Peter J. Butterworth

Alkaline phosphatase of chick kidney.

The alkaline phosphatase prepared from kidneys of domestic chicks is a tetramer (Mr 270,000) consisting of identical subunits (Mr 68,000). The tetramer may be dissociated by detergent treatment to a dimer (Mr 150,000) with no loss of catalytic activity. The tetramer probably represents the in vivo state. The enzyme is stimulated by Mg2+ and inhibited noncompetitively by Zn2+ and levamisole. The stimulation and inhibition show similar pH dependencies. Evidence for an essential histidine is provided by sensitivity of the enzyme to diethyl pyrocarbonate. It is suggested that chick kidney and bone phosphatases could be expressed by different genes.

Effect of estradiol on neurotransmitter sensitive adenylate cyclase. Its possible role in ‘sexual differentiation’

Adenylate cyclase activity is lower in the hypothalamus of 5-day-old male rats than in females. This may be due to the presence of estradiol (E2) in this area of the male but not in the female since castration of the newborn male leads to an enzyme activity in the hypothalamus that is indistinguishable from that of the female and androgenization of the newborn female causes an enzyme activity level comparable to that of the male. In the adult, enzyme activity is highest when the concentration of E2 is at its lowest level; adenylate cyclase activity in the hypothalamus-preoptic area was found to be higher in the metestrus female than in proestrus or in the adult male. In vitro E2 was shown to reduce enzyme activity and this reduction was found to be dependent on induction of protein biosynthesis. In addition, there appears to be a requirement for Ca2+ in the E2-induced reduction of cyclase activity. Although F- activates the enzyme in all of the cases studied, the reduction in enzyme activity brought about by E2 is not reversed by F-, suggesting that the effect of E2 is not on the receptor. As sexual differentiation is brought about by the action of E2 during the first week after birth, it seems plausible to suggest that this interaction between hormone and enzyme is an early step in the sexual differentiation of the brain.

Modulation of phosphate accumulation in isolated chick kidney cells by gluconeogenic substrates

An isolated proximal-tubule preparation is described that accumulates Pi in a saturatable, Na+-dependent manner. The initial rate of Pi accumulation is greater in cells incubated with the gluconeogenic substrates pyruvate and lactate than in cells incubated with glucose. Glucose was produced from the substrates under these conditions. Incubation with either NAD+ or NADH inhibits the initial rate of Pi accumulation. These data provide evidence that the effects of gluconeogenesis on Pi uptake are not mediated by the oxidation state of the nicotinamide coenzymes.

Catalytic and ligand-binding properties of rat intestinal alkaline phosphatase.

Abstract Rat intestinal alkaline phosphatase is a dimeric enzyme with identical subunits and thus possesses two presumably identical active sites. Binding studies with P i and l -phenylalanine and pre-steady-state “burst” titrations confirm the existence of two active sites per molecule of enzyme. The sites appear to be nonequivalent with respect to P i binding, both at low pH, where an enzyme (E)-P i covalent complex is formed, and at high P i , where an E-P i noncovalent complex predominates. The binding affinity of the first site is 100-fold greater than that of the second, i.e., there is negative cooperativity. The K i value for competitive inhibition of substrate hydrolysis by P i corresponds to the higher affinity site. The negative cooperativity appears not to be an artifact resulting from contaminating P i in the purified enzyme preparation. l -Phenylalanine does not bind to the enzyme unless P i is present, as expected from the previously proposed mechanism of uncompetitive inhibition by the amino acid. No negative cooperativity is seen in l -phenylalanine binding, but the number of moles of amino acid bound at saturation depends on the degree of saturation by P i The enzyme is also inhibited uncompetitively by NADH, which can compete with l -phenylalanine for the same site on alkaline phosphatase.

Biochemical studies of the control of renal tubular phosphate reabsorption.

The proximal tubule of the kidney is the site of several important physiological processes including the reabsorbtion of inorganic phosphate from the glomerular filtrate and the production of glucose from intermediate metabolites. The phosphate reabsorption performed by the kidney seems to be the major controlling factor in m aintaining overall phosphate homeostasis.

Reactive thiol groups in rat liver acid phosphatase.

Dimeric rat liver acid phosphatase P1 of Mr 92,000 is inactivated by p-chloromercuribenzoate and fluorescein mercuriacetate (FMA). The enzyme is protected against the mercurials by the substrate analogue Pi. The reaction with FMA is accompanied by changes in absorbance at 495 nm and in fluorescence emission at 520 nm that are characteristic of reaction of this compound with thiol groups. Titration of P1 with FMA monitored by spectrophotometry or by fluorimetry indicated that equivalence is reached at an FMA/P1 ratio of 3. Since FMA can act as a bifunctional reagent, it is likely that P1 contains either 3 or 6 reactive thiol groups per molecule. Analysis of FMA inactivation/modification data by a statistical method suggests that of 6 reactive thiol groups, 2 are essential so that there are probably 3 thiol groups per subunit, one of which is located at the active site. If the total thiol number is 3, analysis suggests 1 essential thiol per subunit.