Rody P. Cox

Hormonal induction of alkaline phosphatase activity by an increase in catalytic efficiency of the enzyme

Abstract Alkaline phosphatase activity of HeLa 65 cells is increased 5- to 20-fold during growth in medium containing cortisol. The kinetics of increase in activity are characterized by a 12- to 24-hour lag period after adding hormone, followed by a linear and rapid increase of activity which reaches a plateau after 60 to 100 hours. Both RNA and protein synthesis are required for “induction”. However, immunological methods show that the amount of enzyme protein in induced cells is not increased; suggesting that there is neither an increased rate of alkaline phosphatase synthesis nor a hormone-mediated decrease in the catabolism of the enzyme. When the kinetics of induction are compared to the kinetics of enzyme synthesis using radioactively labeled enzyme and specific precipitation by antiserum, the alkaline phosphatase is maximally labeled long before the increase of enzyme activity reaches a maximum. This finding suggests that the hormone acts by initiating the synthesis of a modifier molecule which interacts with the enzyme to produce an enhanced catalytic efficiency. The physical and chemical characteristics of the base-level and induced enzyme are similar except the V max of the induced alkaline phosphatase is much greater. The increased catalytic efficiency of induced alkaline phosphatase is not the result of an increase in the zinc ion content of this metalloenzyme. However, the binding of ionic zinc to the induced form of apoenzyme appears to be different from that of the base-level enzyme. It is suggested that the enhanced catalytic activity of induced alkaline phosphatase is the result of an alteration in zinc ion binding which produces an entatic effect, lowering the energy requirements of the enzyme substrate transition state.

A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase.

Abstract The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 m m N 6 , O 2 ′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 m m . Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used.

Enzyme activity in classical and variant forms of maple syrup urine disease

The branched-chain keto acid decarboxylase activity has been determined in skinfibroblasts grown from six subjects with classical maple syrup urine disease and six with variant forms of the disease. The level of activity in the skin fibroblasts reflects the ability of the individual to degrade the amino acids, thus providing an index of the severity of the disease. Observations on sibling pairs indicate that the level of enzyme activity and the severity of the disease are genetically determined. A 3 grade classification is proposed based on tolerance for dietary protein. The metabolic defect in all instances involved the three branched-chain amino acids, providing further support for the concept that this degradative step is under the control of a single gene.

Communication between normal and enzyme deficient cells in tissue culture

Abstract Correction of certain mutant phenotypes by intimate contact with normal cells, i.e. ‘metabolic cooperation’, is an easily studied form of cell communication. Metabolic cooperation between normal cells and mutant cells deficient in hypoxanthine-guanine or adenine phosphoribosyl transferase (HGPRTase and APRTase respectively) appears to be the result of transfer of the enzyme product, nucleotide or nucleotide derivative, from normal to mutant cells. This process shows selectivity in that mutant derivatives of mouse L cells are unable to function as recipients of HGPRTase or APRTase products, while hamster and human fibroblasts with these enzyme deficiencies, exhibit correction of the mutant phenotype, when in contact with normal donor cells. There is also selectivity with respect to substances transferred, since other mutant phenotypes, i.e. G-6 PD deficiency, are not corrected by contact with normal cells. Species specificities do not appear to influence metabolic cooperation, therefore heterospecific cell mixtures provide an opportunity to cytologically distinguish cells and study individual cell interactions. Transfer of nucleotide from normal to mutant cells is less dependent on energy production than is the incorporation of radioactive purines into cellular material. The nucleotide translocation mechanism is not susceptible to sulfhydryl blocking agents.

Production of fetal-like alkaline phosphatase by HeLa cells

Alkaline phosphatase produced by HeLa cells differs in its chemical and physical properties from the enzyme found in adult organs and tissues (Cox and Griffin, 1967). In the present study HeLa cell alkaline phosphatase was compared to a fetal form of the enzyme found in human placenta. Both enzymes have approximately the same molecular weight as judged by sucrose density gradients, and the chemical and physical properties of these alkaline phosphatases are similar. The electrophoretic pattern of the HeLa cell enzyme resembles the placental alkaline phosphatase of the heterozygous FS phenotype except that it is slower moving. Double immunodiffusion using an antibody against HeLa cell alkaline phosphatase and placental and HeLa cell enzymes as antigens shows a single line of partial identity between the two enzymes, with a small spur suggesting additional antigenic sites on the HeLa cell enzyme. The data suggest that malignant cells in culture, HeLa, are producing a fetal-like alkaline phosphatase probably by “derepression of the genome.” However, the electrophoretic and immunological characteristics of the enzyme are altered sufficiently so that it can be distinguished from the normally produced fetal enzyme.

Alkaline phosphatase: I. Comparison of the physical and chemical properties of enzyme preparations from mammalian cell cultures, various animal tissues, and Escherichia coli☆

Abstract Some of the chemical and physical properties of alkaline phosphatase preparations derived from mammalian tissues, cell cultures, and E. coli have been compared. A number of chemical and physical properties of the various enzyme preparations are similar, such as the concentration of cysteine and histidine that inhibits alkaline phosphatase activity, and the phosphotransferase activity of different enzyme preparations. Other properties of the various alkaline phosphatases are markedly different, such as heat stability at 56 °, electrophoretic mobility, and the concentrations of Zn ions, L -phenylalanine, and L -tryptophan required to inhibit enzyme activity by 50%. These differences provide a means of distinguishing between alkaline phosphatases from different tissues and different species. Some of the physical and chemical characteristics of these enzymes appear to be related to, or coincidently associated with the mechanisms of enzyme regulation observed in cell cultures and in vivo.

Disparate Enzyme Activity in Erythrocytes and Leukocytes. A VARIANT OF HYPOXANTHINE PHOSPHORIBOSYL-TRANSFERASE DEFICIENCY WITH AN UNSTABLE ENZYME

A family is reported in which each of two sisters has a son with no detectable hypoxanthine phosphoribosyltransferase (HPRT) (EC 2. 4. 2. 8) in his erythrocytes, a finding considered pathognomonic of Lesch-Nyhan disease. However, neither has the stigmata of the disease. One boy is neurologically normal, and the other is moderately retarded. There was only a slight increase in urinary uric acid, but the amounts of hypoxanthine and xanthine, and their ratios, were similar to those found in Lesch-Nyhan disease, strongly indicating that excesses of these last two oxypurines are not responsible for the symptomatology in that disease. In contrast to the nondetectable HPRT activity in the red blood cells, leukocyte lysates from the two boys have 10-15% of normal activity, possibly reflecting continuing synthesis of an unstable enzyme. This hypothesis is supported by the demonstration that at 4 degrees C HPRT activity was rapidly lost in the propositus while the activity increased in control subjects. The mothers cells were intermediate between the two. The intact and disrupted leukocytes of the hemizygote, in the absence of added phosphoribosyl converted as much hypoxanthine to inosinate as the normal cell, and appropriate tests indicated that under these circumstances enzyme concentration is not rate limiting whereas the concentration of the cosubstrate, phosphoribosyl pyrophosphate, is. The capacity for normal function in the intact mutant cell is more representative of in vivo conditions than the lysate, which may explain the important modification of clinical symptomatology, the relatively mild hyperuricosuria, and the presence of mosaicism in the circulating blood cells of the heterozygotes. A similar explanation may apply to other genetic diseases in which incomplete but severe enzyme deficiencies are found in clinically normal individuals. An associated deficiency in glucose-6-phosphate dehydrogenase in this family permitted confirmation of previous observations on linkage with hypoxanthine phosphoribosyltransferase.

Inhibition of alkaline phosphatase by cysteine and its analogues.

Summary 1. A comparison of the inhibition of human-kidney alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) by cysteine and structurally related compounds shows that two main groups of inhibitors can be distinguished: (a) analogues with free amino and sulfhydryl groups are good inhibitors, (b) analogues with blocked amino or sulfhydryl groups and compounds lacking these groups are poor inhibitors. 2. Studies on the effect of pH on the inhibition reveal that the inhibiting species must have the amino and sulfhydryl group in their basic form. 3. The results indicate that cysteine inhibits alkaline phosphatase by chelation of the Zn atom at the active site of the enzyme. This inhibition seems to be the result of a chelation in situ rather than a removal of the metal from the apoenzyme. 4. Alkaline phosphatase preparations from different tissues, cell cultures and Escherichia coli are all inhibited by the same concentration of L -cysteine.

Enzyme defect in skin fibroblasts in intermittent branched-chain detonuria and in maple syrup urine disease☆

Abstract The enzyme defect in maple syrup urine disease and in its varient form, intermittent branched-chain ketonuria, is demonstrable in skin fibroblast cultures derived from patients with this disease. In both instances there is considerable reduction in the ability to decarboxylate all three branched-chain keto acids. In view of the recent evidence that there are two or three branched-chain decarboxylases, each with a specific substrate, it is suggested that one gene controls the synthesis of a polypeptide that is common to the involved enzymes or to some structural or regulatory part of the enzyme complex.

Hypoxanthine phosphoribosyltransferase activity in intact fibroblasts from patients with X-linked hyperuricemia.

Discordance between clinical phenotype and the level of a mutant enzyme activity may reflect differences between enzyme function in vivo and that measured by the customary enzyme assays on cell extracts. In the present study, the conversion of hypoxanthine to phosphorylated products was measured in intact skin fibroblasts and in cell extracts from seven patients with mutant hypoxanthine-guanine phosphoribosyltransferase (HPRT) and six control subjects. The patients phenotypes ranged from asymptomatic hyperuricemia to the Lesch-Nyhan syndrome. Although there was a general correlation between the HPRT activity in cell extracts assayed by the usual methods and the function of the purine salvage pathway in patients, as reflected by urinary oxypurine excretion, there were notable exceptions. A more accurate appraisal of the functioning of the pathway at the cellular level is achieved by measuring the conversion of substrate to product in the intact cell at physiological concentrations of substrates, activators, and product and metabolite inhibitors, and in a physiological ionic environment. In one of the seven patients, the standard enzyme assay indicated normal function, whereas measurements in the intact cell exposed severe dysfunction of the salvage system. In another, the standard assay suggested a severe deficiency not evident in the intact cell or in the patient.