Immunofluorescent localization of PGK-1 and PGK-2 isozymes within specific cells of the mouse testis

The ubiquitous isozyme of phosphoglycerate kinase, PGK-1, and the testis-specific isozyme, PGK-2, have been localized to specific cells of the testis by indirect immunofluorescence on Bouin's fixed testis sections. The earliest cell of the spermatogenic series in which PGK-2 is detectable by immunofluorescence is the Stage 12 spermatid. The intensity of fluorescence increases as the spermatids progress to later stages and is strong in both released spermatids and their residual bodies. PGK-2 is not detectable in premeiotic germinal cells or somatic cells of the testis. Specific fluorescence for PGK-1 is localized to the somatic cells of the testis: the interstitial and Sertoli cells.     

Developmental program of PGK-1 and PGK-2 isozymes in spermatogenic cells of the mouse: Specific activities and rates of synthesis

The specific activities and synthesis of the ubiquitous isozyme, PGK-1, and the testis-specific isozyme, PGK-2, have been quantitated and localized in spermatogenic cells of the mouse. There is a fivefold increase in total PGK specific activity between immature and adult testes which begins at approximately 30 days of age, coincident with the appearance of late-middle stage spermatids. The increase in total PGK is entirely due to the appearance and increase of the PGK-2 isozyme. Rates of PGK synthesis were measured by labeling testicular cells in vitro with [³H]leucine and purifying the PGK isozymes. When total testicular cells were examined, PGK-2 synthesis was detectable after 22 days of age at very low levels and increased in older testes to a level of 0.5% of total protein synthesis. PGK-1 synthesis remained relatively constant at all ages at a level 100-fold lower (0.005%). Testicular cells were separated into highly enriched fractions of particular spermatogenic stages by centrifugal elutriation. The PGK-1 synthesis rates were, again, very low and not significantly different between the various spermatogenic stages. PGK-2 synthesis was low to nondetectable in pachytene spermatocytes, increased to 0.07% in early spermatids and represented 0.7% of total protein synthesis in late spermatids. This increased rate of PGK-2 synthesis appears to require an increase in the amount of PGK-2 mRNA in late spermatids, cells in which no active RNA synthesis is detectable.     

Immunohistochemical localization of mouse testis-specific phosphoglycerate kinase (PGK-2) by monoclonal antibodies.

Monoclonal antibodies against mouse testis-specific phosphoglycerate kinase (PGK-2) were produced in order to determine immunohistochemically the onset of PGK-2 synthesis in the germinal epithelium of the mouse. PGK-2 was detected in testis sections in spermatids as early as stage 12 and in spermatozoa, but not in earlier stages of spermatogenesis nor in any somatic cells of the testis. During ontogeny, PGK-2 appears within the testis at day 30 post-partum, concomitant with spermatids entering the maturation phase. All three allelic isozymes PGK-2A, -2B, and -2C were detected equally by the monoclonal antibody in testis sections of several inbred mouse strains, each of which expresses a specific PGK-2 variant. Moreover, the monoclonal antibody against mouse PGK-2 reacted with heterologous sperm-specific PGK from rat, rabbit, and bull and, therefore, may serve as a useful immunochemical marker for mammalian spermatogenesis.     

Immunological and structural relatedness of isozymes and genetic variants of 3-phosphoglycerate kinase from the mouse

Isozymes (PGK-1 and PGK-2) and genetic variants (PGK-2A, PGK-2B, and PGK-2C) of 3-phosphoglycerate kinase were purified by affinity chromatography using an 8-(6-aminohexyl)-amino-ATP-Sepharose column as the key step. Antisera raised against purified PGK-1 and PGK-2A were tested for specificity and cross-reactivity by application of double immunodiffusion and enzyme immunoinactivation methods. By double immunodiffusion, no precipitin lines were observed between anti-PGK-2A and PGK-1, but a weak cross-reactivity between anti-PGK-1 and PGK-2A was detected. In addition to specific inhibition of PGK-1 and PGK-2A by their respective antisera, anti-PGK-1 was shown to inhibit PGK-2 activity at high antiserum concentrations, whereas no inhibition of PGK-1 activity by anti-PGK-2A was observed. The amino acid compositions of PGK-1 and PGK-2 revealed a certain degree of homology. However, tryptic peptide maps showed no obvious similarity in the peptide spots between these two 3-phosphoglycerate kinase isozymes. Three electrophoretic variants of PGK-2 were compared biochemically and immunologically. PGK-2C from C57L/J mice, a low activity variant, was shown to be the result of a structural gene mutation that affects the active site of the enzyme.     

The Expression of X-Linked Phosphoglycerate Kinase in the Early Mouse Embryo

Abstract. During early mouse embryogenesis, the activity of X-chromosomally linked maternal and paternal phosphoglycerate kinase (PGK-1) alleles was determined using electrophoretic separation of their gene products and a sensitive fluorometric enzyme assay. In the embryos collected from females homozygous for PGK-1^b mated with PGK-1^a males and vice versa, the paternally derived allozyme was first detected after implantation on day 6. Expression of the maternally inherited allele was studied in embryos from females heterozygous for PGK-1^b and PGK-1^a. From day 1 to day 4, the embryos maintained a constant ratio of enzyme activity of PGK-1B to PGK-1A. Prior to implantation of the embryos between day 4 and day 5, the activity ratio of the two PGK-1 allelic variants changed significantly due to the first appearance of newly synthesized PGK derived from the maternally inherited allele.
Our data demonstrate a temporal difference in the onset of PGK synthesis depending on whether this particular gene product is of maternal or paternal origin. Therefore, we conclude that the maternal PGK-1 locus is already activated during late preimplantation development whereas the paternally inherited gene locus remains silent at the preimplantation stage but is subsequently expressed at approximately the time of X-chromosomal inactivation.     

Sequential expression of maternally inherited phosphoglycerate kinase-1 in the early mouse embryo

Enzyme activities of X-linked phosphoglycerate kinase (PGK-1) and autosomal glucose phosphate isomerase (GPI-1) were determined in intact mouse blastocysts and isolated inner cell masses (ICMs). Blastocysts were recovered from the uterus on day 4 of gestation and cultured overnight in vitro. ICMs were isolated by treatment with calcium ionophore A23187. On day 4, approximately 35% of the total activity of both PGK-1 and GPI-1 was located in the ICM. After overnight culture, the PGK-1 activity of the whole blastocyst nearly doubled, due to the activation of only the maternally derived gene coding for PGK-1. In the ICM, however, a pronounced decrease of PGK-1 activity was measured: only 10% of the total PGK-1 activity was measured in the ICM on day 5. In contrast to PGK-1, GPI-1 activity of the intact blastocyst remained stable from day 4 to day 5. In the ICM, the GPI-1 activity did decline, but to a lesser extent than PGK-1 activity: 20% of total GPI-1 activity was found in the ICM on day 5. These results, when compared with the data of Handyside and Hunter, suggest that the decline in GPI-1 activity in the ICM is due to a change in the ratio of trophectoderm (TE) to ICM cells. The greater reduction of PGK-1 activity in the ICM cannot, however, be explained solely by this mechanism. To explain the observed additional decrease, we postulate that Pgk-1 is not activated in the ICM prior to day 6. This implies that on day 4 maternal Pgk-1 is activated in the TE exclusively.     

Expression of X-linked phosphoglycerate kinase in early mouse embryos homozygous at the Xce locus

The expression of maternally derived X-chromosomal Pgk-1 alleles was investigated in oocytes and early embryos of mice carrying different alleles (Xcea, Xcec) of the X-chromosome controlling element (Xce) locus. Pgk-1 allelic expression was determined by measuring their gene products using Cellogel electrophoresis and a sensitive fluorimetric enzyme assay. In addition to the already existing mouse strain of the genotypes Pgk-1a Xcec and Pgk-1b Xcea, a new line was bred carrying the combination Pgk-1b Xcec. The X chromosomes carrying the combinations Pgk-1a Xcec and Pgk-1b Xcec were of feral origin, whereas Pgk-1b Xcea was derived from a laboratory line. Our results using Xcec homozygous females confirm that maternal Pgk-1 is already expressed on day 4 of embryogenesis, thus substantiating data previously obtained using Xcea/Xcec heterozygous females. This finding also demonstrates that the timing of reactivation of maternal Pgk-1 is not influenced by the Xce locus. Furthermore, we found that oocytes from Xcec homozygous females have a balanced PGK-1 A/PGK-1 B allozyme ratio (50:50), whereas in oocytes obtained from Xcea/Xcec heterozygotes, the PGK-1 allozyme ratio is about 60:40. In tissues of adult Xce homozygous females, the PGK-1 allozymes are also balanced, whereas in Xcea/Xcec heterozygous females, the ratio is about 35:65. In addition to the relative activity of the PGK-1 allozymes, we also measured the absolute activity of PGK-1 in oocytes obtained from three types of Xce homozygous females.(ABSTRACT TRUNCATED AT 250 WORDS)     

PCR derived cDNA clones for X-linked phosphoglycerate kinase-1 in a marsupial, the tammar wallaby (Macropus eugenii).

cDNA clones for the X-linked PGK-1 were obtained from a tammar wallaby liver by PCR and sequenced. The PGK-1 gene published here is the consensus sequence of those clones. The sequence represents an open reading frame of 1251 bp. Sequence comparisons to X-linked and autosomal sequences showed the greatest homology with the X-linked PGK-1 genes in eutherian species. This sequence opens the way for studying the paternal X inactivation phenomenon in marsupials and will assist in defining the time course of mammalian evolution.     

Biochemical and immunological studies of three genetic variants of 3-phosphoglycerate kinase 2 from the mouse

Three electrophoretic variants of 3-phosphoglycerate kinase 2 (PGK-2A, PGK-2B, and PGK-2C) were purified from DBA/2J, C3H/HeJ, and C57L/J mice, respectively. PGK-2C exhibits only 2% of the specific activity of PGK-2A and PGK-2B in the reaction leading to the formation of 1,3-diphosphoglycerate. Compared to PGK-2A and PGK-2B, PGK-2C exhibits broader coenzyme specificity and lower K_ms for substrate and coenzymes. Incubation at 45C revealed that PGK-2B is more heat stable than either PGK-2A or PGK-2C. Enzyme immunoinactivation and double immunodiffusion studies showed that mice carrying any one of these three PGK-2 alleles have similar amounts of proteins for PGK-1 and PGK-2 in testes. The results of these studies suggest that low PGK-2C activity in C57L/J mice is a result of a structural rather than a regulatory gene mutation.     

Distinct temporospatial expression patterns of glycolysis-related proteins in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) represents the sixth most frequent human cancer worldwide and is characterized by rapid progression as well as resistance to systemic chemotherapy. Recently, glycolysis has emerged as a potent driving force of tumor growth and therapy failure. The precise role of glycolysis for the pathogenesis of human HCC has not been elucidated thus far. Therefore, we have conducted a comprehensive analysis of the expression patterns of central glycolysis-related factors [glucose transporter-1 and -2 (Glut-1 and Glut-2), phosphoglycerate kinase-1 (PGK-1) and hypoxia-inducible factor-1α (HIF-1α)] in a large cohort of benign and malignant human liver samples. PGK-1 protein and gene expression was scant in normal liver, elevated in cirrhotic livers and most intense in HCC. Strong immunoreactivity of Glut-2 was noted in cirrhotic livers, whereas in HCC it was only expressed in 50% of examined cases. Strikingly, PGK-1 as well as Glut-2 protein expression was indicative of poor patient prognosis. Glut-1 protein was absent in neoplastic hepatocytes but prominent in tumor-associated endothelial cells. Specific nuclear staining of HIF-1α was noted in only 12% of HCC samples. Our data point toward a tumor-promoting function of glycolysis in HCC and establish PGK-1 as an independent prognostic parameter. Furthermore, the endothelial-specific expression of Glut-1 makes a special dependence of vessels on glucose reasonable to assume. In summary, we believe our analysis warrants the validation of glycolytic inhibitors as innovative treatment approaches of human HCC. Christoph Benckert and Thorsten Cramer have contributed equally to this work.     

Electrophoretic variation for x-chromosome-linked phosphoglycerate kinase (pgk-1) in the mouse

Electrophoretic variation for X-chromosome-linked phosphoglycerate kinase (PGK-1) has been found as a polymorphism in feral mice in Denmark. Males from feral sampling or from a variety of genetic crosses have only a single-banded phenotype of the variant PGK-1A type or of the PGK-1B type commonly found among inbred mice. By contrast, three phenotypes were observed among females; two homozygous single-banded types and a heterozygous double-banded type. The X-chromosome linkage of the Pgk-1 locus was determined from the mode of inheritance in F₁ and backcross generations and confirmed by the linkage of Pgk-1 and the X-linked markers Hq, Ta and Mo. Pgk-1 showed 29/122 recombinations with Hq, 5/185 with Ta and 0/108 recombinants with Mo. Based on these recombination data, a gene order of Hq—Ta—Pgk-1—Mo is suggested.     

Polymorphisms in the coding and noncoding regions of murinePgk-1 alleles

The mouse X-linkedPgk-1 gene encodes phosphoglycerate kinase. When transfected into human cells, thePgk-1^b allele causes the appearance of mouse PGK-1^b enzyme activity. We describe here cloning of mousePgk-1^a, an allele ofPgk-1 which encodes an enzyme, PGK-1^a, with distinct electrophoretic mobility. We constructed recombinants between the DNA encodingPgk-1^b andPgk-1^a and transfected these constructs into human to assess the electrophoretic characteristics of each recombinant. In this way the charge variation between the two proteins was localized to exons 4 or 5. Sequencing of these exons revealed a single base-pair difference between the two alleles at codon 155, which predicts the amino acids lysine and threonine in PGK-1^b and PGK-1^a, respectively. A number of other DNA sequence polymorphisms exist betweenPgk-1^b andPgk-1^a including part of an L1 repeated element unique toPgk-1^a. This work was supported by the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Deutsche Forschungsgemeinschaft, SFB 304.     

The distribution of 4 genes (alpha GAL, PGK-1, HPRT and G6PD) on the X chromosome of the American mink (Mustela vison)

The segregation of X-linked markers (alpha GAL, PGK-1, HPRT and G6PD) was analysed in hybrids between gamma ray-irradiated mink fibroblasts and Chinese hamster cells, or between mink cells and mouse hepatoma cells. Based on the segregation data and the data of cytogenetics analysis of a few hybrids, the order of the mink genes was deduced as alpha GAL--PGK-1--HPRT--G6PD--qter. This order differs from that reported for human and murine genes, in spite of the very obvious similarity between G-banding of the mink and human X chromosomes. Therefore, at least one reversion is responsible for the differences observed for the human and mink X chromosomes.     

Haploid accumulation and translational control of phosphoglycerate kinase-2 messenger RNA during mouse spermatogenesis

The intracellular location of the mRNA for the testis-specific isozyme of phosphoglycerate kinase-2 (PGK-2) has been determined for two spermatogenic cell types. The mRNA activity for PGK-2 from the polysomal and nonpolysomal fractions of pachytene primary spermatocytes or round spermatids has been assayed by cell-free translation with the polypeptide products monitored by immunoprecipitation, followed by one-dimensional or two-dimensional electrophoresis and fluorography. The results reveal that the majority of PGK-2 mRNA activity of round spermatids was present in the polysomal fraction while the relatively less abundant PGK-2 mRNA of pachytene primary spermatocytes was present in the nonpolysomal fraction. No PGK-2 mRNA activity was observed in the cytoplasmic RNA from primitive type A spermatogonia or prepubertal Sertoli cells. These data indicate that mature PGK-2 mRNA first appears in the cytoplasm of spermatogenic cells during the prophase of meiosis and increases in amount after meiosis. Although mature PGK-2 mRNA is present in meiotic cells it is not actively translated until after meiosis has been completed. Thus, mRNA accumulation and translational mechanisms are involved in the control of phosphoglycerate kinase-2 synthesis during spermatogenesis.     

The nucleotide sequence of a cDNA clone containing the entire coding region for mouse X-chromosome-linked phosphoglycerate kinase

A clone containing cDNA for X chromosome-linked phosphoglycerate kinase (PGK-1) was isolated from a mouse myeloma cDNA library. The nucleotide (nt) sequence of the cDNA has been determined, and the amino acid (aa) sequence of the enzyme thereby deduced. At the nt level, the coding region of mouse PGK cDNA has 93% homology with human X-linked cDNA and 60% homology with the yeast gene. Mouse PGK-1 protein contains 416 aa and is 98%, 96% and 64% homologous with human, horse, and yeast enzyme sequences, respectively.