electrophoresis was used throughout. The number of isolates examined in each subspecies varied from 1 (P. y. nigeriensis) to 24 (P. c. chabaudi). Extensive enzyme variation was found among isolates of most of the subspecies from which more than two such isolates were available for study. It is clear that the phenomenon of enzyme polymorphism is of common occurrence among malaria parasites. With the exception of P. berghei and P. yoelii, of which all isolates share an identical electrophoretic form of lactate dehydrogenase, no enzyme forms are shared between any of the 4 species of murine plasmodia. By contrast, within each species common enzyme forms are shared among each of the subspecies. The subspecies are nevertheless, distinguished from each other by the electrophoretic forms of at least one enzyme. The distribution and reassortment of enzyme variation among isolates of a single subspecies is in accordance with the concept of malaria parasites as sexually reproducing organisms. The study of variation among parasites present in individual wild-caught rodent hosts demonstrates that natural malarial infections usually comprise genetically heterogeneous populations of parasites. Nevertheless, the number of genetically distinct types of parasite of any one species present in a single infected host appears to be small. Generally not more than 2 or 3 clones of parasite of distinct genetic constitution are present in a single infected animal.     

Multiple forms of glutamine synthetase in Bacillus brevis AG 4

Glutamine synthetase in Bacillus brevis AG 4, a Gram-positive spore forming bacteria, has been found to exist in multiple molecular forms. It was purified to electrophoretic homogeneity by single-step Blue Sepharose affinity chromatography. The native enzyme has a molecular weight of 600,000 with subunits of 50,000. The enzyme samples purified from different stages of growth differed in Mg2+ sensitivity and other kinetic properties. Four different enzyme samples selected on the basis of Mg2+ sensitivity showed distinct mobilities at pH 6.3 on PAGE using discontinuous buffer system. A correlation amongst Mg2+ sensitivity, electrophoretic mobility, and kinetic properties was highly suggestive of multiple forms of glutamine synthetase in Bacillus brevis arising due to modification.     

Electrophoretic and chromatographic separation of two fructose-1,6-bisphosphatase forms from Synechococcus leopoliensix

d-Fructose-1,6-bisphosphatase (EC 3.1.3.11) activity in crude extracts of the blue-green alga Synechococcus leopoliensis (Anacystis nidulans) has been investigated using high resolving electrophoretic and chromatographic separation techniques. Two catalytically active enzyme forms which exhibited isoelectric points of 4.7–4.8 (designated from A) and 4.5–4.6 (designated form B) were resolved by isoelectric focusing. Both enzyme forms acted specifically on fluctose-1,6-bisphosphate. No interconversion between the A and B forms of fructose bisphosphatase activity was detected after refocusing. The apparent molecular weight of the two enzyme forms was determined by non denaturing polyacrylamide gradient electrophoresis; the values were 67,000–70,000 and 60,000–65,000 for A and B, respectively. Both enzyme forms were separated by preparative scale chromatofocusing. Kinetic measurements performed with the separated and partially purified fructose bisphosphatase forms indicated that both enzyme forms differ in their AMP sensitivity. The two enzymes were completely inactivated by the addition of cysteamine and reactivated by dithiols but the reactivation kinetics were different.Abbreviations DTT dl-Dithiothreithol - MTT 3(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide - PMS phenazine methosulfate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)-aminomethane     

Isolation and some properties of potato 0-diphenol oxidase]

Potato tuber o-diphenoloxidase is found to exist in heterogenous mixture of forms differing from each other in their molecular weight, electrophoretic mobility, substrate specificity. A method is proposed which permits to isolate several molecular forms of the enzyme in a relatively homogenous state. Conditions are studied, in which interconversion of o-diphenoloxidase molecular forms takes place.     

ACETYLCHOLINESTERASE OF RAT SYMPATHETIC GANGLION: MOLECULAR FORMS, LOCALIZATION AND EFFECTS OF DENERVATION

Abstract— In sucrose gradient centrifugation, acetylcholinesterase (AChE, EC 3.1.1.7.) from the rat superior cervical ganglion (SCG) has been found to contain four molecular forms, characterized by their sedimentation coefficients (4 S, 6.5 S, 10 S and 16 S). Homogenization of the ganglia in various media showed that the 4 S enzyme was readily solubilized in water whereas solubilization of the 6.5 S and 10 S forms was quantitative only in media containing Triton X-100. In order to solubilize the 16 S form, high concentrations of salt (NaCl 1 M) and detergent had to be present. AChE analysed by non-denaturing polyacrylamide gel electrophoresis separated into five bands. Although both distribution patterns were stable, i.e. each form or band preserved its characteristic sedimentation or electrophoretic migration when reanalysed, there was no 1:1 correlation between the forms isolated by sedimentation and the bands obtained by electrophoresis: one band might contain more than one form of enzyme, and conversely one form gave rise to several bands. It was therefore impossible to derive molecular weights from electrophoretic migration in non-denaturing gels. However, it could be shown that the results obtained by both methods of analysis were consistent. Acetylcholinesterase from other nervous structures was analysed: in pre- and postganglionic nerves, the main forms were 10 S and 6.5 S, with a small proportion of 4 S; the 16 S form was not detected. In other sympathetic ganglia, the distribution of forms was identical to that of the superior cervical ganglion. In rachidian ganglia, no 16 S form could be found. Following the section of the preganglionic nerve, the acetylcholinesterase activity of the superior cervical ganglion decreased by 50% in 3 days, and then rose again to about 80% of its original value after 2 weeks. These effects mainly reflected variations in the major 4 S and 10 S forms. The 16 S form, in contrast to its disappearance from denervated muscles, increased transiently during the first 2 weeks after denervation, reaching about twice its original activity. A concomitant cytochemical study of normal and denervated ganglia showed that after preganglionic denervation, AChE localized in the sympathetic neurones decreased markedly and remained low even during the recovery phase. During this period a cholinesterasic activity appeared in the perineuronal glia. Controls established that the enzyme synthetized in the glia is AChE.     

Comparison of the properties of two forms of pyruvate kinase in rat liver and determination of their separate activities during development

1. Two forms of hepatic pyruvate kinase, designated type L and type M, were distinguished on the basis of kinetic, chromatographic, electrophoretic and immunological criteria. They were partially purified and their properties compared with each other and with the purified enzyme from skeletal muscle. 2. In contrast with type L, the type M enzyme showed no marked evidence of co-operative interactions with phosphoenolpyruvate and was not stimulated by fructose diphosphate. 3. The activity profiles of type L and type M enzymes were determined in developing rat liver by utilizing differences in the kinetic properties of the two forms. The high activity of type M enzyme in the early foetal rat decreased in late gestation and immediately after birth to reach a low value, which remained essentially constant for the remainder of the developmental period. The activity of type L enzyme, in contrast, was low in the early foetal and neonatal liver but increased markedly at the onset of weaning. 4. Possible roles of the two forms of hepatic pyruvate kinase in the control of glycolysis and gluconeogenesis are discussed.     

Superoxide dismutase in strains of the genus Flavobacterium: isolation and characterization

Two electrophoretically different forms of superoxide dismutase, one of them containing manganese-protein and the other iron-protein, were detected in eleven different strains of the genus Flavobacterium. The activities of the different strains were similar to those described for other bacteria. The two molecular forms of the enzyme differed clearly with regard to activity, electrophoretic behaviour, sensitivity to cyanide and peroxide, and NaCl requirement. Both molecular forms were isolated from Flavobacterium halmephilum. Molecular mass absorption spectra, metal content, optimum pH, heat-sensitivity and stability were described.     

Two apparent molecular weight forms of human and monkey phenylalanine hydroxylase are due to phosphorylation

Two-dimensional polyacrylamide gel analyses of purified human and monkey liver phenylalanine hydroxylase reveal that the enzyme consists of two different apparent molecular weight forms of polypeptide, designated H (Mr = 50,000) and L (Mr = 49,000), each containing three isoelectric forms. The two apparent molecular weight forms, H and L, represent the phosphorylated and dephosphorylated forms of phenylalanine hydroxylase, respectively. After incubation of purified human and monkey liver enzyme with purified cAMP-dependent protein kinase and [gamma-32P]ATP, only the H forms contained 32P. Treatment with alkaline phosphatase converted the phenylalanine hydroxylase H forms to the L forms. The L forms but not the H forms could be phosphorylated on nitrocellulose paper after electrophoretic transfer from two-dimensional gels. Phosphorylation and dephosphorylation of human liver phenylalanine hydroxylase is not accompanied by significant changes in tetrahydrobiopterin-dependent enzyme activity. Peptide mapping and acid hydrolysis confirm that the apparent molecular weight heterogeneity (and charge shift to a more acidic pI) in human and monkey liver enzyme results from phosphorylation of a single serine residue. However, phosphorylation by the catalytic subunit of cAMP-dependent protein kinase does not account for the multiple charge heterogeneity of human and monkey liver phenylalanine hydroxylase.     

Isoenzymes and population genetics of sorbit dehydrogenase (EC: 1.1.1.14) in swine (sus scrofa)

Summary A method is described for detecting the enzyme sorbit dehydrogenase (SDH) after starchgel electrophoresis. Activity of this enzyme occurs predominantly in liver, kidney, thyroid and testis. It was found that this enzyme exists in multiple molecular forms. Three electrophoretic phenotypes were observed apparently representing two different homozygotes and the corresponding heterozygote. These findings can be interpreted in terms of two alleles at one SDH locus.     

Epidemiological typing of Acinetobacter strains by esterase electrophoresis

Fifty-three strains of Acinetobacter, belonging to the species A baumannii, A. haemolyticus and A. johnsonii, were differentiated by electrophoretic typing of their esterases, on the basis of both the enzyme specific activity profiles and their electrophoretic mobilities. Each esterase was defined by its spectrum of hydrolytic activity toward five synthetic substrates and its sensitivity to di-isopropyl fluorophosphate. Since each enzyme was not detected in all strains of a given species, several zymotypes could be defined by the patterns of combinations of esterases. Thus, 24 zymotypes were defined in the 32 A. baumannii strains, 4 were defined in the 10 A. haemolyticus strains and 6 were defined in the 11 A. johnsonii strains. When the electrophoretic mobilities of the various esterases were included, each of the 53 strains of Acinetobacter (with the exception of three A. haemolyticus strains) showed a distinct electrotype.     

Some properties of liver alkaline phosphatase from the goat Capra hircus.

1. Gel-filtration of an extract from the liver of the local Hausa goat Capra hircus indicated the presence of two molecular forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase, E.C. 3.1.3.1.). 2. Cellulose acetate electrophoresis showed that the lower-molecular-weight form had a similar electrophoretic mobility to alpha 2-globulin from goat serum, whereas the higher-molecular-weight form had a similar electrophoretic mobility to gamma-globulin. 3. Only the lower-molecular-weight form was detected on electrophoresis of a liver extract which contained some residual n-butanol used in the extraction procedure, whereas dialysed acetone powder obtained from the liver extract contained both molecular-weight forms. 4. The partially purified enzyme showed maximum activity at pH 9.8, and was stimulated by Mg2+. 5. The enzyme was heat-labile, and was competitively inhibited by phosphate ions but uncompetitively inhibited by L-phenylalanine. 6. These results are discussed in terms of the properties of the enzyme from other sources.     

A new genetic variant of galactose-1-phosphate uridyl transferase

Summary The enzyme galactose-1-phosphate uridyl transferase (E.C. 2.7.7.12), which has an important function in the metabolism of galactose, exists in multiple molecular forms. The different phenotypes are genetically determined. They can be distinguished according to their electrophoretic mobility. The enzymatic activity of the different gene products varies within certain limits. A new phenotype of the enzyme has been detected in the red cells of a healthy individual. The electrophoretic migration of this phenotype is slower compared to the wild type and its enzymatic activity is lower, but still sufficient as not to cause galactosemia. An extensive family study revealed that the rare gene is inherited according to mendelian law. Independently the same gene product has been detected in two other, nonrelated individuals out of a total of 1668 samples tested. The gene frequency can therefore be estimated to 0.0009 in the Swiss population. We suggest that the new type be called Berne variant of galactose-1-phosphate uridyl transferase.     

Enzyme Typing of Malaria Parasites

Analysis of various isolates of Plasmodium falciparum from East and West Africa and from South-east Asia showed that some of the parasite enzymes can exist in more than one electrophoretic form. At least one form of each enzyme was common to parasites from all three regions. The enzyme forms could be used to differentiate morphologically indistinguishable samples of P. falciparum.