Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Precursor limitation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources
PubMed2004

Treatment of spinal cord injury by transplantation of fetal neural precursor cells engineered to express BMP inhibitor.

Spontaneous recovery after spinal cord injury is limited. Transplantation of neural precursor cells (NPCs) into lesioned adult rat spinal cord results in only partial functional recovery, and most transplanted cells tend to differentiate predominantly into astrocytes. In order to improve functional recovery after transplantation, it is important that transplanted neural precursor cells appropriately differentiate into cell lineages required for spinal cord regeneration. In order to modulate the fate of transplanted cells, we advocate transplanting gene-modified neural precursor cells. We demonstrate that gene modification to inhibit bone morphogenetic protein (BMP) signaling by noggin expression promoted differentiation of neural precursor cells into neurons and oligodendrocytes, in addition to astrocytes after transplantation. Furthermore, functional recovery of the recipient mice with spinal cord injury was observed when noggin-expressing neural precursor cells were transplanted. These observations suggest that gene-modified neural precursor cells that express molecules involved in cell fate modulation could improve central nervous system (CNS) regeneration.

Animals↗
PubMed1992

Evidence that umbilical cord blood contains a higher frequency of HLA class II-specific alloreactive T cells than adult peripheral blood. A limiting dilution analysis.

Umbilical cord blood has been used to effect hematological reconstitution and there are sufficient stem cells available in the cord blood obtainable from a single placenta to reconstitute an adult patient. Umbilical cord blood might therefore, have widespread potential use in the field of bone marrow transplantation. We compared alloreactivity of paired samples of adult and cord peripheral blood mononuclear cells, by measuring frequencies of both alloreactive T helper cells and cytotoxic T cell precursors (CTLp), using limiting dilution analysis. In addition we compared the phenotype of adult and neonatal PBMC, using monoclonal antibody staining. Cord PBMC in general showed higher frequencies of alloreactive Th than adult PBMC, with statistically significant differences in 6 out of 10 experiments. There was no statistically significant difference between adult and cord CTLp frequencies. Adult and cord PBMC surface phenotype was similar, except that cord blood contained fewer lymphocyte function-associated-3 (LFA-3) positive (memory) cells.

Adult↗
PubMed1986

Long-lasting deficit of functional T cell precursors in human bone marrow transplant recipients revealed by limiting dilution methods.

We have applied limiting dilution methods suitable for the estimation of mitogen-reactive helper (pHTL) and cytotoxic (pCTL) T cell frequencies to the analysis of immune function in patients 1 mo to 6 yr after allogeneic bone marrow transplantation (BMT). Although the majority of these patients have regained normal levels of Leu-3+ (helper) and Leu-2+ (killer/suppressor) cells by 6 to 12 mo after BMT as assessed by cytofluorimetry, the fraction of these cells that can function in limiting dilution cultures is substantially below normal levels in nearly all patients. Although some BMT patients eventually recover normal frequencies of pCTL and pHTL, values typically remain greatly depressed even in patients transplanted as many as 4 to 6 yr previously. In contrast, recovery of precursors able to proliferate (without expressing either helper or cytotoxic function) in response to phytohemagglutinin (PHA) and interleukin 2 occurs in many patients by 1 yr after transplant. In spite of the decreased frequency of functional precursor cells found after BMT, each precursor is capable of giving rise to the same amount of function at limiting dilutions as that produced by cells from normal controls. In many BMT patients, proliferation in conventional PHA-stimulated cultures returns to near-normal levels even though precursor frequencies remain low. The limiting dilution method is sensitive to residual immune dysfunction in BMT recipients not easily quantitated by other, more conventional techniques.

Adolescent↗
PubMedDate not supplied

[Epidermal growth factor-responsive precursors with similar properties isolated from embryonic rat septum and striatum].

INTRODUCTION: The potential correspondence of precursor cells isolated from different brain regions is partially unknown. Since models and culture conditions used in several studies vary, comparison of precursor characteristics has been limited. OBJECTIVE: In this paper epidermal growth factor (EGF)-responsive precursors from the striatum and septum were isolated and their growth pattern in vitro were determined. We also evaluated the influence of fibroblast growth factor (FGF) and nerve growth factor (NGF) on the proliferation of these cells. MATERIAL AND METHODS: Dissociated cells from rat septum and striatum were cultivated in suspension with 20 ng/ml of EGF. Total cells quantification, immunocytochemical staining and neuron counts were used to evaluate cell proliferation and cellular phenotypes produced by EGF-generated cells. RESULTS: Considering both culture evolution and cellular growth we demonstrated a similar growth pattern of septal and striatal EGF-responsive precursors. Furthermore, the proliferation of both cells populations was supported by FGF. On the contrary, NGF neither had a proliferative effect nor affected cell survival. Upon differentiation, a small proportion of precursor cells differentiated into neuronal phenotype.

Animals↗
PubMed2000

A cell-intrinsic timer that operates during oligodendrocyte development.

Multicellular organisms develop on a predictable schedule that depends on both cell-intrinsic timers and sequential cell-cell interactions mediated by extracellular signals. The interplay between intracellular timers and extracellular signals is well illustrated by the development of oligodendrocytes, the cells that make the myelin in the vertebrate central nervous system. An intrinsic timing mechanism operates in each oligodendrocyte precursor cell to limit the length of time the cell divides before terminally differentiating. This mechanism consists of two components, a timing component, which depends on the mitogen platelet-derived growth factor (PDGF) and measures elapsed time, and an effector component, which depends on thyroid hormone and stops cell division and initiates differentiation at the appropriate time. The cell-cycle inhibitor p27/Kip1 accumulates in the precursor cells as they proliferate and is part of both components of the timer. It seems likely that similar timing mechanisms operate in other cell lineages. BioEssays 22:64-71, 2000.

Animals↗
PubMed1997

Ontogeny of prohormone convertases in rat prenatal development.

It has been well established that peptide precursors usually undergo limited proteolysis at pairs or single basic amino acids during their biosynthetic process. This posttranslational modification paradigm is common for numerous membrane-spanning and secreted proteins, neuropeptides, and peptide hormones of physiological significance, in which endoproteolytic cleavage is invariably essential for the accurate biosynthesis and full activity of the mature products. Establishment of an effective peptide profile is dependent on not only the presence of peptide precursor, but also the presence and the enzymatic specificities of cleavage enzymes. We have, therefore, characterized the spatial and temporal patterns of six subtilisin-like serine endoproteases known to be involved in proprotein processing, including furin, PC1, PC2, PC4, PC5, and PACE4, in rat prenatal development and related the results to the expression patterns of several peptide precursors. We have observed largely distinct and sometimes complementary expression patterns of individual PCs in various embryonic structures, suggesting PCs may be functionally distinct in processing different sets of proprotein substrates in development. From these studies, numerous tentative enzyme-substrate relationships in various embryonic structures have been proposed and should encourage more studies to test the in vitro cleavage potentialities of individual PCs toward these precursors. In the future, knowledge gained from these studies, when combined with insights gained from in vivo perturbation and genetic ablation studies, should lead to final comprehensive understanding of specific precursors cleaved by specific enzymes at specific cleavage sites in known spatial and temporal expression patterns during development.

Animals↗
PubMed2001

Diversity of Arabidopsis genes encoding precursors for phytosulfokine, a peptide growth factor.

Phytosulfokine-alpha (PSK-alpha), a unique plant peptide growth factor, was originally isolated from conditioned medium of asparagus (Asparagus officinalis) mesophyll cell cultures. PSK-alpha has several biological activities including promoting plant cell proliferation. Four genes that encode precursors of PSK-alpha have been identified from Arabidopsis. Analysis of cDNAs for two of these, AtPSK2 and AtPSK3, shows that both of these genes consist of two exons and one intron. The predicted precursors have N-terminal signal peptides and only a single PSK-alpha sequence located close to their carboxyl termini. Both precursors contain dibasic processing sites flanking PSK, analogous to animal and yeast prohormones. Although the PSK domain including the sequence of PSK-alpha and three amino acids preceding it are perfectly conserved, the precursors bear very limited similarity among Arabidopsis and rice (Oryza sativa), suggesting a new level of diversity among polypeptides that are processed into the same signaling molecule in plants, a scenario not found in animals and yeast. Unnatural [serine-4]PSK-beta was found to be secreted by transgenic Arabidopsis cells expressing a mutant of either AtPSK2 or AtPSK3 cDNAs, suggesting that both AtPSK2 and AtPSK3 encode PSK-alpha precursors. AtPSK2 and AtPSK3 were expressed demonstrably not only in cultured cells but also in intact plants, suggesting that PSK-alpha may be essential for plant cell proliferation in vivo as well as in vitro. Overexpression of either precursor gene allowed the transgenic calli to grow twice as large as the controls. However, the transgenic cells expressing either antisense cDNA did not dramatically decrease mitogenic activity, suggesting that these two genes may act redundantly.

Amino Acid Sequence↗
PubMed1990

Limiting dilution analysis of lymphokine-activated killer cell precursor frequencies in peripheral blood lymphocytes of cancer patients receiving interleukin-2 therapy.

Eleven patients receiving weekly cycles of therapy with recombinant interleukin-2 (IL-2) were evaluated with a sensitive limiting dilution analysis to determine lymphokine-activated killer (LAK) cell precursor frequencies in peripheral blood lymphocytes. An increase in LAK precursor frequency above baseline was suggested by day 6 of this protocol and was clearly significant by day 20, indicating an expansion of the circulating precursor pool results from in vivo IL-2 administration. Correlations were not significant between LAK precursor frequency during IL-2 therapy and the total number of circulating lymphocytes, the percentage of CD56+ lymphocytes, IL-2 proliferative responses, or LAK activity of peripheral blood lymphocytes, indicating that the precursor frequency identification based on functional testing of individual cells is not accurately reflected by these analyses of heterogeneous bulk populations. Selective cell depletion analyses revealed that the majority of LAK precursors after in vivo IL-2 therapy were cells with the natural killer phenotype. Analysis of LAK precursors may help define the in vivo IL-2 administration, alone or in combination with other hematopoietic or immunodifferentiative cytokines, necessary to further augment in vivo effector cell numbers and activity for patients with cancer.

Cell Division↗
PubMed1988

Limiting dilution analysis of the response of murine L3T4+ spleen cells to alloantigen.

Cell sorter-purified small splenic L3T4+ cells from B6 mice were clonally expanded under limiting dilution (LD) conditions by coculture for 4-6 days with irradiated allogeneic stimulator cells in culture medium supplemented with various growth factor preparations. Proliferating L3T4+ cell clones were detected by [3H]thymidine uptake; interleukin 2 (IL-2) production of restimulated L3T4+ cell clones was measured in a sensitive colorimetric assay. IL-3 (but not IL-1 or IL-4) supported clonal expansion in vitro of many L3T4+ cell clones produced IL-2. The data were consistent with the hypothesis that only a single titrated precursor cell was limiting in the system. In the response to class II (bm12) H-2 alloantigen, 1 in 40-200 L3T4+ cells was induced to clonal growth; in the response to class I (bm1) H-2 alloantigen, a tenfold lower frequency (1 in 600-800) of inducible L3T4+ B6 cells was measured. A fraction of the generated L3T4+ cell clones showed IL-2-independent growth: anti-IL-2 receptor monoclonal antibodies (MoAb) (7D4 and PC61.5) blocked the proliferation of about 80% of the IL-2-producing L3T4+ cell clones, while about 20% of these clones seemed resistant to inhibition of proliferation by these MoAb. We have thus defined an LD system with high cloning efficiency for L3T4+ cells that does not depend on exogenous IL-2 supplements.

Animals↗
PubMed2003

Regulation of anthraquinone biosynthesis in cell cultures of Morinda citrifolia.

Cell cultures of Morinda citrifolia L. are capable of accumulating substantial amounts of anthraquinones. Chorismate formed by the shikimate pathway is an important precursor of these secondary metabolites. Isochorismate synthase (EC 5.4.99.6), the enzyme that channels chorismate into the direction of the anthraquinones, is involved in the regulation of anthraquinone biosynthesis. Other enzymes of the shikimate pathway such as deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) and chorismate mutase (EC 5.4.99.5) do not play a regulatory role in the process. The accumulation of anthraquinones is correlated with isochorismate synthase activity under a variety of conditions, which indicates that under most circumstances the concentration of the branchpoint metabolite chorismate is not a rate-limiting factor. Anthraquinone biosynthesis in Morinda is strongly inhibited by 2,4-D, but much less by NAA. Both auxins inhibit the activity of isochorismate synthase proportionally to the concomitant reduction in the amount of anthraquinone accumulated. However, the correlation between enzyme activity and rate of biosynthesis is less clear when the activity of the enzyme is very high. In this case, a limiting concentration of precursor may determine the extent of anthraquinone accumulation. Partial inhibition of chorismate biosynthesis by glyphosate leads to less anthraquinone accumulation, but also to a reduction in ICS activity. The complexity of the interference of glyphosate with anthraquinone biosynthesis is illustrated by the effect of the inhibitor in cell cultures of the related species Rubia tinctorum L. in these cells, glyphosate leads to an increase in anthraquinone content and a concomitant rise in ICS activity. All data indicate that the main point of regulation in anthraquinone biosynthesis is located at the entrance of the specific secondary route.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1982

Limit-dilution analysis of weak influenza-immune T cell responses associated with H-2Kb and H-2Db.

A limiting-dilution analysis has been made of influenza-immune cytotoxic T lymphocyte (CTL) response patterns associated with H-2Kb and H-2Dd in the B10.A(5R), and H-2Kk and H-2Db in the B10.A(2R) and B10.A(4R) recombinant mouse strains. In previous in vivo experiments the H-2Kb allele has been shown to be associated with low responsiveness or nonresponsiveness, whereas Db-restricted influenza-specific responses were found to be weak in recombinant strains that possessed the Kk allele. Influenza-immune CTL restricted to the more "dominant" H-2Dd and H-2Kk alleles were found consistently when primed T cells were restimulated in vitro under limiting-dilution conditions. Precursors were present at frequencies of at least twice background levels (lysis of normal targets) in the great majority of experiments, performed with or without added helper factors. The H-2Kb- and H-2Db-restricted responses were much more variable and, for H-2Kb but not for H-2Db, were completely absent when the culture medium did not contain added interleukin preparations. Even so, experiments in which individual microcultures were tested on more than one target demonstrated conclusively both that H-2Kb-restricted CTL can be generated under limiting-dilution conditions and that most, if not all, of these T cells do not cross-react with influenza virus presented in the context of H-2Dd. The characteristics of these "weak" responses are thus that they are more help dependent and show evidence of greater variability between individual mice than the "dominant" CTL clones do.

Animals↗
PubMed1989

Recovery of activated cytotoxic T cells from minor H incompatible tumor graft rejection sites.

In this study we determined whether minor H-specific cytotoxic T cells and their precursors (pTc) are present at the site of rejection of minor H disparate tumor allografts. Lymphocytes were retrieved from eyes of BALB/c mice that received subconjunctival injections of minor H-incompatible P815 tumor cells. The lymphocytes were then assayed for direct cytotoxic activity as well as precursor frequency by limiting dilution. Similar assays were conducted on cells obtained from the draining lymph nodes and from the spleen. As expected, tumor rejection was accompanied by significant clonal expansion of minor H-specific pTc within the draining lymph node and the spleen. A correspondingly high frequency of pTc was also detected at the graft site. More importantly, fully functional cytotoxic T cells were recovered from the tumor graft site during rejection, but no similarly active cells were found in either the draining nodes or spleen. We conclude that, after Ag stimulation, pTc are generated in draining central lymphoid compartments. From this generative site, the precursor cells then disseminate systemically, gradually reaching and infiltrating the tumor graft site. A further activation step, dependent upon Ag and T cell help, permits these cells to mature into fully active cytotoxic cells which can then effect tumor rejection. We propose that the terminal stage(s) of pTc activation is promoted by lymphokines released locally from TDH cells that are also generated during the alloimmune response and simultaneously infiltrate the site.

Animals↗
PubMed2001

Transcriptional regulation of the two sterol esterification genes in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae transcribes two genes, ARE1 and ARE2, that contribute disproportionately to the esterification of sterols. Are2p is the major enzyme isoform in a wild-type cell growing aerobically. This likely results from a combination of differential transcription initiation and transcript stability. By using ARE1 and ARE2 promoter fusions to lacZ reporters, we demonstrated that transcriptional initiation from the ARE1 promoter is significantly reduced compared to that from the ARE2 promoter. Furthermore, the half-life of the ARE2 mRNA is approximately 12 times as long as that of the ARE1 transcript. We present evidence that the primary role of the minor sterol esterification isoform encoded by ARE1 is to esterify sterol intermediates, whereas the role of the ARE2 enzyme is to esterify ergosterol, the end product of the pathway. Accordingly, the ARE1 promoter is upregulated in strains that accumulate ergosterol precursors. Furthermore, ARE1 and ARE2 are oppositely regulated by heme. Under heme-deficient growth conditions, ARE1 was upregulated fivefold while ARE2 was down-regulated. ARE2 requires the HAP1 transcription factor for optimal expression, and both ARE genes are derepressed in a rox1 (repressor of oxygen) mutant genetic background. We further report that the ARE genes are not subject to end product inhibition; neither ARE1 nor ARE2 transcription is altered in an are mutant background, nor does overexpression of either ARE gene alter the response of the ARE-lacZ reporter constructs. Our observations are consistent with an important physiological role for Are1p during anaerobic growth when heme is limiting and sterol precursors may accumulate. Conversely, Are2p is optimally required during aerobiosis when ergosterol is plentiful.

Acyltransferases↗
PubMed1985

Differential resistance to growth of a tumor expressing incompatible minor alloantigens reflects regulatory influences rather than differences in anti-minor-CTL-P frequencies.

In previous studies it was found that BALB/c (H-2d) was more susceptible than (BALB/c X A)F1 (H-2d X H-2a) to a tumor bearing multiple mismatched minor histocompatibility antigens, the DBA/2 (H-2d) mastocytoma P815, and that this resistance was H-2 linked. In the present studies the immunologic basis of this effect was examined by comparing the cytotoxic-T-lymphocyte (CTL) responses of BALB/c with those of (BALB/c X A)F1. Despite the BALB/c X A)F1's 34-fold greater resistance to P815 in vivo, the numbers of effector cell precursors were found to be similar in the two hosts as shown by (a) similar anti-P815 CTL responses in vitro with T-cell growth factor, (b) similar secondary anti-DBA/2 MiHA responses after in vivo priming with irradiated P815, and (c) similar frequencies of anti-DBA/2 CTL precursors by limiting-dilution analysis. However, priming with proliferating P815 in vivo revealed a defect in the BALB/c animals: Spleen cells from such animals were unable to control the growth of contaminating P815 cells in vitro or to mount strong secondary CTL responses to DBA/2 antigens. The defective priming of BALB/c could be corrected when DBA/2 spleen cells were added to the P815 inoculum. This impaired priming by living tumor cells was not seen in (BALB/c X A)F1. It is concluded that the use of living P815 tumor cells revealed a defect in immunoregulation in BALB/c mice, which rendered them susceptible to tumor growth in spite of apparently adequate numbers of anti-minor-CTL precursors. How the additional H-2 products expressed in the (BALB/c X A)F1 might correct this defect is discussed.

Animals↗
PubMed1994

Prothrombin synthesis and degradation in rat hepatoma (H-35) cells: effects of warfarin.

Vitamin K is a substrate for the enzyme catalyzing the carboxylation of specific glutamyl residues to gamma-carboxyglutamyl residues in hepatic precursors of a limited number of plasma proteins, including prothrombin. The gamma-carboxylation of these proteins can be blocked by the anticoagulant warfarin; and in the bovine and human, warfarin treatment results in the secretion of under-gamma-carboxylated forms of prothrombin into plasma. In the rat, this response is not seen, but plasma prothrombin concentrations are drastically decreased. This response has now been studied in rat hepatoma (H-35) cells in which prothrombin secretion is decreased 90% by incubation in the presence of warfarin. Neither prothrombin mRNA levels nor the apparent rate of prothrombin message translation were decreased when cells were cultured in the presence of warfarin rather than of vitamin K. The pool of intracellular prothrombin precursors is increased threefold by warfarin treatment, and this pool is rapidly secreted when vitamin K is administered. In contrast, continued incubation in the presence of warfarin resulted in the degradation of 60% of this pool in 24 hours. When transport of secretory proteins to the golgi apparatus was blocked with Brefeldin A, this precursor pool was gamma-carboxylated in the presence of vitamin K and no degradation occurred. Lysosomal enzyme inhibitors did not block the degradation, and the data suggest that, in rat hepatocytes, under-gamma-carboxylated prothrombin is specifically targeted to a pathway of protein degradation located in the endoplasmic reticulum.

Animals↗
PubMed2001

Human vascular endothelial cells stimulate a lower frequency of alloreactive CD8+ pre-CTL and induce less clonal expansion than matching B lymphoblastoid cells: development of a novel limiting dilution analysis method based on CFSE labeling of lymphocytes.

We have previously shown that human endothelial cells (EC) are less efficient than professional APC, e.g., B lymphoblastoid cells (BLC), at stimulating allogeneic CD8(+) T cells to develop into CTL. In this study we describe FACS-based limiting dilution analyses using the dilution of the intracellular dye CFSE as an indicator of CD8(+) T cell alloactivation and expansion with significantly increased sensitivity compared with conventional, cytotoxicity-based assays. In addition, this assay permits the relative size of clonal CTL populations that are generated in individual CD8(+) T cell cultures to be determined (clonal burst size). We have applied this method to quantitatively compare the generation of CTL at the clonal level following stimulation of allogeneic CD8(+) T cells by either BLC or HUVEC derived from the same donor. CD8(+) T cells expanded by allostimulation were identified as CD8(+), CFSE(low) cells and were categorized as CTL by the expression of intracellular perforin and IFN-gamma. Precursor frequencies for EC-stimulated CTL were 5- to 40-fold (mean, 7.5-fold) lower compared with BLC-stimulated CTL (p < 0.01). Concomitantly, the average clonal burst sizes in EC-stimulated CTL cultures were significantly smaller than those in conventional CTL cultures, primarily due to the occurrence of some very large clone sizes exclusively with BLC stimulation. Although EC-stimulated CTL were generated only from the memory subset of CD8(+) T cells, BLC-stimulated very large burst sizes of CTL were observed from both naive and memory CD8(+) T cell precursors. These data establish that both a lower frequency of reactive precursors and more limited clonal expansion, but not regulatory T cells, contribute to the reduced capacity of EC to promote alloreactive CTL differentiation compared with that of professional APC.

Antigens, CD↗
PubMed1996

Natural killer cell precursors in the CD44neg/dim T-cell receptor population of mouse bone marrow.

Natural killer (NK) cells develop from the nonadherent cell component of NK long-term bone marrow (BM) cultures (NK-LTBMC). Because these nonadherent cells are depleted of mature NK cells and T cells, but appear to enriched for NK precursors, they were used as a starting population to begin to define the NK precursors that function in NK-LTBMC. As the stromal cell component of NK-LTBMC has been shown to support interleukin (IL)-2-induced, CD44 dependent, NK cell development from nonadherent NK precursors, NK-LTBMC stroma was used in a limiting dilution assay (LDA) to quantitate the precursors. NK-LTBMC in 96-well plates were irradiated (20 Gy) to kill hematopoietic cells (including the NK precursors), seeded with limiting dilutions of the cells to be quantitated, cultured with 500 U/mL IL-2 for 13 days and assayed for development of NK activity by adding 51Cr-labeled YAC-1 cells to the wells and evaluating the release of 51Cr after 4 hours. Flow cytometric analysis, sorting, and quantitation of the nonadherent cell component of NK-LTBMC showed that NK precursors were concentrated in the CD44neg/dim subset that comprised 10% of the "lymphoid" gated cells. When the CD44neg/dim subset was sorted from BM of mice treated with 5-fluorouracil (5-FU) day before (-1FUBM), there were about 30% T cells, but no NK-1.1+ cells. When the T cells were removed by sorting and the CD44neg/dim, alphabeta, gammadelta T-cell receptorneg (TCR-) subpopulation was seeded onto irradiated stroma with IL-2, they proliferated, developed NK activity, became NK-1.1+ and CD44bright and remained alphabeta, gammadelta TCR-. The frequency of NK precursors in this population as estimated from the LDA was about 1/500.

Animals↗
PubMed1986

Tyrosinase isozyme heterogeneity in differentiating B16/C3 melanoma.

The B16/C3 murine melanoma is a pigmented tumor that is rich in the copper-containing enzyme, tyrosinase. This enzyme, which converts tyrosine to melanin precursors, is largely associated with membrane fractions of cells and exists in a number of discrete isozymic forms ranging in molecular mass from 58,000 to 150,000 daltons and pI from 3.4 to 5.2. One of these isozymes (Mr = 58,000, pI 3.4) has been purified to homogeneity. The purified enzyme catalyzes the hydroxylation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) and the conversion of L-DOPA to dopaquinone. Ascorbic acid, tetrahydrofolate, and dopamine can serve as cofactors in the hydroxylase reaction. The Michaelis constants for the purified enzyme were 7 X 10(-4) M for L-tyrosine and 6 X 10(-4) M for L-DOPA. The Vmax for L-DOPA was much greater than the Vmax for L-tyrosine indicating that tyrosine hydroxylation is rate-limiting in melanin precursor biosynthesis. Two putative copper chelators, phenylthiourea and diethyldithiocarbamide inhibited both the tyrosine hydroxylase and L-DOPA oxidase activities of the enzyme. Phenylthiourea was a noncompetitive inhibitor while diethyldithiocarbamide was a competitive inhibitor indicating that these agents act by different mechanisms. When digested with proteases and glycosidases, higher molecular weight forms of tyrosinase co-migrated with the purified enzyme in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggesting that the isozyme was derived from larger precursors. Thus, post-translational processing of tyrosinase may underlie isozyme diversity and this may be important in the control of melanogenesis in this tumor model.

Amino Acids↗