Concentrations and anti-Haemophilus influenzae activities of beta-casein phosphoforms in human milk.

BACKGROUND: The distribution and concentrations of six phosphorylated forms of human beta-casein, a major source of nutrition among breast-fed infants, have not been examined in milk samples without prior fractionation. In this study, the levels of beta-casein phosphoforms in untreated human milk samples were analyzed and their antiadhesion activities determined against Haemophilus influenzae, a pathogen implicated in middle ear infection in infants. METHODS: Human milk samples were analyzed using urea-polyacrylamide gel electrophoresis of whole-milk samples and scanning densitometry to determine the concentrations of beta-casein and its phosphoforms. A nontypable H. influenzae strain was radiolabeled to monitor its attachment to human pharyngeal cells in microtiter plates. Purified phosphoforms of beta-casein were preincubated for 15 minutes with radiolabeled bacteria to determine their antiadhesion activities. RESULTS: The average beta-casein concentration in 151 human milk samples was 5.37+/-2.26 mg/ml. On average, the phosphoforms in untreated milk are present in the following order ranked by concentration: tetra- > di- > non- > mono- > tri- > pentaphosphorylated beta-casein. The tri-, tetra-, and pentaphosphorylated forms of human beta-casein exhibited more than 60% inhibition of H. influenzae in the antiadhesion assay when used at a concentration of 0.6 to 0.9 mg/ml. CONCLUSION: The beta-casein level in untreated human milk is significantly higher than previously reported. The phosphoform distribution of beta-casein in individual donors varies widely. Anti-H. influenzae activity was detected in vitro among human beta-casein molecules with three or more phosphate groups.

Expression and characterization of phosphorylated recombinant human beta-casein in Escherichia coli.

Specific serine and threonine residues of recombinant human beta-casein produced in Escherichia coli were shown to be phosphorylated in vivo when human casein kinase II was coexpressed in the same plasmid. All of the phosphorylated forms found in the native protein were also detected in the recombinant protein. The phosphorylation of recombinant human beta-casein was confirmed by immunoblots, fast protein liquid chromatography, urea-polyacrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis, and liquid chromatography-mass spectrometry. The results indicate that the substrate specificity of casein kinase II in vivo was unaffected in its recombinant form. This is the first demonstration of in vivo phosphorylation of specific residues of a multiphosphorylated protein produced in E. coli with a single plasmid.

Resolution of the intermediates of de novo purine biosynthesis by ion-pair reversed-phase high-performance liquid chromatography.

An ion-pair reversed-phase high-performance liquid chromotographic procedure capable of resolving twelve of the fourteen intermediates of de novo purine biosynthesis is presented. The method utilizes isocratic elution and detection by ultraviolet light absorption. Separation of all twelve intermediates can be achieved in 90 min.

Variation of glycinamide ribonucleotide synthetase levels during in vitro aging of human fibroblasts: implications for gene dosage studies.

The levels of glycinamide ribonucleotide synthetase were monitored in four human fibroblast cell lines, two diploid, one monosomy 21 and one trisomy 21, from early passage number to senescence. In all four lines, enzyme activity increased as the cells aged. Enzyme levels for the diploid versus trisomy 21 cells did not consistently exhibit the expected gene dosage effect.

Assignment of a third purine biosynthetic gene (glycinamide ribonucleotide transformylase) to human chromosome 21.

Using a series of human-hamster hybrid cell lines, a gene coding for glycinamide ribonucleotide transformylase was mapped to human chromosome 21. The availability of hybrids containing only portions of chromosome 21 allowed the gene to be assigned to the region between the q11.2 and the q22.2 bands, inclusive. Differentiation of human and hamster glycinamide ribonucleotide transformylase was accomplished via an immunoprecipitation assay that employed a polyclonal antibody raised against the human enzyme.

Measurement of glycinamide ribonucleotide synthetase activity in intact human fibroblasts and Chinese hamster ovary cells.

An intact cell assay system based on Tween 80 permeabilization was used to investigate glycinamide ribonucleotide (GAR) synthetase activity in human fibroblasts and Chinese hamster ovary cells. Optimal conditions for the assay of the enzyme were determined with regards to ATP, MgCl2, NH4Cl and ribose-5'-phosphate concentrations as well as pH. Using the optimal assay conditions, the Vmax values as determined by Lineweaver-Burke double reciprocal plots were found to be 5.19 nmol GAR formed/5 X 10(5) cells/30 min for the fibroblasts and 13.4 nmol GAR formed/5 X 10(5) cells/30 min for the Chinese hamster ovary cells.

Regulation of ribonucleotide reductase activity in intact mammalian cells.

An intact cell assay system based upon Tween-80 permeabilization was used to investigate the regulation of ribonucleotide reductase activity in Chinese hamster ovary cells. Models used to explain the regulation of the enzyme have been based upon work carried out with cell-free extracts, although there is concern that the properties of such a complex enzyme would be modified by extraction procedures. We have used the intact cell assay system to evaluate, within whole cells, the current model of ribonucleotide reductase regulation. While some of the results agree with the proposals of the model, others do not. Most significantly, it was found that ribonucleotide reductase within the intact cell could simultaneously bind the nucleoside triphosphate activators for both CDP and ADP reductions. According to the model based upon studies with cell-free preparations, the binding of one of these nucleotides should exclude the binding of others. Also, studies on intracellular enzyme activity in the presence of combinations of nucleotide effectors indicate that GTP and perhaps dCTP should be included in a model for ribonucleotide reductase regulation. For example, GTP has the unique ability to modify through activation both ADP and CDP reductions, and synergistic effects were obtained for the reduction of CDP by various combinations of ATP and dCTP. In general, studies with intact cells suggest that the in vivo regulation of ribonucleotide reductase is more complex than predicted from enzyme work with cell-free preparations. A possible mechanism for the in vivo regulation of ribonucleotide reductase, which combines observations of enzyme activity in intact cells and recent reports of independent substrate-binding subunits in mammalian cells is discussed.

Ribonucleotide reductase activity in intact mammalian cells: stimulation of enzyme activity by MgCl2, dithiothreitol, and several nucleotides.

An intact cell assay system based on Tween-80 permeabilization was used to investigate ribonucleotide reductase activity in Chinese hamster ovary cells. Dithiothreitol, a reducing agent, is required for optimum activity. Analysis of dithiothreitol stimulation of CDP and ADP reductions indicated that in both cases the reducing agent served only to increase the reaction rate without altering the affinity of the enzyme for substrates. Magnesium chloride significantly stimulated the reduction of CDP but not ADP; this elevation in CDP reduction was due to an increase in both the affinity of the enzyme for substrate and the Vmax. In addition to ATP and dGTP, well-known activators of CDP and ADP reductase activities, it was found that dCTP and GTP were also able to activate CDP and ADP reductase activities, respectively. For the dCTP-activated reaction the Vmax was 0.158 nmol dCDP formed 5 X 10(6) cells-1 h-1 and the Km was 0.033 mM CDP, while for the GTP-activated reduction a Vmax of 0.667 nmol dADP formed 5 X 10(6) cells(-1) h-1 and Km of 0.20 mM ADP were observed. Kinetic analysis revealed that dCTP, dGTP, and GTP stimulate ribonucleotide reduction solely by increasing the affinity of the enzyme for substrate without affecting the Vmax of the respective reactions. ATP behaves in a different manner as it stimulates CDP reduction by altering both the affinity of the enzyme for substrate and the Vmax. Cellular concentrations of ribo- and deoxyribonucleoside di- and triphosphate pools were measured to help evaluate the relative physiological importance of the nucleotide activators. These determinations, along with the reaction kinetic studies, strongly imply that ATP is a much more important regulator of CDP reduction that dCTP, whereas GTP may serve as well or better than dGTP as the in vivo activator of ADP reduction.

A series of N-carbamoyloxyurea resistant cell lines with alterations in ribonucleotide reductase: lack of coordination in pyrimidine and purine reductase activity.

N-Carbamoyloxyurea is cytotoxic for cells in culture and, like hydroxyurea and guanazole, the drug is an effective inhibitor of mammalian ribonucleotide reductase and thus DNA synthesis. In addition to ribonucleotide reductase, N-carbamoyloxyurea has a second site of action which also appears to be in the pathway of DNA synthesis. A series of drug-resistant cell lines, which contain alterations in ribonucleotide reduction, have been sequentially selected in the presence of increasing concentrations of N-carbamoyloxyurea. CDP and ADP reductase activities in these drug-resistant lines have been investigated and two types of alterations have been identified: elevated levels of enzyme activity with wild-type sensitivity to drug and altered levels of reductase with reduced drug sensitivity, probably owing to structural modification of the enzyme. Furthermore, N-carbamoyloxyurea resistant lines contain another alteration as well, presumably at a second site of drug action. They are also cross-resistant to hydroxyurea and guanazole, and studies on enzyme activity levels support our previous findings with cells selected for resistance to hydroxyurea, which showed changes in CDP reductase activity are not always coordinated with changes in ADP reductase. Although several possibilities exist, these observations are most easily explained by the existence of independent enzyme substrate binding subunits which are regulated by different mechanisms. Moreover, increases in cellular resistance were accompanied by significant increases in CDP but not ADP reductase, suggesting that an ability to maintain an adequate level of CDP reductase activity is especially important to achieve resistance to DNA synthesis inhibitors like N-carbamoyloxyurea, hydroxyurea, and guanazole.

An iron-dependent 5'-nucleotide phosphoribohydrolase activity in the venom of Crotalus atrox and its effect upon the determination of mammalian ribonucleotide reductase.

There is an iron-dependent activity in the venom of Crotalus atrox which appears to hydrolyze the N-glycosidic sugar-base bond of pyrimidine and purine nucleotides. Maximal activation of this activity occurred at 1.0 mmol/l and 0.8 mmol/l FeCl3 for cytidine diphosphate (CDP) and ADP substrates, respectively. The release of free base affects the background values of control experiments carried out during nucleotide-labelled assays of ribonucleotide reductase which require the conversion of nucleotides to nucleosides by snake venom treatment. When the reductase is examined in intact cells, a situation closely resembling normal physiological conditions for the enzyme, FeCl3 was found to be an inhibitor of ADP reductions and varied from a mild stimulator to a significant inhibitor of CDP reductions depending upon FeCl3 concentration. Possible explanations for previously observed variability in ribonucleotide reductase activity in the presence of iron are suggested.

N-carbamoyloxyurea-resistant Chinese hamster ovary cells with elevated levels of ribonucleotide reductase activity.

We describe the isolation and characterization of a Chinese hamster ovary cell line selected for resistance to N-carbamoyloxyurea. Using the mammalian cell permeabilization assay developed in our laboratory, a detailed analysis of the target enzyme, ribonucleotide reductase (EC 1.17.4.1), was carried out. Both drug-resistant and parental wild-type cells required the same optimum conditions for enzyme activity. The Ki values for N-carbamoyloxyurea inhibition of CDP reduction were 2.0 mM for NCR-30A cells and 2.3 mM for wild-type cells, while the Ki value for ADP reduction was 2.3 mM for both cell lines. Although the Ki values remained essentially unchanged, the Vmax values for NCR-30A cells were 1.01 nmoles dCDP formed/5 X 10(6) cells/hour and 1.83 nmoles dADP/5 X 10(6) cells/hour, while those for the wild-type cells were 0.49 nmoles dCDP produced/5 X 10(6) cells/hour and 1.00 nmoles dADP/5 X 10(6) cells/hour. This approximate twofold increase in reductase activity as least partially accounts for a 2.6-fold increase in D10 value for cellular resistance to N-carbamoyloxyurea exhibited by NCR-30A cells. The NCR-30A cell line was also cross-resistant to the antitumor agents, hydroxyurea and guanazole. No differences in Ki values for inhibition of CDP and ADP reduction by these two drugs were detected and cellular resistance could be entirely accounted for by the elevation in activity of the reductase in the NCR-30A cell line. The properties of N-carbamoyloxyurea-resistance cells indicate they should be useful for further investigations into the regulation of mammalian enzyme activity.