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.     

A comparative study of the age-related patterns of decay of some nucleoside monophosphate kinases in human red cells

The nucleoside monophosphate kinases, adenylate kinase (AK), guanylate kinase (GUK), and uridine monophosphate kinase (UMPK), were studied electrophoretically and quantitatively in density gradient fractions of human red cells from normal adults which contain red cells of differing mean age. The enzymes were found to differ both in their rates and patterns of decay and in secondary isozyme formation during the life of the red cell in the circulation. AK showed no appreciable enzyme decay and slight genetation of secondary isozymes; UMPK showed a rapid monophasic decline and no secondary isozyme formation; GUK showed intermediate overall loss of activity with a biphasic pattern of decay and marked secondary isozyme formation. A comparative study of the two common phenotypes of UMPK (UMPK 1 and UMPK 2-1) and of AK (AK 1 and AK 2-1) was made. The UMPK 2 isozyme showed a more rapid decay than the UMPK 1 isozyme, whereas no difference was observed between the AK 1 and AK 2 isozymes.     

Intramolecular interactions regulate SAP97 binding to GKAP

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.     

Cloning and expression of the essential gene for guanylate kinase from yeast.

Guanylate kinase catalyzes the reversible transfer of the terminal phosphoryl group of ATP to the acceptor molecule GMP. Detailed analysis of the in vivo function of this enzyme has been limited by the lack of any genetic data. Using oligonucleotides based on amino acid sequence information of the yeast enzyme, the Saccharomyces cerevisiae gene, GUK1, was isolated and characterized. The gene is present in single copy and maps to chromosome IV. Insertional mutagenesis of the GUK1 locus caused recessive lethality, indicating that this enzyme is necessary for vegetative cell growth. Using inducible expression systems, guanylate kinase was produced in large amounts both in S. cerevisiae and in Escherichia coli.     

Genetic and molecular analysis of nonrandom dimer assembly of the creatine kinase isozymes of fishes

Species within many families of actinopterygian bony fishes (class Osteichthyes) have a two-banded allelic isozyme phenotype in individuals heterozygous at the creatine kinase A locus. This two-banded pattern is formed by the presence of the two homodimeric isozymes and the absence of the expected heterodimer. Sharks and amphibians have retained the ability to form all three allelic isozymes in individuals which are heterozygous. Reversible denaturation procedures were able to assemble the different allelic CK-A subunits within a species to form CK-A₂ heterodimers. Furthermore, heterodimers were formed from different CK-A subunits from highly divergent species after this in vitro molecular hybridization process. It is concluded from these studies that the polypeptidebinding sites of creatine kinase are structurally conservative in most fishes and that the absence of a heterodimer in heterozygous individuals is not due to a structural incompatibility between the different A subunit types or to an instability of the heterodimer during electrophoresis. A temporal and/or spatial isolation of allelic CK-A subunit synthesis and assembly, within differentiated skeletal muscle, appears to have evolved in the actinopterygian bony fishes.This research was supported by NSF Grant PCM76-08383 to G. S. W. and by a NIH Cell and Molecular Biology Traineeship to S. D. F.     

The inheritance of rye seed peroxidases

Summary Genetic analyses were conducted on peroxidase of the embryo and endosperm of seeds of one open pollinated and six inbred lines of cultivated rye (Secale cereale L.), and one line of Secale vavilovii Grossh. The analyses of the individual parts of the S. cereale seed yield a total of 14 peroxidase isozymes. Isozymes m, a, b, c, d, e, f and g (in order from faster to slower migration) were found in the embryo plus scutellum, while isozymes 1, 2, 3, 4, 5 and 6 (also from faster to slower migration) were peculiar of the endosperm. S. vavilovii has isozymes m, c₁, d, e, f and g in its embryo plus scutellum, and isozyme 2 in the endosperm. Segregation data indicated that at least 13 different loci would be controlling the peroxidase of S. cereale. Isozymes a and b are controlled by alleles of the same locus, all the other loci have one active and dominant allele coding for one isozyme, and other null and recessive allele. The estimation of linkage relationships shows that five endosperm loci are linked, and tentative maps are shown. A possible dosage effect and the existence of controlling gene(s) for endosperm isozyme 4 is reported. All these data and the high frequency of null alleles found are discussed in relation to recent reports.     

Isotope effects in the study of enzymatic phosphoryl transfer reactions

CASK, a member of the membrane-associated guanylate kinase (MAGUK) superfamily, binds to the carboxyl-terminus of beta-neurexins on the intracellular side of the presynaptic membrane. The guanylate kinase-like (GUK) domains of MAGUKs lack kinase activities, but might be important for mediating specific protein-protein interaction. By a yeast two-hybrid approach, we identified an interaction between the GUK domain of CASK and the C2B domain of rabphilin3a, a presynaptic protein involved in synaptic vesicle exocytosis. The interaction was confirmed by in vitro GST pull-down and co-immunoprecipitation assays. It was proposed that presynaptic vesicles might be guided to the vicinity of points of exocytosis defined by beta-neurexins via the interaction between rabphilin3a-CASK-beta-neurexins.     

三种蜘蛛酯酶同工酶的比较研究

应用聚丙烯酰胺凝胶电泳对狼蛛科的拟水狼蛛、蟹蛛科的三突花蛛和肖蛸科的鳞纹肖蛸3种蜘蛛的酯酶同工酶进行了比较研究。结果表明,不同科的蜘蛛酯酶同工酶种问差异性大并有着明显的种簇特异性,推测它们的酯酶同工酶酶谱中的区带组受不同的基因位点控制,且各自的基因位点数不等;同种蜘蛛的雌蛛和雄蛛之间也有各自的酯酶同工酶谱型,但差异小,其控制基因位点大体相同。这样,我们从分子的水平上讨论了酯酶同工酶的差异性可以用来作为识别物种的附加指标。     

Pyruvate kinase isozymes in man

Anti human M2 type and anti human L type pyruvate kinase sera allowed us to distinguish two groups of pyruvate kinase in man. Erythrocyte and liver (L type) enzymes on the one hand were inhibited by anti L and not all by anti M2 serum; pyruvate kinase from all the other tissues on the other hand were inhibited by anti M2 and not at all by anti L serum. This latter group represent the M type pyruvate kinase isozymes. The M type isozymes have been studied by electrofocusing in thin layer acrylamide-ampholine gel. In adult tissues 4 types of isozymes were found, designated, from acid to alkaline pH, as M2 (predominant form in spleen, leukocytes, lung...), M3, M4 and M1 (predominant form in muscle and brain). In foetal tissues an extra band M2, called M2f, more anodic than M2, was added to the previously described isozymes. Except in brain (in which the isozymes M2, M3, M4 and M1 were found), the most anodic bands (M2f, M2 and M3) were predominant in all the foetal tissues. The isozymes M2f and M2 seem therefore to be the original M type pyruvate kinase forms from which the other isozymes issue. The rate of each isozyme seems to depend on tissue factors characterizing the state of differentiation of some tissues, as indicated by the ability of adult muscle extracts to change the isozymes M2 and M3 into more cathodic forms.     

Pyruvate kinase isozymes in adult tissues and eggs of Rana pipiens. Comparative studies on the properties of the different isozymes

The properties of the isozymes of pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) found in unfertilized frog egg have been compared to those found in adult tissues of Rana pipiens. Chromatographic, kinetic, and electrophoretic data indicate that, of the five electrophoretic forms found in egg, the isozyme with the least anodic mobility (isozyme I) is the same molecular species as the only isozyme found in heart, and the egg isozyme with the greatest anodic mobility (isozyme V) is identical to the major isozyme found in liver.The activity of egg isozyme I was markedly inhibited by the antibody to the skeletal muscle enzyme, which has been shown previously to cross-react with the cardiac enzyme, but was unaffected by the antibody to liver isozyme V; the opposite effects were observed with egg isozyme V. The antibody to the skeletal muscle enzyme inhibited egg isozymes II > III > IV whereas the antibody to the liver enzyme gave the reverse inhibitory pattern, e.g., isozyme IV > III > II.In vitro dissociation-reassociation of mixtures of isozyme I and V led to the formation of the other three isozymes. Similar experiments performed individually with either egg isozyme III or IV resulted in the production of predominantly isozymes III, II, and I due to the instability of isozyme V during the hybridization procedure.The above results indicate that isozymes I and V are tetramers of the respective parental subunits and that isozymes II, III, and IV are hybrid molecules with subunit assignments of (I₃V₁), I₂V₂), and (I₁V₃), respectively.     

Parietal cell protein kinases. Selective activation of type I cAMP-dependent protein kinase by histamine

cAMP-dependent protein kinases have been characterized in parietal cells isolated from rabbit gastric mucosa. Both Type I and Type II cAMP-dependent protein kinase isozymes are present in these cells. Type II isozymes were detected in 900, 14,000, and 100,000 X g particulate fractions as well as 100,000 X g cytosolic fractions; Type I isozymes were found predominately in the cytosolic fraction. When parietal cells were stimulated with histamine, an agent that elevates intracellular cAMP content and initiates parietal cell HCl secretion, cAMP-dependent protein kinase activity was increased in homogenates of these cells as measured by an increase in the cAMP-dependent protein kinase activity ratio. Histamine activation of cAMP-dependent protein kinase was correlated with parietal cell acid secretory responses which were measured indirectly as increased cellular uptake of the weak base, [14C]aminopyrine. These results suggest that cAMP-dependent protein kinase(s) is involved in the control of parietal cell HCl secretion. The parietal cell response to histamine may be compartmentalized because histamine appears to activate only a cytosolic Type I cAMP-dependent protein kinase isozyme, as determined by three different techniques including 1) ion exchange chromatography; 2) Sephadex G-25 to remove cAMP and allow rapid reassociation of the Type II but not the Type I isozyme; and 3) 8-azido-[32P]cAMP photoaffinity labeling. Forskolin, an agent that directly stimulates adenylate cyclases, was found to activate both the Type I and Type II isozymes. Several cAMP-dependent protein kinases were also detected in parietal cell homogenates, including a Ca2+-phospholipid-sensitive or C kinase and two casein kinases which were tentatively identified as casein kinase I and II. At least two additional protein kinases with a preference for serine or lysine-rich histones, respectively, were also detected. The function of these enzymes in parietal cells remains to be shown.     

hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions

Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane. All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK). We showed previously that one MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes. We now report that hCASK binds another MAGUK, human discs large (hDlg). Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine. These proteins co-precipitate from lysates of an intestinal cell line, Caco-2. The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells. In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding. The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK. Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo. These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding.     

Human red cell acid phosphatase: purification and properties of the A, B and C isozymes

Human red cell acid phosphatase isozymes encoded by three alleles (ACP1*A, ACPI*B and ACP1*C), each of which generates two isozymes, (f) and (s), were purified to homogeneity. The molecular mass of the six isozymes (Af, As, Bf, Bs, Cf and Cs) was estimated to be 17-18 kDa, the mass of the f isozymes probably being slightly higher than that of the s isozymes. It was indicated that the isozymes react with p-nitrophenyl phosphate in the mono anionic state, and that a group with a pKa value of about 6, which may be histidine, is of importance for the catalytic function of the s isozymes. Significant differences between the f and s isozymes were observed with respect to specific activity. Km (p-nitrophenyl phosphate), Ki (p-aminobenzylphosphonic acid), amino acid composition, stability in the presence of urea, thermal stability, retention time in size-exclusion chromatography of the native isozymes and migration in sodium dodecyl sulphate polyacrylamide gel electrophoresis, In contrast, identical or similar properties were observed for the three genetically different f isozymes, and the same was the case for the three s isozymes. It is suggested that the f and s isozymes serve different functions in the cell.     

Species Specificity of Brain Cyclic AMP Receptor Proteins

The cyclic AMP binding proteins present in mouse, rat, bovine, and sheep brains were compared. Extracts were isotopically labeled with 8-azido-cyclic [32P]AMP, a photoaffinity analog specific for cyclic AMP binding sites, and then subjected to two-dimensional gel electrophoresis. The resulting autoradiographic patterns were generally similar, but showed consistent species variations. Proteins identified by their size and phosphorylatability as regulatory subunits of Type II protein kinase isozymes were present in all species, but with slight variations in pI. A series of charge variants identified as regulatory subunits of Type I kinase isozymes on the basis of their size was also ubiquitously present, as were several smaller proteins postulated to be proteolytic fragments derived from the regulatory subunits. The major species difference was a series of labeled proteins found only in rodent brains and not in the brains of any ruminant, or in other rodent tissues. These proteins had a molecular weight of 54,000 and a pI range of 5.89--6.26, and could not be endogenously phosphorylated. The identities of these proteins and their relationship to the protein kinase regulatory subunits are unknown.     

Linkage of two enzyme loci in fishes of the genus Xiphophorus (Poeciliidae). Guanylate kinase-2 (GUK2) and glyceraldehyde-3-phosphate dehydrogenase-1 (GAPD1) designated as linkage group III

Electrophoretic variation ascribable to two enzyme loci, coding for a guanylate kinase (GUK2) and a glyceraldehyde-3-phosphate dehydrogenase (GAPD1), was observed in three species of fishes of the genus Xiphophorus. Electrophoretic patterns in F1 hybrid heterozygotes suggested a monomeric subunit structure for GUK2 and confirmed a tetrameric structure for GAPD1. Variant alleles at the two loci exhibited normal Mendelian segregation in backcross hybrids. Linkage analyses indicate estimated recombination of GUK2-7.6 percent-GAPD 1. This group (designated linkage group III) was shown to assort independently from the 7 loci comprising linkage groups I and II and from 26 other informative markers, within the limits of the data. Difficulties inherent in establishing homology with linkage groups in other species in cases involving presumed gene duplication are discussed.     

Homology of Melanoma-Inducing Loci in the Genus Xiphophorus

Several species of the genus Xiphophorus are polymorphic for specific pigment patterns. Some of these give rise to malignant melanoma following the appropriate crossings. For one of these pattern loci from the playfish Xiphophorus maculatus the melanoma-inducing gene has been cloned and found to encode a novel receptor tyrosine kinase, designated Xmrk. Using molecular probes from this gene in Southern blot analyses on single fish DNA preparations from 600 specimens of different populations of various species of the genus Xiphophorus and their hybrids, either with or without melanoma-predisposing pattern, it was shown that all individuals contain the Xmrk gene as a proto-oncogene. It is located on the sex chromosome. All fish that carry a melanoma-predisposing locus which has been identified by Mendelian genetics contain an additional copy of Xmrk, closely linked to a specific melanophore pattern locus on the sex chromosome. The melanoma-inducing loci of the different species and populations are homologous. The additional copy of Xmrk obviously arose by a gene-duplication event, thereby acquiring the oncogenic potential. The homology of the melanoma-inducing loci points to a similar mechanism of tumor suppression in all feral fish populations of the different species of the genus Xiphophorus.     

Arabidopsis AtDGK7, the smallest member of plant diacylglycerol kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration: the DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alters plant growth and development

Diacylglycerol kinase (DGK) regulates the level of the second messenger diacylglycerol and produces phosphatidic acid (PA), another signaling molecule. The Arabidopsis thaliana genome encodes seven putative diacylglycerol kinase isozymes (named AtDGK1 to -7), structurally falling into three major clusters. So far, enzymatic activity has not been reported for any plant Cluster II DGK. Here, we demonstrate that a representative of this cluster, AtDGK7, is biochemically active when expressed as a recombinant protein in Escherichia coli. AtDGK7, encoded by gene locus At4g30340, contains 374 amino acids with an apparent molecular mass of 41.2 kDa. AtDGK7 harbors an N-terminal catalytic domain, but in contrast to various characterized DGKs (including AtDGK2), it lacks a cysteine-rich domain at its N terminus, and, importantly, its C-terminal DGK accessory domain is incomplete. Recombinant AtDGK7 expressed in E. coli exhibits Michaelis-Menten type kinetics with 1,2-dioleoyl-sn-glycerol as substrate. AtDGK7 activity was affected by pH, detergents, and the DGK inhibitor R59022. We demonstrate that both AtDGK2 and AtDGK7 phosphorylate diacylglycerol molecular species that are typically found in plants, indicating that both enzymes convert physiologically relevant substrates. AtDGK7 is expressed throughout the Arabidopsis plant, but expression is strongest in flowers and young seedlings. Expression of AtDGK2 is transiently induced by wounding. R59022 at approximately 80 mum inhibits root elongation and lateral root formation and reduces plant growth, indicating that DGKs play an important role in plant development.     

Associations of genes encoding allozymes peroxidase and superoxide dismutase in poplar and spruce species

Summary Isozymes of peroxidase (PER) and superoxide dismutase (SOD) were analyzed in vegetative buds or very young leaves of seven species and two interspecific hybrids of Populus, in progenies of seven controlled crosses of three Populus species, and in needles of five Picea species and one putative hybrid. One to three PER, and one or two SOD zones of activity were observed. Electrophoretic mobility (EM) and banding phenotypes of isozymes of one PER locus were identical to those of one SOD locus in vegetative buds of five Populus species and hybrid. In leaves of the four Populus species and hybrid and progenies of controlled crosses, EM and phenotypes of isozymes of two PER loci were identical to those of two SOD loci. In Picea species, EM of isozymes of the only SOD locus was somewhat similar but not identical to that of one PER locus, and isozyme phenotypes of all individuals at the SOD locus were not identical to those at a PER locus. Chi-square tests verified the single-gene Mendelian control of the segregating allozyme variants at each of Per-L1 and Sod-1 in the three Populus species. The results of joint two-locus segregation tests indicated a very tight linkage and no recombination between Per-L1 and Sod-1 in three Populus species. Genes coding for isozymes of one or two PER loci are either presumably the same as, or very tightly linked to, the genes coding for isozymes of one or two SOD loci in the Populus species.     

Implication of tissue expression of lactate dehydrogenase-C gene in phylogenetic study of euteleosts

An electrophoretic investigation of the lactate dehydrogenase isozymes in twelve euteleostean species was conducted. Expression of the LDH-C locus and association of the A and B subunits in these fishes is discussed. InChanos chanos this locus is found prevalent in the liver suggesting a close relation to otophysans. Presence of four iso-spaced A-B polymers in this species is a character different from otophysans which are provided with five iso-spaced A-B tetramers. Absence of tissue specificity of C4 band in all holocentrid species suggests a possible primitive phylogenetic status of this family in the Beryciformes. However, expression of the LDH-C locus provides no strong evidence for the resolution of the phylogenetic positions of the Polymixiidae and the other groups examined.     

Features of glycogen phosphorylase from the body wall musculature of the lugworm Arenicola marina and the mode of activation during anoxia

The activities of glycogen phosphorylases a and b from the body wall musculature of the marine worm Arenicola marina (Annelida, Polychaeta) were determined after various periods of anoxia. Already under normoxic conditions one third of the total activity was produced from the a form. During anoxia the ratio of both forms as well as the total activity did not change. The activity of soluble phosphorylase kinase was comparatively low in this tissue 4.3 +/- 1.2 nmol . min-1 . (g wet wt.)-1; the fast twitching tail muscle of shrimps, e.g., had a 10-fold higher phosphorylase kinase activity, whereas phosphorylase activities in both tissues were about the same 2.3 +/- 0.5 mumol . min-1 . (g wet wt.)-1. Glycogen phosphorylase b was purified from the body wall tissue of the marine worm in one step by 5'-AMP-Sepharose resulting in a single protein band in SDS-PAGE. This preparation was accepted as substrate by the phosphorylase kinase from rabbit muscle but a complete phosphorylation could not be achieved. The molecular mass of native phosphorylase was approximately 216 kDa, that of subunits 95 kDa indicating that the enzyme exists as a dimer. There were no isozymes in this preparation, the RF-value (0.17) of the single band in PAGE ranged between those of the isozymes from mice hearts. The activities of phosphorylases b and a were similarly dependent on pH and temperature but differed drastically in the affinities to phosphate and AMP. In presence of 1 mM AMP the app. Km of phosphorylase a for phosphate was 16 mM, that of phosphorylase b above 100 mM.(ABSTRACT TRUNCATED AT 250 WORDS)