Extracellular ATP induces assembly and activation of the myosin light chain phosphatase complex in endothelial cells.

OBJECTIVES: Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile machinery of endothelial cells. This inactivation relies on dephosphorylation of the regulatory myosin light chain (MLC) due to an activation of the MLC phosphatase (MLCP). To date, activation and function of MLCP in endothelial cells are only partially understood. METHODS: Here, the mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human endothelial cells from umbilical veins. Cells were transfected with the endogenous protein phosphatase 1 (PP1)-specific inhibitor-2 (I-2). RESULTS: Overexpression of I-2 led to inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing the MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP recruits the PP1delta catalytic subunit and the myosin phosphatase targeting subunit (MYPT1) to form a complex. ATP stimulated dephosphorylation of MYPT1 at the inhibitory phosphorylation sites threonine 850 and 696. However, extracellular ATP failed to stimulate MYPT1 dephosphorylation in I-2-overexpressing cells. CONCLUSIONS: The present study shows for the first time that, in endothelial cells, extracellular ATP causes activation of MLCP through recruitment of PP1delta and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1.

Immunodetection of nmt55/p54nrb isoforms in human breast cancer.

BACKGROUND: We previously identified and characterized a novel 55 kDa nuclear protein, termed nmt55/p54nrb, whose expression was decreased in a subset of human breast tumors. The objective of this study was to determine if this reduced expression in human breast tumors was attributed to the regulation of mRNA transcription or the presence of altered forms of this protein. RESULTS: Northern blot analysis and ribonuclease protection assay indicated that nmt55/p54nrb mRNA is expressed at varying levels in estrogen receptor positive (ER+) and estrogen receptor negative (ER-) human breast tumors suggesting that reduced expression of nmt55/p54nrb protein in ER- tumors was not due to transcriptional regulation. To determine if multiple protein isoforms are expressed in breast cancer, we utilized Western blot and immunohistochemical analyses, which revealed the expression of an nmt55/p54nrb protein isoform in a subset of ER+ tumors. This subset of ER+ human breast tumors expressed an altered form of nmt55/p54nrb that was undetectable with an amino-terminal specific antibody suggesting that this isoform contains alterations or modifications within the amino terminal domain. CONCLUSIONS: Our study indicates that nmt55/p54nrb protein is post-transcriptionally regulated in human breast tumors leading to reduced expression in ER- tumors and the expression of an amino terminal altered isoform in a subset of ER+ tumors. The potential involvement of nmt55/p54nrb in RNA binding and pre-mRNA splicing may be important for normal cell growth and function; thus, loss or alteration of protein structure may contribute to tumor growth and progression.

Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture.

Epidemiological evidence suggests tea (Camellia sinensis L.) has chemopreventive effects against various tumors. Green tea contains many polyphenols, including epigallocatechin-3 gallate (EGCG), which possess anti-oxidant qualities. Reduction of chemically induced mammary gland carcinogenesis by green tea in a carcinogen-induced rat model has been suggested previously, but the results reported were not statistically significant. Here we have tested the effects of green tea on mammary tumorigenesis using the 7,12-dimethylbenz(a)anthracene (DMBA) Sprague-Dawley (S-D) rat model. We report that green tea significantly increased mean latency to first tumor, and reduced tumor burden and number of invasive tumors per tumor-bearing animal; although, it did not affect tumor number in the female rats. Furthermore, we show that proliferation and/or viability of cultured Hs578T and MDA-MB-231 estrogen receptor-negative breast cancer cell lines was reduced by EGCG treatment. Similar negative effects on proliferation were observed with the DMBA-transformed D3-1 cell line. Growth inhibition of Hs578T cells correlated with induction of p27(Kip1) cyclin-dependent kinase inhibitor (CKI) expression. Hs578T cells expressing elevated levels of p27(Kip1) protein due to stable ectopic expression displayed increased G1 arrest. Thus, green tea had significant chemopreventive effects on carcinogen-induced mammary tumorigenesis in female S-D rats. In culture, inhibition of human breast cancer cell proliferation by EGCG was mediated in part via induction of the p27(Kip1) CKI.

Expression of the aryl hydrocarbon receptor/transcription factor (AhR) and AhR-regulated CYP1 gene transcripts in a rat model of mammary tumorigenesis.

Exposure to ubiquitous environmental chemicals, such as polycyclic aromatic hydrocarbons (PAH), may contribute to human breast cancer. In animals, PAH induce tumors in part by activating the aryl hydrocarbon receptor (AhR)/transcription factor. Historically, investigations into AhR-regulated carcinogenesis have focused on AhR-dependent transcriptional regulation of cytochrome P450 (CYP) enzymes which oxidize PAH to mutagenic intermediates. However, recent studies suggest that the AhR directly regulates cell growth. Given the postulated role of the AhR in carcinogenesis, we predicted that: (1) tissue predisposed to PAH tumorigenesis would express the AhR and (2) aberrant AhR and/or AhR-regulated gene expression would accompany malignant transformation. To test these hypotheses, AhR and CYP1 protein and/or mRNA levels were evaluated in rat mammary tumors induced with 7, 12-dimethylbenz[a]anthracene (DMBA), a prototypic PAH and AhR ligand. Results indicate modest AhR expression in normal mammary myoepithelial and ductal epithelial cells. In contrast, high AhR levels were detected in DMBA-induced tumors. Nuclear AhR localization in tumors suggested constitutive AhR activation. In situ hybridization and quantitative RT-PCR assays indicated high AhR mRNA levels in neoplastic epithelial cells. While both AhR-regulated CYP1A1 and CYP1B1 mRNAs were induced in breast tissue within 6 h of DMBA gavage, only CYP1B1 mRNA remained elevated in tumors. These results: (1) help explain targeting of breast tissue by carcinogenic PAH, (2) imply that AhR and CYP1B1 hyper-expression represent molecular biomarkers for, at least, PAH-induced mammary cell transformation, and (3) suggest mechanisms through which the AhR may contribute to carcinogenesis well after exogenous AhR ligands have been eliminated.

Immunochemical analyses of estrogen receptors in human breast tumors by a novel monoclonal estrogen receptor antibody (EVG F9).

The objective of this study was to assess the potential utility of a new site-directed, monoclonal anti-estrogen receptor antibody (EVG F9) in detection and analyses of human breast tumor estrogen receptor (ERalpha), using immunoblotting and immunohistochemical assays. Using Western Blot analyses, we demonstrated that EVG F9 monoclonal antibody binds specifically to ERalpha and does not cross-react with ERbeta. Furthermore, binding of EVG F9 to ERalpha was effectively displaced with the immunogenic peptide in Western Blots and in immunohistochemical analyses. In Western Blot analyses, EVG F9 detected ERalpha at low concentrations approaching 5 to 10 fmol/sample. Determination of ERalpha status of a series of human breast tumor samples by Western Blot analyses or immunohistochemistry using EVG F9 correlated well with ERalpha values measured by ligand binding assays. These observations suggest that EVG F9 monoclonal anti-ERalpha antibody is a valuable immunochemical tool for detection and analyses of ERalpha in human breast tumors.

Activation of NF-kappaB/Rel occurs early during neoplastic transformation of mammary cells.

NF-kappaB/Rel is a family of transcription factors which are expressed in all cells; however, in most non-B cells, they are sequestered in the cytoplasm in inactive complexes with specific inhibitory proteins, termed IkappaBs. We have recently shown that NF-kappaB/Rel factors are aberrantly activated in human breast cancer and rodent mammary tumors, and function to promote tumor cell survival and proliferation. Here, we have examined the time-course of induction of NF-kappaB/Rel factors upon carcinogen treatment of female Sprague-Dawley (S-D) rats in vivo and in human mammary epithelial cells (HMECs) in culture. We observed that NF-kappaB/Rel activation is an early event, occurring prior to malignant transformation. In S-D rats, increased NF-kappaB/Rel binding was detected in nuclear extracts of mammary glands from 40% of animals 3 weeks post-treatment with 15 mg/kg 7,12-dimethylbenz[a]anthracene (DMBA); this is prior to formation of tumors which normally begin to be detected after 7-9 weeks. In non-tumorigenic MCF-10F cells, in vitro malignant transformation upon treatment with either DMBA or benzo[a]pyrene (B[a]P) resulted in a 4- to 12-fold increase in activity of classical NF-kappaB (p65/p50). NF-kappaB induction was corrrelated with a decrease in the stability of the NF-kappaB-specific inhibitory protein IkappaB-alpha. Ectopic expression of the transactivating p65 subunit of NF-kappaB in MCF-10F cells induced the c-myc oncogene promoter, which is driven by two NF-kappaB elements, and endogenous c-Myc levels. Furthermore, reduction mammoplasty-derived HMECs, immortalized following B[a]P exposure, showed dysregulated induction of classical NF-kappaB prior to malignant transformation. Together these findings suggest that activation of NF-kappaB plays an early, critical role in the carcinogen-driven transformation of mammary glands.

Dietary fat, body weight, and cancer: contributions of studies in rodents to understanding these cancer risk factors in humans.

Understanding diet and energy balance as risk factors for breast, colon, and other cancers requires information on the contribution of each factor and of interactions among factors to cancer risk. Rodent models for breast cancer provide extensive data on effects of dietary fat and calories, energy balance, body weight gain, and physical activity on tumor development. Analyses of the combined data from many studies have shown clearly that quality and quantity of dietary fat and energy balance contribute independently to increased mammary gland tumorigenesis. These findings were seen in female rats fed diets high in fat (35-40% of calories) compared to rats fed control diets, with approximately 10% of calories as fat (Fay and Freedman, 1997, Breast Cancer Res. Treat. 46, 215-223). The methods used permit comparison of experimental and epidemiological data, and they may be useful in extrapolating between species and developing public health recommendations. In addition to the contributions of lifetime-diet composition, intake, energy balance, and physical activity to cancer risk, there are questions about the timing and duration of alterations in these factors and about the "dose-response" characteristics of cancer risk to the factors. Endocrine mechanisms may be significant in mammary gland tumor risk, but experimental and epidemiological data indicate that cancers at other sites, such as colon and liver, also are influenced by the factors listed. Other diet and lifestyle factors that influence energy, or specifically fat, metabolism may also affect risk for cancers that are promoted by increased intake of fat and calories. Studies of separate and interactive effects of dietary fat, black tea, weight gain, and mammary gland tumorigenesis (Rogers, et al, 1998, Carcinogenesis 19, 1269-1273) have been analyzed. Using adjustment of carcinogenesis endpoints for body weight, tumor burden, and latency, they were found to be related to weight gain within treatment groups in 2 of 3 experiments.

Demonstration of aromatase activity and its regulation in breast tumor and benign breast fibroblasts.

Breast tumors from post-menopausal women contain higher amounts of estradiol than would be predicted from levels circulating in plasma. This observation raised the hypothesis that tumors may synthesize estradiol in situ and increase their tissue estradiol levels via this mechanism. The key enzyme involved in tissue estrogen synthesis, aromatase, is present in breast tumors but, according to some investigators, not in sufficient concentration to be biologically meaningful. We postulated that foci of cells in breast tumors might contain high amounts of aromatase and this locally produced estrogen might act in a paracrine or autocrine fashion. To test this hypothesis, we utilized immunohistochemistry to localize the aromatase enzyme, an histological scoring system to quantitate it, and culture of isolated breast cells to demonstrate its potential regulation. In 26 archival breast tumors, 16 (62%) contained aromatase by radiometric assay. With the immunohistochemical method, we detected areas with staining in the stroma as well as tumor epithelial cells. Staining ranged from the intensity approaching that seen in placenta to levels just distinguishable from background. We adopted an histological scoring system (H-score) from that used to quantitate progesterone receptor levels in tissue and used it to quantitate aromatase activity. A higher histologic score was found in stromal spindle cells (13) than in tumor epithelial cells (4.8). The biochemical aromatase results correlated with the H-score of stromal but not epithelial cells. To further study stromal cells from tumors, we isolated stromal cells from breast tumors and the benign areas of breast distal to the tumor and grew them in culture. Addition of dexamethasone, phorbol esters, and cyclic AMP analogues stimulated aromatase enzyme and messenger RNA levels substantially. Use of aromatase enzyme inhibitors such as letrozole blocked estrogen production but did not alter aromatase message levels. Epithelial cells, whether nonmalignant or cancer derived, exhibited no regulation by dexamethasone, phorbol esters, or cAMP analogues. These data, taken together, suggest that stromal cells may be more important than epithelial cancer cells for estrogen production in breast tumors. The ability to stimulate aromatase activity substantially with various enhancers of aromatase provides further credence for an important biologic role of estrogen production in tumor tissue.

Black tea and mammary gland carcinogenesis by 7,12-dimethylbenz[a]anthracene in rats fed control or high fat diets.

Epidemiological studies suggest that tea may reduce cancer risk, and in laboratory rodents, chemopreventive effects of tea or purified extracts of tea have been demonstrated in lung, gastrointestinal tract and skin. There is some evidence of chemoprevention by tea in the mammary gland, but the data are not conclusive. In order to evaluate more fully the possible influence of black tea on 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary gland tumors in the female S-D (Sprague-Dawley) rat, three large studies were performed: experiment 1, tumorigenesis in rats fed AIN-76A diet and given 25 mg/kg DMBA and 1.25 or 2.5% whole tea extract or water to drink; experiment 2, tumorigenesis in rats given 15 mg/kg DMBA and the same diet and fluids as in experiment 1; experiment 3, tumorigenesis in rats fed control or HF (high fat, corn oil) diet and given 15 mg/kg DMBA and 2% tea or water to drink. Tea was given throughout the experiment; DMBA was given by gastric gavage at 8 weeks of age. There was no consistent effect of tea on tumorigenesis in rats fed AIN-76A diet; there was, however, evidence in experiment 3 of a reduction of tumorigenesis by tea in rats fed the HF diet. In experiment 3, rats fed the HF diet and given water showed the expected increase in tumor burden (number and weight) compared with rats fed control diet. However, rats fed the HF diet and given 2% tea showed no increase in tumor burden; their tumor burden was significantly lower than in rats fed the HF diet and given water (P < 0.01) and was not different from rats fed control diet and given water or tea. In addition, in experiment 3, the number of malignant tumors per tumor-bearing rat was increased by the HF diet in water-drinking rats (P < 0.01) but not in tea-drinking rats. Therefore, it appears that tea partially blocked the promotion of DMBA-induced mammary tumorigenesis by the HF diet.

Induction of PreB cell apoptosis by 7,12-dimethylbenz[a]anthracene in long-term primary murine bone marrow cultures.

Numerous studies demonstrate that polycyclic aromatic hydrocarbons (PAH) suppress immunity by modifying the function of both B and T cells. Relatively few studies have assessed the effects of these common environmental chemicals on immature lymphocytes. In the present study, long-term primary bone marrow cultures were employed to investigate the effects of a prototypic PAH and aryl hydrocarbon receptor (AhR) agonist, 7,12-dimethylbenz[a]anthracene (DMBA), on immature B lymphocytes. In this system, immature preB cells are maintained in a supportive microenvironment provided by bone marrow stromal cells. Results presented here demonstrate that (1) exposure of primary bone marrow cultures to DMBA results in preB cell death by apoptosis; (2) notably low doses of DMBA (> or = 10(-8) M) induce preB cell apoptosis; (3) in long-term cultures, bone marrow stromal cells, but not preB cells, express AhR mRNA and protein as determined by in situ hybridization, RT-PCR, and immunoblotting; (4) freshly isolated unfractionated bone marrow cells, but not purified bone marrow B cells, express AhR protein as assessed by immunohistochemistry; (5) alpha-naphthoflavone, a competitive AhR inhibitor and cytochrome P450 antagonist, completely blocks DMBA-induced preB cell apoptosis in primary bone marrow cultures; and (6) DMBA or benzo[a]pyrene injection in vivo results in bone marrow cell apoptosis consistent with the death of hematopoietic cells clustered around stromal elements. The results implicate programmed cell death as a mechanism underlying DMBA-mediated immunosuppression and suggest that preB cell death is influenced by local interactions with AhR+ bone marrow stromal cells.

Diagnosis of dermatitis herpetiformis by an avidin-biotin-peroxidase method.

BACKGROUND AND DESIGN: Immunofluorescence detection of stippled IgA in dermal papillae has been considered the gold standard in the diagnosis of dermatitis herpetiformis (DH). We have developed an immunohistochemical technique using the avidin-biotin-peroxidase complex that is equally effective as direct immunofluorescence in detecting IgA. We retrospectively studied 43 paraffin-embedded biopsy specimens obtained from patients with DH and a variety of other diseases for the presence of IgA along the basement membrane zone. RESULTS: Eleven immunofluorescence-proved cases of DH were found positive for IgA with the avidin-biotin-peroxidase method. One biopsy specimen originally classified as DH was identified and reclassified as linear IgA bullous disease based on the immunoperoxidase findings. All the samples that were positive on direct immunofluorescence were positive with the avidin-biotin-peroxidase method. Control samples of bullous pemphigoid, discoid lupus erythematosus, pemphigus vulgaris, and dermatitis were all negative for IgA deposition. CONCLUSION: The diagnosis of DH on formalin-fixed tissue is possible with the use of an avidin-biotin-peroxidase method, which is convenient and cost-effective.

Intraoperative immunocytochemistry of cytologic scrape specimens. A rapid immunoperoxidase method for triage and diagnosis.

We investigated a rapid immunoperoxidase (IPX) method utilizing a wide variety of antibodies for use on cytologic scrapings of fresh tissue that were submitted for intraoperative frozen section. The handling, fixation and IPX staining of these cytologic scrape specimens are more rapid and convenient than frozen tissue sections and spare tissues that may be of small quantity for appropriate special diagnostic studies (flow cytometry, cell markers, genetic studies, electron microscopy, and so forth). Fresh tissues from normal organs and tumors were scraped with a scalpel blade, smeared on an uncoated glass slide and immersed in 95% alcohol for one minute. Antibodies investigated by this method included: CAM 5.2, AE1/3, keratin 903, desmin, vimentin, HHF-35, CEA, PLAP, NSE, S-100, HMB45, LCA, L26 and UCHL-1. Twenty-three fresh epithelial and mesenchymal tissues from the uterus, colon, tonsil, lymph node, kidney and adrenal gland were examined, along with 29 tumors. There were 21 carcinomas, 3 melanomas, 3 sarcomas and 2 lymphomas. Using the streptavidin-biotin technique with aminoethylcarbazole localization, IPX results were available in 15-20 minutes, comparable to the time required for the reporting of a frozen section diagnosis. Keratins, vimentin, desmin and LCA gave the best results. HMB45 was weakly reactive in one case. This rapid cytologic IPX method may be used as an adjunct to frozen sections and cytologic imprints for intraoperative diagnosis or for efficient triage of poorly differentiated tumors for special studies at the time of frozen section.

Stromal spindle cells contain aromatase in human breast tumors.

In situ synthesis of estrogens by breast cancer tissue provides a potential explanation for the high concentrations of estradiol in mammary neoplasms in postmenopausal women. A major metabolic pathway for estrogen biosynthesis is the conversion of androstenedione to estrone via the enzyme aromatase. Biochemical studies have demonstrated aromatase in tumor tissue, but at relatively low and not clearly biologically significant levels. The present study tested the hypothesis that tumor levels of aromatase, albeit low, could be biologically important if present in high concentrations in focal clusters of specific cell types. A pilot study used an immunohistochemical method in frozen sections of fresh breast tumors as an optimal means to detect aromatase. Twelve of 18 tumors contained aromatase-positive cells, some with highly intense staining. A follow-up study then attempted to precisely define the types of cells containing aromatase and correlate the immunohistochemical findings with biochemical aromatase activity. A modified H-score (histological scoring system) was used to semiquantitate the amount of aromatase staining in tumor epithelial, stromal spindle, stromal inflammatory, and normal breast epithelial cells. We found that immunohistochemical staining for aromatase predominated in stromal spindle cells with a median H-score of 13, whereas tumor epithelial, stromal inflammatory, and normal breast elements contained lesser amounts (median H-scores of 4.8, 0.03, and 0.5, respectively). The H-score for stromal spindle cells, but not those for other cell types, correlated highly with the biochemical aromatase assay (P < 0.01). Using a cut-off parameter estimated by a sensitivity/specificity (receiver operating curve) analysis, 62% of tumors were classified as aromatase positive based on stromal spindle cell staining. A similar number were also positive by biochemical assay, with concordance between the two methods of 77%. These observations provide substantial evidence for the presence of aromatase in human breast tumors, particularly in stromal spindle cells, and support the biological importance of aromatase for in situ production of estradiol.

Increased expression of intercellular adhesion molecules in biliary atresia.

The expression of the inflammatory adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1, was studied in six infants with biliary atresia using an immunoperoxidase technique on frozen sections. Controls consisted of five patients with various conditions including total parenteral nutrition-induced cholestasis, choledochal cyst, viral hepatitis, metastatic carcinoma, and thrombotic thrombocytopenic purpura. None of the patients were in liver failure. Bile ducts from the control subjects did not express any of the inflammatory adhesion molecules on ductal epithelium. In marked contrast, all of the biliary atresia specimens demonstrated strong intercellular adhesion molecule-1 expression and occasional vascular cell adhesion molecule-1 staining on epithelial cell membranes of both intra- and extrahepatic ductal structures. Hepatocytes and sinusoidal lining cells including Kupffer cells showed a pattern of intense intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in all specimens with active inflammation that could not differentiate the biliary atresia cases from the control group. Lymphocyte function-associated antigen-1 intensely stained the inflammatory cell infiltrate in the biliary atresia and inflamed control specimens. The strong expression of intercellular adhesion molecule-1 on biliary ductal epithelium in patients with biliary atresia suggests a potential role for this adhesion molecule in the pathogenesis of this devastating neonatal hepatic disorder.

CA-549 is not diagnostically useful as an immunohistochemical marker for breast carcinoma.

We evaluated BC4E549, a monoclonal antibody to CA-549, for use as an immunohistochemical marker to determine (1) differences in staining patterns between breast carcinoma and nonmalignant breast tissue specimens, (2) sensitivity and specificity of staining patterns for CA-549 in breast carcinoma vs other carcinomas, and (3) conservation of primary staining patterns in tumor metastases. We studied paraffin-embedded tissue specimens from 382 cases including nonmalignant breast tissue, endometrium, lung, pancreas, and thyroid gland; breast carcinoma; and adenocarcinomas of the colon, endometrium, lung, ovary, pancreas, and thyroid gland. Membranous staining was present in all nonmalignant breast tissue specimens and in most breast carcinoma tissue specimens. Diffuse cytoplasmic staining was more extensive in breast carcinomas than in benign processes. However, similar staining was also seen in normal and neoplastic tissue specimens obtained from a variety of other sites. Sensitivity and specificity of diffuse cytoplasmic staining for CA-549 in breast carcinoma relative to carcinomas from other sites studied were 78.6% and 77.4%, respectively. Staining patterns were not preserved from primary tumor to metastases in 67.4% of cases evaluated. We conclude that BC4E549 antibody to CA-549 is not a sufficiently sensitive or specific immunohistochemical marker in routinely processed tissue specimens to be useful in the clinical-diagnostic laboratory setting.

Sodium and potassium saturation kinetics of Na+K+-ATPase in plasma membranes from corneal endothelium: fresh tissue vs. tissue culture.

The maximal velocity (Vmax) and the apparent dissociation constant (K0.5) of Na+K+-ATPase have each been estimated with respect to sodium and potassium ion activation. These estimations were made from the enzymatic activity of plasma membrane preparations derived from bovine corneal endothelial cells. The determinations were made on cells obtained from fresh tissue and from secondary tissue cultures. Two methods were used to obtain the estimates: the first used a combination of Eadie-Hofstee and Hill plots; the second used Eisenthal-Cornish-Bowden plots. The Vmax for sodium was 5.58-5.60 mumol Pi/mg protein/30 min for fresh tissue vs. 2.00-1.80 mumoles Pi/mg protein/30 min for tissue cultures. The corresponding K0.5 values were 62-57 mM (fresh tissue) vs. 7.9-6.7 mM (tissue culture). Vmax for potassium was 4.28-4.00 mumoles Pi/mg protein/30 min for fresh tissue vs. 1.37-1.34 mumoles Pi/mg protein/30 min for tissue cultures. The corresponding K0.5 values were 3.3-3.1 mM (fresh tissue) and 1.7-1.7 mM (tissue culture). The results indicate a lowered activity and change in affinity of the enzyme for the two ions in tissue cultures compared to fresh tissues. The detergent Lubrol W-X increased activity in both tissue sources. Sonication had no significant effect on the activity. Variations in pH (7-9) indicated that the highest activity was obtained at pH 7.8 for the enzyme in tissue culture while activity was highest at pH 8.0 for the enzyme in fresh tissues. These differences in kinetic activity suggest a response to changes in the ion requirements of these cells due to their environment, developmental stage or some other parameter.