Enolase isozymes from Ricinus communis: Partial purification and characterization of the isozymes

The plastid and cytosolic isozymes of enolase from developing endosperm of castor oil seeds, Ricinus communis L. cv. Baker 296, were separated and partially purified. Each purified isozyme had a specific activity of approximately 200 μmol min^?1 mg protein. The isozymes have similar pH optima for the forward reaction, but different optima for the reverse reaction. The divalent metal specificity is the same for both isozymes. In addition to differences in charge, the isozymes can be distinguished by their different kinetic constants, thermostability and sensitivity to fluoride inhibition. Antibodies against yeast enolase isozyme I cross-react with Ricinus plastid enolase but not with the cytosolic isozyme.     

Cloning of alkaline phosphatase isozyme gene (iap) of Escherichia coli

In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions. A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits. A chromosomal fragment which complemented the Iap^? phenotype was cloned into pBR322 by a shotgun method. Transducing phage λiap was constructed in vitro from the chromosomal fragment containing the iap gene and λtna DNA. The integration site of the phage on chromosome was identified as the iap locus by PI transduction, which meant that the cloned chromosomal DNA contained authentic iap gene.The restriction map of the hybrid plasmid was constructed. Based upon this information, several iap deletion plasmids as well as smaller iup⁺ plasmids were constructed. Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA.The cells carrying the iap⁺ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion. This indicates overproduction of the iap gene product.     

Identification of a selective inhibitor of murine intestinal alkaline phosphatase (ML260) by concurrent ultra-high throughput screening against human and mouse isozymes

Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In humans, four AP isozymes have been identified—one tissue-nonspecific (TNAP) and three tissue-specific—named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endotoxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with specificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.     

Immunochemical characterization of human red cell acid phosphatase isozymes

An immunological study was performed on human red cell acid phosphatase (ACP1) isozymes encoded by different alleles, each of which is expressed as an electrophoretically fast (f) isozyme and a slow (s) isozyme. These isozymes reacted as two immunochemically different groups. Allele-specific reactions were not detected between either the f isozymes or the s isozymes. Quantitation of ACP1 isozymes in red cells by crossed immunoelectrophoresis revealed a phenotype-dependent variation in the concentration of isozyme protein. A simple gene dosage effect was indicated and the ordering of the ACP1 alleles (ACP1*A < ACP1*B < ACP1*C < ACP1*E) was identical to that found for enzyme activity levels. Also, an allele effect on the proportion between s and f isozymes (s/f) was observed; the ordering here was ACP1* B < ACP1*A < ACP1*, which is the same as that reported for the susceptibility to modulation with purines. These variations in isozyme protein levels appear to account for the phenotypic differences in the intensity of the isozyme bands, when activity-stained after electrophoresis, and in the red cell enzyme activity levels. Investigation of two carriers of a Null allele showed no evidence of an aberrant protein product, and half-normal concentrations of enzyme protein were observed in the red cells of these individuals.     

Characterization of two different alkaline phosphatases in mouse teratoma: Partial purification,electrophoretic, and histochemical studies

Alkaline phosphatase (E.C.3.1.3.1.) has been used as a marker for embryonal carcinoma cells which constitute the multipotential stem cells of the mouse teratoma. Studies by other investigators based on kinetics of thermal inactivation and L-phenylalanine inhibition have shown that the alkaline phosphatase of the teratoma differs from the mouse intestinal and liver isozymes, but resembles the isozymes of kidney and placenta. Since functional characterization of nonpurified enzymes is not the most accurate means for distinguishing different molecular forms of an enzyme, we have partially purified the enzymes from the ascitic (embryoid body) and solid tumor forms of the OTT-6050 teratoma line, and utilized the technique of electrophoresis in polyacrylamide gels to compare the teratoma enzyme with isozymes from kidney and placenta. Covalent ³²PO₄-labeling of the alkaline phosphatases and polyacrylamide gel electrophoresis in sodium dodecylsulfate was also used to compare the subunit molecular weights of the enzymes. The results indicate that the mouse teratoma enzyme is distinct from the kidney and placental isozymes. Since histochemical studies have localized the enzyme to the stem cell population of the teratoma, the results imply that stem cell alkaline phosphatase is a distinct isozyme. The embryoid bodies contain a second alkaline phosphatase which may correspond to the placental isozyme. This enzyme may be attributed to the outer cell layer of embryoid bodies of the ascitic tumor, since this cell type histochemically demonstrates alkaline phosphatase activity.     

Further studies on the genetic control of chicken plasma alkaline phosphatase isozyme

The effects of neuraminidase treatment on the electrophoretic pattern of alkaline phosphatase (AP) isozymes and AP activity were investigated in chicken plasma. AP comprised three isozymes. The zymogram of an individual chicken plasma had two bands, either the faster (F) or the slower (S) moving band by isozyme types and the B band irrespective of isozyme types. Mobility of the S band and AP activity in chicken plasma were not affected by neuraminidase treatment. The treatment has a reduced migration rate of the F band equal to that of the S band and the B band of both types closer to the origin. The genetic control of these bands is discussed.     

Further studies on the genetic control of chicken plasma alkaline phosphatase isozyme.

The effects of neuraminidase treatment on the electrophoretic pattern of alkaline phosphatase (AP) isozymes and AP activity were investigated in chicken plasma. AP comprised three isozymes. The zymogram of an individual chicken plasma had two bands, either the faster (F) or the slower (S) moving band by isozyme types and the B band irrespective of isozyme types. Mobility of the S band and AP activity in chicken plasma were not affected by neuraminidase treatment. The treatment has a reduced migration rate of the F band equal to that of the S band and the B band of both types closer to the origin. The genetic control of these bands is discussed.     

Thermostability of acid phosphatase inSelinum vaginatum Clarke andAcer caesium Wall. grown at low and high altitudes

Acid phosphatase isolated from low altitude grown plants of two high altitude plant species,Selinum vaginatum Clarke andAcer caesium Wall, displayed higher thermostability than that from plants of the same species grown at high altitude. The isozyme composition, however, remained unchanged inSelinum vaginatum. InA. caesium, one of four isozymes, was thermolabile in the samples from high altitude and was lost after 10 min heating of the extracts at 60 °c. In the samples from low altitude, this isozyme was not detected and a band with slightly lower Rf value was present which was thermostable. The described changes in the thermal properties of acid phosphatase reflect an adaptive step towards high temperature acclimation at low altitude.     

Genetic control of alkaline phosphatase isozymes in chicken duodenum

A genetic control of alkaline phosphatase (AP) in chicken duodenum was studied in a White Plymouth Rock strain. Unpurified chicken duodenum AP heated in an extraction procedure comprised either F or S band by isozyme types. On the other hand, chromatographically purified intestinal AP (NBCo, USA) had three bands, i.e., F', S' and B' bands. Characterization by urea, heat and neuraminidase treatments suggested that the genetic control of plasma AP isozymes may be applicable to the duodenum AP isozymes.     

Biosynthesis of two developmentally distinct acid phosphatase isozymes in Dictyostelium discoideum

The presence of a common antigenic determinant on the Dictyostelium discoideum acid phosphatase isozyme 1 (ap 1), and the absence of this determinant on the isozyme ap2 enables separation of the two isozymes. This separation is accomplished by removal of ap1 from samples with a common antigen monoclonal antibody followed by immunoprecipitation of ap2 with an acid phosphatase monoclonal antibody. Application of this separation scheme on cells pulse-labeled early (2 h) and late (18 h) in the developmental cycle reveal that ap1 protein synthesis occurs only early in development and that the protein remains stable throughout development, whereas ap2 protein synthesis occurs only late in development. Furthermore, pulse-chase experiments during both early and late development reveal that both isozymes of acid phosphatase are initially synthesized as precursor molecules (Mr = 60,000) which are then processed to mature forms (Mr = 58,000). The processing event(s) for acid phosphatase begin in less than 5 min compared to 25-30 min for Dictyostelium alpha-mannosidase and 10-15 min for Dictyostelium beta-glucosidase. Endoglycosidase H and Endoglycosidase F treatment of both isozymes reveals identical cleavage patterns for ap1 and ap2, indicating that the amount of carbohydrate on both molecules is equivalent. Preliminary studies to identify modification differences reveal that fucose is not present on either isozyme; however, sulfate is present on the ap1 isozyme and absent on the ap2 isozyme. These results suggest that differences in the modification of newly synthesized acid phosphatase at different times during the Dictyostelium life cycle result in the appearance of two distinct acid phosphatase isozymes.     

Origin of the triosephosphate isomerase isozymes in humans: genetic evidence for the expression of a single structural locus.

A genetic approach is used to ascertain that a single structural locus for triosephosphate isomerase (TPI) (E.C.5.3.1.1.) is expressed in rapidly dividing human lymphoblasts. This approach is made possible through the identification of a rare electrophoretic variant of human TPI. The variant phenotype is expressed by the TPI-B isozyme in both erythrocytes and peripheral lymphocytes. The variant phenotype is also expressed in the thermostability and electrophoretic pattern of the TPI-A isozyme in mitogen-stimulated lymphoblasts, indicating that TPI-A and TPI-B are products of the same structural locus. These findings are in contrast to the recent conclusions of Yuan et al. based upon structural analysis, suggesting that the TPI-A and TPI-B isozymes are products of distinct structural loci.     

Escherichia coli mutants deficient in the production of alkaline phosphatase isozymes.

Escherichia coli K-12 mutants showing an altered isozyme pattern of alkaline phosphatase were isolated. Whereas wild-type strains synthesized all three isozymes in a synthetic medium supplemented with Casamino Acids or arginine but synthesized only isozyme 3 in a medium without supplement, the mutant strains synthesized isozyme 1 and a small amount (if any) of isozyme 2, but no isozyme 3, under all growth conditions. The mutation responsible for the altered isozyme pattern, designated iap, was mapped by P1 transduction in the interval between cysC and srl (at about 58.5 min on the E. coli genetic map). It was cotransducible with cysC and srl at frequencies of 0.54 and 0.08, respectively. The order of the genes in this region was srl-iap-cysC-argA-thyA-lysA. Three more independent mutations were also mapped in the same locus. We purified isozymes 1' and 3' from iap and iap+ strains and analyzed the sequences of four amino acids from the amino terminus of each polypeptide. They were Arg-Thr-Pro-Glu (or Gln) in isozyme 1' and Thr-Pro-Glu (or gln)-Met in isozyme 3', which were identical with those of corresponding isozymes produced by the wild-type phoA+ strain (P.M. Kelley, P.A. Neumann, K. Schriefer, F. Cancedda, M.J. Schlesinger, and R.A. Bradshaw, Biochemistry 12:3499-3503, 1973; M.J. Schlesinger, W. Bloch, and P.M. Kelley, p. 333-342, in Isozymes, Academic Press Inc., 1975). These results indicate that the different mobilities of isozymes 1, 2, and 3 are determined by the presence or absence of amino-terminal arginine residues in polypeptides.     

Patterns of gene expression during teleost embryogenesis: Lactate dehydrogenase isozyme ontogeny in the medaka (Oryzias iatipes)

The ontogeny of the lactate dehydrogenase (LDH; EC 1.1.1.27) isozymes during medaka (Oryzias latipes) embryogenesis was determined after the genetic and molecular bases of this multilocus isozyme system were established. Three LDH loci are differentially expressed among the tissues of the adult medaka. The LDH-A locus was expressed almost exclusively in the white skeletal muscle, the LDH-B locus in all tissues examined, and the LDH-C locus in the eye and brain. The contribution of each of these LDH loci was quantitatively determined throughout early medaka embryogenesis by using a combination of electrophoretic, immunochemical, and spectrophotometric procedures. LDH-B₄ is present throughout embryogenesis and is the predominant LDH isozyme during this period. LDH-C subunit activity was first detected 146 hr after fertilization (26°C), 142 hr prior to hatching. LDH-A subunit activity, however, was not detected until after hatching and, then, only as heterotetramers containing LDH-B subunits. The pattern of LDH gene expression during medaka embryogenesis was compared with the patterns of LDH gene expression during early development in five other teleost species. Some common patterns of differential LDH gene expression appear to exist among the teleosts. In all species examined, isozymes encoded in at least one LDH locus, A and/or B, were present throughout development. Those isozymes present continually during embryogenesis also tend to be active in a wide variety of differentiated tissues in the adult fish. Conversely, LDH isozymes which are active in a restricted number of adult tissues are detected only later in embryogenesis. The initiation of LDH-C gene expression, however, is closely coupled with morphological and functional differentiation of those cells in which this locus is predominantly expressed in the adult.     

Subcellular localization of isozymes of NAD-dependent malate dehydrogenase in sugar beet <Emphasis Type="Italic">Beta vulgaris</Emphasis> L.

Subcellular localization of isozymes of NAD-dependent malate dehydrogenase (MDH) in sugar beet was studied. Isozymes ss and ll controlled by loci Mdh2 and Mdh3, respectively, were shown to locate in mitochondria, whereas isozyme pp controlled by locus Mdh1, in microbodies. All examined samples lack hybrid MDH isozymes, which could testify to the interaction between products of nonallelic Mdh genes. This can be explained by the localization of nonallelic isozymes in various compartments of the cell and organelles.     

Liver-like alkaline phosphatase in the tissue-unspecific type enzyme found in rabbit organs

Rabbit liver and kidney tissues are known to produce an intestinal-like alkaline phosphatase (IAP-like enzyme) as a dominant isozyme, with a minor isozyme of tissue-unspecific type (UAP), unlike humans and other mammalians. We investigated immunohistochemically and biochemically these unique isozymes in the rabbit liver and bone, and compared them with the human isozyme. In rabbit liver, UAP was found to be localized only in the apical part of the membrane of cells lining the bile duct, whereas IAP-like enzyme was found in the sinusoidal membrane of hepatocytes. Rabbit liver UAP was separated from IAP-like enzyme by DEAE-cellulose column chromatography. Rabbit bone tissue contained only one UAP isozyme. The two UAPs were biochemically and physicochemically compared with human liver AP. Both UAPs reacted with an anti-human liver AP monoclonal antibody, not with an anti-human bone AP monoclonal antibody, indicating that both enzymes have the same antigenicity as human liver AP. Rabbit liver and bone UAPs had similar N-linked sugar-chain heterogeneities to the respective human enzymes. In addition, rabbit bone AP also had an O-linked sugar chain, as did human bone AP, unlike rabbit and human liver APs.     

Identification of pulmonary surfactant that bears intestinal-type and tissue-nonspecific-type alkaline phosphatase in endotoxin-induced rat bronchoalveolar fluid

The characteristics of lipopolysaccharide (LPS)-induced alkaline phosphatase (AP) isozymes on the various pulmonary surfactant subtypes were investigated. We used continuous sucrose-gradient centrifugation to separate surfactant into subtypes. The density of each surfactant subtype isolated from LPS-instilled rats was greater than that of the subtypes from the control rats; and the proportion of light surfactant was lower, thereby decreasing the ratio of light to heavy surfactant. The results of an inhibition study revealed the main AP isozyme in bronchoalveolar fluid (BAF) to be tissue-nonspecific AP (TNAP), but some of the activity was characteristic of intestinal-type AP (IAP). IAP, in addition to TNAP and surfactant-associated protein A (SP-A), was detected on heavy surfactant, and LPS induced both APs. To examine the expression of IAP in the lungs, we prepared primers to detect the cDNAs of two types of rat IAP mRNA, IAP-I and -II, and amplified their cDNAs. LPS instillation induced IAP-I mRNA, but not IAP-II mRNA or TNAP mRNA. Immunohistochemical localization of IAP and TNAP revealed reaction products for both in type II cells. The present study thus demonstrated that, in rats, type II cells produce both IAP and TNAP and that these surfactants bearing AP isozymes are secreted into the alveolar space following induction by intratracheal instillation of LPS.     

Studies on the properties and tissue distribution of the isozymes of guanylate kinase in man.

The isozyme patterns of guanylate kinase were examined in fetal and adult tissues, in cultured cells and also in red cells separated by density gradient fractionation. Results from fetal and cultured cells inidcated that there are three primary isozymes a, c, and e among the seven isozymes of guanylate kinase in man. Serial secondary isozyme production in red cells in vivo showed that isozyme a produces b, c produces d, and e produces f and g. The three sets of isozymes were found to differ in the following properties: activation/inhibition by EDTA; thermostability, and molecular weights. Isoelectric points of several of the isozymes were estimated by isoelectric focusing. It was concluded that the isozymes of guanylate kinase are determined by three separate gene loci.     

Further studies of primer-independent phosphorylase isozymes in the algae

Both Oscillatoria princeps and Cyanidium caldarium contain phosphorylase isozymes that can cause the synthesis of polyglucan from glucose-1-phosphate in the absence of added maltodextrin ‘primer’. In addition, O. princeps contains a primer-dependent phosphorylase isozyme. When the phosphorylase fractions isolated from extracts of the algae were treated with α-amylase, the primer-independent isozyme became primer-dependent and shifted from the position it was normally found at after polyacrylamide gel electrophoresis. This primer-independent isozyme became less mobile towards the anode, and was found at the locus usually occupied by the primer-dependent isozyme. It was not possible to restore its mobility towards the anode and its primer-independent properties by preincubation with maltoheptaose. The indication is that this isozyme is a glucoprotein and that the glucan component is chemically bonded to the protein.