Martin J. Griffin

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.

The invivo effect of benzamide and phenobarbital on liver enzymes: Poly(ADP-ribose) polymerase, cytochrome P-450, styrene oxide hydrolase, cholesterol oxide hydrolase, glutathione S-transferase and UDP-glucuronyl transferase

Rats fed a synthetic diet containing 0.25% benzamide, 0.1% phenobarbital, separately or in combination, for two weeks showed a significant augmentation in the activity of nuclear poly(ADP-ribose) polymerase as well as changes in various nuclear, microsomal and cytosolic liver enzymes involved in the metabolism of xenobiotics. A selective depression of microsomal styrene oxide hydrolase activity by benzamide feeding, and a contrasting augmentation by phenobarbital, were confirmed by immunological titration of the enzyme-protein content suggesting actual enzyme repression and induction. The NAD content of these livers is not altered significantly as a result of benzamide and phenobarbital feeding, indicating that the changes in enzymes are not a result of non-specific toxic effects.

Synchronization of some human cell strains by serum and calcium starvation

SummaryA technique was investigated for producing parasynchronous growth of some established, aneuploid human cell strains. Removal of both serum and calcium from exponentially growing monolayer cells tended to inhibit their growth. After 20 hr, a high percentage of the cell population was arrested in or near mitosis. Readdition of serum and calcium caused parasynchronous growth of the cells of three human strains studied. All three strains incorporated tritiated thymidine maximally 10 to 15 hr after serum and calcium were added, and cell numbers increased rapidly 17 to 25 hr after the growth medium was reconstituted. Population-doubling ranged from 80% to 100% of the theoretical. The yield of parasynchronous cells is high with this technique and may produce a significant amount of nontemporally distorted biological material upon which direct biochemical analysis can be performed at various times within the generation cycle.

Cell cycle variations in HeLa 65 plasma membrane alkaline phosphatase.

Abstract HeLa plasma membranes from M, G 1 , and S phase cells were isolated from growing synchronous cell cultures. It was found that the specific activity of plasma membrane alkaline phosphatase was over three times higher in the M phase cell than in the G 1 and S phase cell. However, sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis showed that the S phase plasma membrane contained 5.5 times more alkaline phosphatase protein than did the plasma membrane from mitotic cells, and 11.0 times more than the G 1 phase plasma membrane. This would indicate that the high specific activity in mitosis was due to modification of the alkaline phosphatase protein resulting in increased enzymatic activity.

Cyclic AMP inhibition of protein kinase activity in hela 65 plasma membranes

Abstract Protein kinase activity was found associated with isolated HeLa 65 plasma membranes. This Mg++ dependent kinase activity was found to be inhibited by 10−4 M cyclic AMP, 2 × 10−2 M NaF and 2 × 10−2 M theophylline. The product of the endogenous phosphorylation was a protein, which was phosphorylated on serine residues. The in vivo phosphorylation of HeLa 65 cell plasma membrane was negligible in G1 and highest in the S phase of the cell cycle. There was no significant difference in kinase activity of isolated plasma membranes from M, G1, or S phase cells. Thus cyclic AMP inhibition of plasma membrane kinase during G1 may be physiologically important.