Isozymes of bovine intestinal alkaline phosphatase

Alkaline phosphatases from calf and bovine small intestines have been isolated in homogeneous form from both mucosa and luminal contents. The detergent-solubilized calf enzyme resolves into two peaks of activity, C-1 and C-2, on chromatofocusing. Only one of these activity peaks is present in the enzyme from the adult animal. Amino acid compositions, N-terminal sequences, and tryptic peptide maps show that C-1 and C-2 are isozymes of differing primary structure and that the adult form of the enzyme is identical to C-2. The developmentally controlled expression of the two isozymes reported here suggests a molecular basis for the previous indications that functional changes in intestinal alkaline phosphatase occur with tissue maturation. The sugar composition of the carbohydrate chains of these isozymes has been determined and enzymatic deglycosylation with endo-beta-N-acetylglucosaminidase-F indicates two N-linked and one or more O-linked glycoconjugates/monomer.     

Putting plant hexokinases in their proper place

Hexokinases (HXKs), catalysts of the first essential step in glucose metabolism, have emerged as important enzymes that mediate sugar sensing in many organisms, including plants. The presence of several types of plant HXK isozymes, located in different intracellular locations, has been suggested. However, recent studies have indicated that most plants have only two types of HXKs, a plastidic stromal isozyme and membrane-associated isozymes located mainly adjacent to the mitochondria, but also in the nucleus. The membrane-associated isozymes are involved in sugar sensing and regulate gene expression. The central role of HXKs in plant development and the increasing interest in their role necessitate the correction of inaccuracies that have spread concerning the substrate specificity and intracellular localization of HXK isozymes, as these inaccuracies are affecting the hypothesized roles presented for these isozymes and shaping future research in this active field.     

Genetics of rye phosphatases: evidence of a duplication

Summary Genetic analyses were conducted on alkaline phosphatases of the endosperm of dry kernels and leaf acid phosphatases in four open pollinated and one inbred line of cultivated rye (Secale cereale L.). A total of seven alkaline phosphatase isozymes were observed occurring at variable frequencies in the different cultivars analyzed. We propose that at least five loci control the alkaline phosphatases of rye endosperm — Alph-1, Alph-2, Alph-3, Alph-4 and Alph-5 — all of which have monomeric behaviour. The leaf acid phosphatases are controlled by one locus and have a dimeric quaternary structure. All loci coding for alkaline phosphatase isozymes showed one active, dominant allele and one null, recessive allele, except for the locus Alph-3 which showed two active, dominant alleles and one null, recessive one. The linkage analyses suggest the existence of two linkage groups for alkaline phosphatases: one of them would contain Alph-2, Alph-4, Alph-5 and the locus/loci coding isozymes 6 and 7. This linkage group is located in the 7RS chromosome arm. The other group would include Alph-1 and Alph-3 loci, being located in the 1RL chromosome arm. Leaf acid phosphatases have been previously located in the 7RL chromosome arm. Our data also support an independent relationship between loci controlling the endosperm alkaline phosphatases and leaf acid phosphatases.     

Serine/threonine phosphatases in the nervous system.

The cloning and sequence determination of cDNAs encoding different types of serine/threonine protein phosphatases has provided a molecular basis for the protein phosphatase classification proposed by Ingebritsen and Cohen. Each of the phosphatases, phosphatase-1, -2A, -2B and -2C, exists as multiple isozymes raising the possibility that isozymes selectively expressed in different tissues may perform specific functions. The recent discovery of potent toxin inhibitors specific for protein phosphatase-1 and -2A will undoubtedly play an important role in the elucidation of the role of these enzymes in neuronal function.     

Protein tyrosine phosphatases: regulatory mechanisms

Protein-tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post-translational modifications affecting intrinsic catalytic activity, ligand binding and dimerization. Here, we review the regulatory mechanisms found to control the classical protein-tyrosine phosphatases.     

Evolutionary radiation pattern of novel protein phosphatases revealed by analysis of protein data from the completely sequenced genomes of humans, green algae, and higher plants

In addition to the major serine/threonine-specific phosphoprotein phosphatase, Mg(2+)-dependent phosphoprotein phosphatase, and protein tyrosine phosphatase families, there are novel protein phosphatases, including enzymes with aspartic acid-based catalysis and subfamilies of protein tyrosine phosphatases, whose evolutionary history and representation in plants is poorly characterized. We have searched the protein data sets encoded by the well-finished nuclear genomes of the higher plants Arabidopsis (Arabidopsis thaliana) and Oryza sativa, and the latest draft data sets from the tree Populus trichocarpa and the green algae Chlamydomonas reinhardtii and Ostreococcus tauri, for homologs to several classes of novel protein phosphatases. The Arabidopsis proteins, in combination with previously published data, provide a complete inventory of known types of protein phosphatases in this organism. Phylogenetic analysis of these proteins reveals a pattern of evolution where a diverse set of protein phosphatases was present early in the history of eukaryotes, and the division of plant and animal evolution resulted in two distinct sets of protein phosphatases. The green algae occupy an intermediate position, and show similarity to both plants and animals, depending on the protein. Of specific interest are the lack of cell division cycle (CDC) phosphatases CDC25 and CDC14, and the seeming adaptation of CDC14 as a protein interaction domain in higher plants. In addition, there is a dramatic increase in proteins containing RNA polymerase C-terminal domain phosphatase-like catalytic domains in the higher plants. Expression analysis of Arabidopsis phosphatase genes differentially amplified in plants (specifically the C-terminal domain phosphatase-like phosphatases) shows patterns of tissue-specific expression with a statistically significant number of correlated genes encoding putative signal transduction proteins.     

Multiplicity of protein serine-threonine phosphatases in PC12 pheochromocytoma and FTO-2B hepatoma cells

Protein purification and molecular cloning have defined five classes of protein serine-threonine phosphatase catalytic subunits referred to as types 1, 2A, 2B (calcineurin), 2C, and X. Protein serine-threonine phosphatases 1, 2A, 2B, and X appear to have significant sequence homologies, whereas the 2C enzyme is more divergent. We have used the polymerase chain reaction to define the multiplicity of the closely related types 1, 2A, 2B, and X phosphatase catalytic subunits in two clonal cell lines, rat PC12 pheochromocytoma and rat FTO-2B hepatoma. RNAs for all four related phosphatase types were expressed in both cell lines. In addition to the phosphatase X enzyme, four phosphatase 1, two phosphatase 2A, and three phosphatase 2B isoforms were identified in PC12 and FTO-2B cells. The results indicate a large multiplicity of protein serine-threonine phosphatases within clonal cells of different tissue origin, suggesting that their role in cell regulation will be as divergent as that for the protein serine-threonine kinases.     

Intersyngenic variations in the esterases and acid phosphatases of Tetrahymena pyriformis

The esterase and acid phosphatase isozymes were surveyed in strains of syngens 2–12 under conditions found to be optimal for syngen 1. Both intersyngenic and intrasyngenic variations were found. Comparisons of the esterases suggest that homologous enzymes are present in certain syngens and that some ordering of the variations with respect to syngen differences is possible. The acid phosphatases are highly polymorphic in different strains even within a syngen, and the variations cannot be ordered with respect to syngen differences. These results are discussed in terms of other types of studies directed at assessing syngen relationships and in terms of the sources of variation. It was concluded that only characters less vulnerable to intraclonal variation will be capable of revealing syngen relationships.Supported by a Research Grant, GM-15879, from the National Institute of General Medical Sciences, U.S. Public Health Service.     

Purification and properties of two distinct groups of ADH isozymes from Chinese hamster liver

Two major classes of alcohol dehydrogenase isozymes have been purified from Chinese hamster liver. The two isozyme classes have the same subunit molecular weights but different electrophoretic mobilities. They have a similar range of substrates but different KROM values and sensitivities to the inhibitor pyrazole. The ADHs are immunologically related as determined by Ouchterlony double diffusion experiments. These results suggest that the isozymes are encoded by different structural gene loci derived from a common ancestral gene.Financial support was provided by the National Cancer Institute of Canada and the Medical Research Council of Canada, Grant MT4860. J.-P. Thirion is a Research Scholar of the Science Research Council of Quebec.     

Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307,000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44,400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417,000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1% (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321,000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.     

Further studies on bovine serum amylase. 3. Affinity chromatography

The major bovine serum isoamylases controlled by the AmI locus have been examined by gel filtration. On Sephadex G-200 the isoamylases can be resolved into two classes. The AmI A and AmI B have apparent molecular weights of 307 000 daltons whilst the AmI C isozyme has an apparent molecular weight of 44 400 daltons. The separation of the isozymes into two classes according to their elution behaviour on Sephadex G-200 has been shown to be an affinity separation. All three AmI isozymes are eluted from a non-dextran media (BioGel A1.5m) with apparent molecular weights of 417 000 daltons. The affinity separation on Sephadex G-200 has been shown to be inhibited by the addition of 1% (w/v) maltose to the elution buffer. In the presence of 1 % (w/v) maltose all three AmI isozymes are coeluted from Sephadex G-200 with apparent molecular weights of 321000 daltons. The maltase and amylase activities of the AmI isozymes were eluted coincidentally under all the conditions studied.     

Glucose 6-phosphate dehydrogenase from brook,lake, and splake trout: An isozymic and immunological study

There are five G6P-specific G6PD isozymes in both brook and lake trout. The most anodal isozyme in each species has the same electrophoretic mobility; however, the five lake trout isozymes are more widely spaced on polyacrylamide gels than are those from brook trout. In hybrids, i.e., splake trout, nine forms of G6PD can be resolved. These results can be explained by a model in which we assume that each isozyme is a tetramer and that two different subunit types are produced. In splake trout, three electrophoretically distinct subunits yield 15 tetramers. That only nine are detected is a consequence of coincident electrophoretic mobility of some of the possible subunit combinations. Our results indicate that the G6PD isozymes in these trout are the products of two codominant autosomal gene loci. The hypothesis that G6PD and H6PD arose from a common ancestral type G6PD is supported by microcomplement fixation data which show an immunochemical relatedness between these enzymes. The relationship of G6PD and H6PD in trout is discussed from an evolutionary standpoint.This research was supported in part by National Science Foundation Grant GB-7271 and by United States Public Health Predoctoral Fellowship 4 FO1 GM 43657-03.     

A Comparative Study of Multiple: Acid Phosphatases in Cellular Fractions of Bean Hypocotyl

Six kinds of acid phosphatases were solubilized with TritonX-100 from the cell wall (W-I, W-II), mitochondrial (M-I, M-II,M-III) and microsomal (Ms) fractions of bean hypocotyl, andthey were partially purified by using Sephadex gel filtrationand DEAE-cellulose column chromatography. Acid phosphatasesfrom the soluble fraction were also fractionated into 12 isozymesby electrophoresis, and the properties of the isozymes werecompared. The soluble isozymes showed pH optima at 5·0,5·3 and 5·6; the isozymes possessed high affinityto p-nitrophenyl phosphate and ADP, and their mol. wts rangedfrom 30000 to 45000. Among the solubilized phosphatases, W-I,M-I and M-III showed maximum activity at pH 5·0 and theirmol. wts were between 50000 and 110000. W-I proved to have highaffinity to ATP and bis-p-nitrophenyl phosphate as M-I did top-nitrophenyl phosphate and M-III to phenyl phosphate. The characteristicsof these solubilized isozymes were different from those of thesoluble isozymes. On the contrary, W-II, M-II and Ms were quitesimilar to those of the solubles in pH optima, substrate specificity,K_m value, affinity to DEAE-cellulose and gel electrophoreticpatterns. These results suggest that W-II, M-II and Ms werederived from the soluble isozymes. Phaseolus vulgaris L., bean, hypocotyl, acid phosphatases, Michaelis constant     

Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations.

In all eucaryotic cell types analyzed, proliferations of the endoplasmic reticulum (ER) can be induced by increasing the levels of certain integral ER proteins. One of the best characterized of these proteins is HMG-CoA reductase, which catalyzes the rate-limiting step in sterol biosynthesis. We have investigated the subcellular distributions of the two HMG-CoA reductase isozymes in Saccharomyces cerevisiae and the types of ER proliferations that arise in response to elevated levels of each isozyme. At endogenous expression levels, Hmg1p and Hmg2p were both primarily localized in the nuclear envelope. However, at increased levels, the isozymes displayed distinct subcellular localization patterns in which each isozyme was predominantly localized in a different region of the ER. Specifically, increased levels of Hmg1p were concentrated in the nuclear envelope, whereas increased levels of Hmg2p were concentrated in the peripheral ER. In addition, an Hmg2p chimeric protein containing a 77-amino acid lumenal segment from Hmg1p was localized in a pattern that resembled that of Hmg1p when expressed at increased levels. Reflecting their different subcellular distributions, elevated levels of Hmg1p and Hmg2p induced sets of ER membrane proliferations with distinct morphologies. The ER membrane protein, Sec61p, was localized in the membranes induced by both Hmg1p and Hmg2p green fluorescent protein (GFP) fusions. In contrast, the lumenal ER protein, Kar2p, was present in Hmg1p:GFP membranes, but only rarely in Hmg2p:GFP membranes. These results indicated that the membranes synthesized in response to Hmg1p and Hmg2p were derived from the ER, but that the membranes were not identical in protein composition. We determined that the different types of ER proliferations were not simply due to quantitative differences in protein amounts or to the different half-lives of the two isozymes. It is possible that the specific distributions of the two yeast HMG-CoA reductase isozymes and their corresponding membrane proliferations may reveal regions of the ER that are specialized for certain branches of the sterol biosynthetic pathway.     

Guinea pig liver alcohol dehydrogenase: isolation and characterization of two distinct classes of isozymes

1. Two distinct classes of alcohol dehydrogenase (ADH) isozymes were purified from guinea pig liver. 2. While the two classes of isozymes have similar subunit weight and electrophoretic mobility on starch gel, they differ markedly in catalytic properties. 3. The class A ADH oxidizes rapidly, exhibits saturated kinetics with both primary and secondary alcohols and is inhibited very effectively by 4-methylpyrazole (Ki = 0.58 microM) and o-phenanthroline (I50 = 0.1 mM). 4. The class B isozyme does not oxidize secondary alcohols, exhibits saturated kinetics only with long chain primary alcohols and is less sensitive to the ADH inhibitors 4-methylpyrazole (Ki = 15 mM) and o-phenanthroline (I50 greater than 10 mM).     

Distribution and classification of acylphosphatase isozymes

Distributions of acylphosphatase isozymes among organs of several animal species were investigated. Organ extracts of pig and chicken were treated with isozyme-specific antibodies, subjected to electrophoresis on a polyacrylamide gel, then the gel was stained for acylphosphatase activity. Both animals showed three activity bands; one band was named common type isozyme because of its wide distribution in testis, muscle, brain, heart, spleen, kidney, liver, and erythrocyte, and the other two bands were named muscle type isozymes because of their localization in skeletal muscle. This classification was supported by selective and quantitative reactions of the isozymes to the isozyme-specific antibodies. Because the two bands of the muscle type have the same amino acid sequence and differ only in modifications on an -SH group, it is suggested that pig and chicken have only the two major types of acylphosphatase. This conclusion was supported by similar experiments on dog, human, rabbit, and pigeon.     

Genetic relationships between the multiple alcohol dehydrogenases of maize

There are three electrophoretically separable sets of alcohol dehydrogenase isozymes in maize. Previous work has shown that two of these isozymes (Sets I and II) share a subunit in common, since mutations in one of the Adh genes, Adh ₁, alter both isozymes. A mutation in the second Adh gene, Adh ₂, has now been induced and recovered. This mutant allele also alters two of the three isozymes—Sets III and II. Adh ₁ and Adh ₂ appear to segregate independently. Gel filtration data show that all ADH isozymes are indistinguishable in size. These findings support the hypothesis that the two Adh genes specify promoters which homo- and heterodimerize, yielding three types of ADH isozymes.This research was supported by National Science Foundation Grant GB 25594. M.F. is a recipient of Public Health Service Genetics Training Grant GM 82-12.     

Isozymes delta of phosphoinositide-specific phospholipase C.

Phospholipase C (PLC, EC 3.1.4.11) is the major starting point in the phosphatidylinositol pathway, which generates intracellular signals that regulate protein kinase C and intracellular calcium concentration. To date, three major types of phosphoinositide-specific PLC species named beta, gamma and delta, have been characterized. This article reviews recent studies on isozymes delta of PLC. Four such isozymes have been cloned and termed delta1-4. Their structural organization, regulation of activity and the interaction with membrane lipid are considered. The intracellular localization of delta isozymes and distribution in various tissues are presented. Attention is given to the pathological conditions in which an abnormal protein level of PLC delta or its activity have been observed.     

Inhibitor profiles of alkaline phosphatases in bovine preattachment embryos and adult tissues.

The alkaline phosphatases are a small family of isozymes. Bovine preattachment embryos transcribe mRNA for two tissue-specific alkaline phosphatases (TSAP2 and TSAP3) beginning at the 4- and 8-cell stages. Whereas no mRNA has been detected in oocytes, there is maternally inherited alkaline phosphatase activity. It is not known which isozyme(s) is responsible for the maternal activity or when TSAP2 and TSAP3 form functional protein. No antibodies are available that recognize the relevant bovine alkaline phosphatases. Therefore, sensitivity to heat and chemical inhibition was used to separate the different isozymes. By screening tissues, it was determined that the bovine tissue-nonspecific alkaline phosphatase (TNAP) is inactivated by low temperatures (65C) and low concentrations of levamisole (<1 mM), whereas bovine tissue-specific isozymes require higher temperatures (90C) and levamisole concentrations (>5 mM). Inhibition by L-homoarginine and L-phenylalanine was less informative. Cumulus cells transcribe two isozymes and the pattern of inhibition suggested heterodimer formation. Inhibition of alkaline phosphatase in bovine embryos before the 8-cell stage indicated the presence of only TNAP. At the 16-cell stage the pattern was consistent with TNAP plus TSAP2 or -3 activity, and in morulae and blastocysts the pattern indicated that the maternal TNAP is fully supplanted by TSAP2 or TSAP3.