Changes in sperm plasma membrane lipid diffusibility after hyperactivation during in vitro capacitation in the mouse.

We have used the technique of fluorescence recovery after photobleaching to measure the diffusibility of the fluorescent lipid analogue, 1,1'-dihexadecyl 3,3,3',3'-tetramethylindocarbocyanine perchlorate on the morphologically distinct regions of the plasma membranes of mouse spermatozoa, and the changes in lipid diffusibility that result from in vitro hyperactivation and capacitation with bovine serum albumin. We found that, as previously observed on ram spermatozoa, lipid analogue diffusibility is regionalized on mouse spermatozoa, being fastest on the flagellum. The bovine serum albumin induced changes in diffusibility that occur with hyperactivation are also regionalized. Specifically, if we compare serum incubated in control medium, which maintains normal motility, with those hyperactivated in capacitating medium, we observe with hyperactivation an increase in lipid analogue diffusion rate in the anterior region of the head, the midpiece, and tail, and a decrease in diffusing fraction in the anterior region of the head.

Identification of amino-acid residues linked to different properties of phosphoribosylpyrophosphate synthetase isoforms I and II.

The catalytic subunit of rat liver phosphoribosylpyrophosphate synthetase is composed of two isoforms, PRS I and PRS II. The amino-acid sequences differ only by 13 residues, out of which two Lys residues of PRS I at positions 4 and 152 give net additional positive charges to PRS I. Previous work has shown that PRS I is more sensitive to inhibition by ADP and GDP and more stable to heat treatment than is PRS II. To identify amino-acid residues responsible for the different properties, five chimeric enzymes between rat PRS I and PRS II and two mutated enzymes with a single point mutation at position 152 were constructed; these enzymes were produced in Escherichia coli. Changing Lys-4 of PRS I to Val, together with Ile-5 to Leu, completely abolished sensitivity to GDP inhibition of PRS I, indicating that Lys-4 in PRS I is critical for GDP inhibition. The substitutions at position 152 had little effect on GDP inhibition. Characterization of the chimeric enzymes revealed that residues between residues 54-110 and 229-317, namely, Val-55 and/or Ala-81, and Arg-242 and/or Cys-264 of PRS I also contribute to the strong GDP inhibition. Lys-4 was also important for the strong ADP inhibition of PRS I. Regarding the physical properties, chimeric enzymes bearing residues 12-53 of PRS I were stable at 49 degrees C and with digestion with papain and proteinase K. Our observations suggest that Lys-17, Ile-18, and/or Cys-40 of PRS I contribute to stability of the enzyme.

Cloning and sequencing of rat cDNA for the 41-kDa phosphoribosylpyrophosphate synthetase-associated protein has a high homology to the catalytic subunits and the 39-kDa associated protein.

Rat liver phosphoribosylpyrophosphate synthetase is a complex aggregate of 34-kDa catalytic subunits (PRS I and II) and 39- and 41-kDa associated proteins (PAP39 and 41). When the rat cDNA encoding PAP41 was isolated, the deduced protein sequence was seen to contain 369 amino acids with a calculated molecular mass of 41130. PAP41 has a 79 and 49% identity with PAP39 and PRSs, respectively. When conservative substitutions are included, PAP41 and the three other components have a 66% homology. PAP41 shares some common features with PAP39 and the two proteins form the PAP subfamily. The mRNA of PAP41 is present in all rat tissues we examined.

Partial reconstitution of mammalian phosphoribosylpyrophosphate synthetase in Escherichia coli cells. Coexpression of catalytic subunits with the 39-kDa associated protein leads to formation of soluble multimeric complexes of various compositions.

Rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of 34-kDa catalytic subunits (PRS I and PRS II) and homologous 39- and 41-kDa proteins termed PRPP synthetase-associated proteins (PAPs). While a negative regulatory role was indicated for PAPs, the physiological function of PAPs is less well understood. We attempted to prepare recombinant 39-kDa PAP (PAP39) and to reconstitute the enzyme complex. Free PAP39 was poorly expressed in Escherichia coli, while expression of protein fused with glutathione S-transferase was successful. The purified fusion protein had no PRPP synthetase activity, and bound to dissociated PRS I and PRS II, with a similar affinity. A free form of PAP39 prepared from the fusion protein formed insoluble aggregates. The enzyme complex was then partially reconstituted in situ by coexpression of PAP39 with PRS I or PRS II in E. coli cells. This coexpression led to formation of soluble complexes of various compositions, depending on the conditions. When the relative amount of PAP39 was higher, specific catalytic activities, in terms of the amount of the catalytic subunit, were lowered. PAP39 complexed with PRS I was more readily degraded by proteolysis than seen with PRS II, in vivo and in vitro. These results provide additional, strong evidence for that PAP39 has no catalytic activity in the enzyme complex, but does exert inhibitory effects in an amount-dependent manner, and that composition of the enzyme complex varies, depending on the relative abundance of components present at the site of aggregate formation.

Rat liver phosphoribosylpyrophosphate synthetase is activated by free Mg2+ in a manner that overcomes its inhibition by nucleotides.

Phosphoribosylpyrophosphate synthetase is activated by Pi and free Mg2+ as an essential activator and inhibited by nucleotides, especially ADP and GDP. The rat liver enzyme is a complex aggregate of two highly homologous catalytic subunits (PRS I and PRS II) and two associated proteins (PAP39 and PAP41). PRS I is more sensitive to inhibition by ADP and GDP than is PRS II. The native liver enzyme showed a weaker sensitivity to inhibition by nucleotides than expected from its composition. To further understand the regulation of the liver enzyme, kinetic studies of each subunit component and the liver enzyme regarding Mg2+ activation and inhibition by ADP and GDP were carried out. Assay conditions were designed to keep free Mg2+ at constant concentrations. (1) GDP, as MgGDP, did not affect the apparent Km values of PRS I for MgATP and ribose-5-phosphate but did dramatically increase the apparent Ka value for free Mg2+. (2) In contrast, ADP, as MgADP, increased the Km value for MgATP of PRS I as well as the Ka value for free Mg2+. (3) High concentrations of free Mg2+ almost completely nullified the inhibitory effect of MgGDP and partly that of MgADP on PRS I. (4) At low free Mg2+ concentrations within the physiological range, inhibition by the nucleotides is of physiological significance and conversely, variation in free Mg2+ concentrations critically affects the enzyme activity in the presence of inhibitory nucleotides. (5) The response of PRS II and the native liver enzyme is similar to that of PRS I, while the effects of MgGDP and MgADP were smaller than that on PRS I. (6) We propose that MgGDP binds to a regulatory site of PRS I and PRS II and MgADP to the substrate MgATP site and also the regulatory site. The allosteric interaction of the regulatory site and the Mg2+ binding site is also considered.

Promoter regions of the human X-linked housekeeping genes PRPS1 and PRPS2 encoding phosphoribosylpyrophosphate synthetase subunit I and II isoforms.

The 5' regions of the human phosphoribosylpyrophosphate synthetase subunit I and II genes (PRPS1 and PRPS2, respectively) were isolated and sequenced. A comparison of the nucleotide sequences between human and rat PRPS1 genes revealed that the sequences around the transcription initiation sites were conserved over 56 nucleotides, and that a TATA-like sequence, a CCAAT box and three putative Sp1 binding sites were present at almost the same positions in the GC-rich sequences. Two major transcription initiation sites were localized in the human PRPS1, one of the two was located 27 nucleotides downstream from the TATA-like sequence, while the upstream initiation site was in the TATA-like sequence. The promoter region of the human PRPS2 gene was also GC-rich and contained a TATA-like sequence, four Sp1 binding sites and a homopyrimidine stretch. The initiation sites were localized at 90 nucleotides upstream from the ATG initiation codon. Chloramphenicol acetyltransferase (CAT)/promoter fusion assays showed that a 2.0 kb region (human PRPS1) and a 1.1 kb region (human PRPS2) possessed the promoter activities in four cell lines. The CAT activities in the three human cell lines tended to correlate with the steady-state mRNA levels of the PRPS1 and PRPS2 genes. These results suggest that the 5' flanking regions cloned contribute to the cell-differential expression of these two genes.

Cloning and sequencing of human complementary DNA for the phosphoribosylpyrophosphate synthetase-associated protein 39.

A human cDNA encoding a human homologue of the rat phosphoribosylpyrophosphate synthetase-associated protein of 39 kDa was isolated. The deduced protein contains 356 amino acids and has calculated molecular mass of 38561. The amino acid sequence is 98% identical to that of the rat. The corresponding mRNA is present in all human tissues examined.

Molecular cloning and sequencing of human cDNA for phosphoribosyl pyrophosphate synthetase subunit II.

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit II (PRS II) were isolated. The five overlapping clones contained 2457 base pairs (bp) covering a 954-bp complete coding region for 318 amino acid residues. Homologies between human and rat PRS II were 99% of the amino acids and 88% of the nucleotides in the coding region. This amino acid homology seems to be the highest so far reported for enzymes involved in nucleotide metabolism and glycolysis. The highly conserved structure may be required for unique catalysis and rigid regulation of this enzyme.

Understanding the mechanism of the age-change of thymic function to promote T cell differentiation.

Immunological functions peak at around puberty and gradually decline thereafter with advancing age. The immunological decline mainly occurs in the T cell-dependent immune system and is generally associated with an increase in not only susceptibility to infections but also incidence of autoimmune phenomena. The age-related changes in T-cell dependent immune functions can be mainly ascribed to the physiological thymic involution which starts in the early phase of life. The age-related thymic involution can be ascribed to either extrinsic or intrinsic factors. Bone marrow stem cells can be one of the extrinsic factors for the thymic involution, but their role is estimated to be marginal as compared with alteration of the thymic microenvironment. With advancing age, the thymic capacity to promote T-cell differentiation declines together with a change in the composition of T-cell subsets produced. Such an alteration of the thymic environment is responsible for the age-related change in peripheral T cells in number and in composition. Age change is observed in several intrinsic factors in the thymic environment which influence proliferation of thymocytes. These thymic intrinsic factors can either promote or inhibit proliferation of thymocytes, and promoting factors generally decrease with age with a concomitant increase in inhibitory factors. Various endocrine hormones are important extrinsic factors influencing the thymic function. In fact, physiological thymic involution can be intervened by manipulation of the endocrine system, sometimes resulting in rejuvenation of immune functions to a certain extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid mobilization of intracellular Mg2+ by bombesin in Swiss 3T3 cells: mobilization through external Ca(2+)- and tyrosine kinase-dependent mechanisms.

We examined the effects of growth factors on intracellular free Mg2+ concentrations ([Mg2+]i) in single Swiss 3T3 fibroblasts, using microfluorometry of a Mg(2+)-sensitive dye mag-fura-2. We had already noted an increase in [Mg2+]i after exposure to bombesin for 30-60 min [Ishijima, S., Sonoda, T., & Tatibana, M. (1991) Am. J. Physiol. 261 (Cell Physiol. 30), C1074-C1080]. In the present work, we found that bombesin also induced early changes in [Mg2+]i. The [Mg2+]i reached peak values within 15 s in most cells, and the significant rise lasted only for 1-2 min. The extent of the increase varied from cell to cell (0-600 microM above basal). On the average, the [Mg2+]i was increased from basal 0.33 to 0.54 mM. Since the time course was similar to that of [Ca2+]i changes, and the dye mag-fura-2 also binds Ca2+, we evaluated Ca2+ interference with measurement of [Mg2+]i. The contribution of Ca2+ binding would be below 20% of the mag-fura-2 signal. The bombesin-induced [Mg2+]i increase was not dependent on external Mg2+, but the omission of external Ca2+ decreased by 60% the [Mg2+]i increase, and the Ca2+ channel blocker, nicardipine inhibited by 90% the [Mg2+]i response. This inhibition was partially reversed by raising the concentration of external Ca2+. Two structurally distinct tyrosine kinase inhibitors, genistein and lavendustin A, almost completely inhibited the [Mg2+]i response. These results suggest that bombesin rapidly induces Mg2+ mobilization from the intracellular pool, through external Ca(2+)- and tyrosine kinase-dependent mechanisms.

Phosphoenolpyruvate carboxylase of Escherichia coli K-12. N- and C-terminal sequences and tentative assignment of the catalytically essential cysteine residue.

The N- and C-terminal amino acid sequences of phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were determined to establish the primary structure deduced from the nucleotide sequence of the cloned gene for the enzyme (Fujita, N., Miwa, T., Ishijima, S., Izui, K., & Katsuki, H. (1984) J. Biochem. 95, 909-916). As predicted from the nucleotide sequence, two polypeptides were produced upon treatment with hydroxylamine, which specifically cleaves the Asn-Gly bond, and their amino acid compositions were also in accordance with those predicted. The tryptic peptides which contained cysteine residues labeled with a fluorescent reagent, N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide, were isolated by high-performance liquid chromatography and partially sequenced. All of them could be assigned on the deduced primary structure. The modified cysteine residues were Cys-157, Cys-385, Cys-458, Cys-568, Cys-665, and Cys-754. Furthermore, the essential cysteine residue which is presumably located at or near the active site was tentatively identified as Cys-568, since it was consistently protected against the modification by 2-phospholactate, a substrate analog.

The primary structure of phosphoenolpyruvate carboxylase of Escherichia coli. Nucleotide sequence of the ppc gene and deduced amino acid sequence.

The nucleotide sequence of the ppc gene, the structural gene for phosphoenolpyruvate carboxylase [EC 4.1.1.31], of Escherichia coli K-12 was determined. The gene codes for a polypeptide comprising 883 amino acid residues with a calculated molecular weight of 99,061. The amino acid sequence deduced from the nucleotide sequence was entirely consistent with the protein chemical data obtained with the purified enzyme, including the NH2- and COOH-terminal sequences and amino acid composition. The coding region is preceded by two putative ribosome binding sites, and is followed closely by a good representative of rho-independent terminator. The codon usage in the ppc gene suggests a moderate expression of the gene. The secondary structure of the enzyme was predicted from the deduced amino acid sequence.

Complete nucleotide sequence of human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) cDNA and a comparison with human and rat PRPS gene families.

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit I (PRS I) were isolated from a glioblastoma cell line MGC 1 cDNA library. The longest clone contained 2,075 base pairs (bp) almost covering the 2.3-kb mRNA and the base sequence of the coding region (954 bp) had a 92.0% sequence homology with that of rat PRS I cDNA. The deduced amino acid sequences were identical between human and rat PRS I. This perfect conservation has heretofore not been reported for other enzymes involved in nucleotide metabolism and glycolysis. A comparison with other isoforms of this enzyme, PRS II and PRS III, showed that the human PRS I was 79.9 and 92.2% homologous in the coding sequence and 95.3 and 94.0% in the deduced amino acid sequence to human PRS II and PRS III, respectively. The high value of the synonymous difference between PRS I and PRS II cDNAs places their time of divergence long before that of the radiation of mammals. Based on the evolutionary rate of amino acid substitution, the PRS I and II genes probably diverged about 760 million years ago.

Evidence for early mitogenic stimulation of metabolic flux through phosphoribosyl pyrophosphate into nucleotides in Swiss 3T3 cells.

Various mitogens activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent fibroblasts. Increased synthesis of 5-phosphoribosyl 1-pyrophosphate (PRPP) may precede or underlie these activations, but little direct evidence has been presented for this notion, due to lack of suitable analytical methods. To preferentially label intracellular ribose phosphate and quantitatively follow metabolic flux through PRPP into nucleotides, we prepared [ribosyl-14C]inosine and used it as a tracer. Evidence showed the validity of this method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor plus insulin for 45-60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl-14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [14C]Uracil incorporation into nucleotides, a measure of PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that epidermal growth factor plus insulin stimulates the metabolic flux through PRPP. Similar extents of stimulation were induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone, suggesting the presence of an unknown signaling pathway common to these mitogens. This system is highly useful for studies of the mechanisms that stimulate in situ activity of PRPP synthetase.

Preparation and characterization of monoclonal antibodies against phosphoenolpyruvate carboxylase of Escherichia coli.

Twelve hybridoma clones which secrete monoclonal antibodies (mAb) against purified phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were obtained. These 12 mAb were prepared from the ascites fluids of mice. Six among the 12 mAb formed precipitin lines with the enzyme on immunodiffusion. Four mAb inhibited the activity of the enzyme and 2 mAb enhanced it. Four mAb altered the sensitivity of the enzyme to allosteric effectors. Competitive enzyme-binding experiments among the 12 different mAb were also performed. The results showed that the 12 mAb can be classified into at least 8 groups.

Kinetic and regulatory properties of rat liver phosphoribosylpyrophosphate synthetase complex are partly distinct from those of isolated recombinant component catalytic subunits.

Rat liver phosphoribosylpyrophosphate (PRPP) synthetase exists as complex aggregates composed of two catalytic subunits (PRS I and II, in a ratio of approximately 4:1) and two catalytically inactive PRPP synthetase-associated proteins. To better understand the significance of the complex structure, the properties of the native liver enzyme were compared with those of homologous aggregates of recombinant PRS I and PRS II (rPRS I and rPRS II). (1) The specific activity per catalytic subunits of the liver enzyme was about 2.5 times lower than that of rPRS I over a wide pH range. Km values for substrates and Ka values for Pi and Mg2+ of the three enzymes were similar. (2) Specific activity of the liver enzyme for the reverse reaction was about 2 times lower than those of rPRSs. Km values for substrates of the three enzymes were comparable. (3) The liver enzyme was more stable than were rPRSs when incubated at a high temperature or in the absence of stabilizing agents. (4) The liver enzyme was markedly less sensitive to inhibition by nucleotides compared to rPRS I. GDP at 1 mM inhibited the liver enzyme and rPRS I by 32 and 93%, respectively. This effect is not ascribable to molecular interaction between rPRS I and II, as reconstitution of the two did not alter the sensitivity to nucleotide inhibition. (5) Our observations suggest that complex aggregation states of the native enzyme not only suppress the activities but also stabilize the catalytic subunits and the associated proteins and remarkably reduce the sensitivity to inhibition by nucleotides.

Ultrastructure of cell wall of the cps8 actin mutant cell in Schizosaccharomyces pombe.

A Schizosaccharomyces pombe cps8 mutant, of which the gene encodes a mutant actin with an amino acid substitution of Asp for Gly(273) [J. Ishiguro and W. Kobayashi (1996) FEBS Lett. 392, 237-241], was used to determine the role of the actin cytoskeleton in cell wall formation. In the cps8 mutant cells, atomic force microscopic and scanning electron microscopic images showed abnormal depolarized and branched morphology. Fibrous material covered a part of the surface of growing cps8 cells. Transmission electron microscopic images showed variable thickness of the cell wall due to multilayering of cell wall materials, and aberrant multisepta due to diagonal growth of the primary septum, whereas the normal primary septum grows at a right angle from the cortex. This abnormal septum formation may induce abnormality of the cell with multinuclei and/or multisepta, caused by non-separation of daughter cells. These results indicate that actin plays an important role in cell wall and septum formation.

Morphological effects of itraconazole on murine macrophage.

Morphological effects of itraconazole (ITCZ) on murine macrophages were examined by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and compared with the effects of other antifungal agents. Thioglycolate-induced peritoneal macrophages were prepared from C3H/He J mice and cultured for 20 h in the presence of the antifungal azoles econazole, fluconazole, miconazole, ketoconazole, ITCZ, hydroxy-itraconazole (ITCZ-OH), and a polyene antibiotic amphotericin B (AMPH). Among these reagents, only ITCZ and its derivative ITCZ-OH were effective in causing morphological changes of murine macrophage as determined by LM and SEM. Macrophages treated with 2 microg/ml ITCZ or ITCZ-OH were stretched out bidirectionally, their surface was smooth and their 'ruffles' decreased. TEM observation showed that the bundles of the filamentous structure existed along the cell shape in the cytoplasm. These findings suggest that ITCZ and ITCZ-OH affect the morphology of macrophages.

Early mitogenic stimulation of metabolic flux through phosphoribosyl pyrophosphate into nucleotides in Swiss 3T3 cells and requirement of external magnesium for the response.

5-Phosphoribosyl 1-pyrophosphate (PRPP) is a common precursor for the synthesis of all nucleotides and also serves as a critical regulator for the synthesis. In spite of a number of studies in vitro on mammalian PRPP synthetase, our understanding of the regulation of PRPP synthesis in situ is very limited. Various mitogens are known to activate purine and pyrimidine de novo biosynthesis and purine base phosphoribosylation as an early response in quiescent mouse fibroblasts. We aimed at elucidation of the underlying mechanism for the possible increase in PRPP synthesis in mitogen-stimulated mouse fibroblasts in culture. In order to quantitatively follow metabolic flux through PRPP into nucleotides, [ribosyl-14C]inosine was enzymatically prepared and used as a tracer to preferentially label intracellular ribose phosphate. The radioactivity incorporation into cellular nucleotides was measured. Evidence supported the validity of the method. Prior exposure of quiescent Swiss 3T3 cells in culture to epidermal growth factor (EGF) plus insulin for 45-60 min enhanced approximately 2-fold the radioactivity incorporation from [ribosyl-14C]inosine into nucleotides, without increasing the specific radioactivity of intracellular free ribose 5-phosphate. [14C]Uracil incorporation into nucleotides, a measure for PRPP-independent ribose phosphate utilization for nucleotide synthesis, was not increased. These and other results indicate that EGF plus insulin stimulates the metabolic flux through PRPP. A similar stimulation was induced by bombesin and melittin in combination with insulin and by fibroblast growth factor alone. Quiescent Swiss 3T6 cells and human fetal fibroblasts showed a similar stimulation of nucleotide synthesis in response to exposure to serum. For characterization of intracellular signaling pathways, we examined effects of several inhibitors and agents on the stimulation. The divalent cation ionophore A23187 mimicked the response to EGF and insulin in Swiss 3T3 cells, thereby suggesting involvement of divalent cation mobilization in this increase. The effect of the ionophore was not additive to that of the growth factors. Omission of Ca2+ from the incubation medium did not affect the response to EGF and insulin, whereas the omission of Mg2+ did abolish the response. Furosemide, an inhibitor of Mg2+ influx, partially inhibited the stimulated synthesis of nucleotides. Thus, the entry of external Mg2+ into the cells may play a critical role in this signal transduction. These results provided an important access to elucidation of the intracellular mechanisms for the mitogen-induced increase in PRPP and nucleotide syntheses.

Mammalian phosphoribosyl-pyrophosphate synthetase.

PRPP synthetase from rat liver exists as large molecular weight aggregates composed of at least three different components. Cloning of cDNA for the catalytic subunit revealed the presence of two highly homologous isoforms of 34 kDa, designated as PRS I and PRS II. Northern blot analysis showed tissue-differential expression of the two isoform genes. cDNA was expressed in E. coli and studies on the recombinant isoforms showed differences in sensitivity to inhibition by ADP and GDP and to heat inactivation. The rat gene for PRS I has 22 kb and is split into 7 exons. cDNAs for human enzymes were also cloned. Human genes for PRS I and PRS II are localized at different regions on the X-chromosome and their promoter regions were examined. Another component, PRPP synthetase-associated protein of 39 kDa (PAP39), was cloned from cDNA library of the rat liver. The deduced amino acid sequence of PAP39 is remarkably similar to those of PRS I and PRS II. Evidence indicated molecular interaction between PAP39 and the catalytic subunits and an inhibitory effect of PAP39 on the catalytic activity. Expression of the PAP39 gene is tissue-differential like the PRS genes, indicating that the composition of PRPP synthetase may differ with the tissue, hence properties of the enzyme would differ. Further studies on these components and their interaction are expected to reveal various mechanisms governing mammalian PRPP synthetase.