Alkaline phosphatases heat sensitivity

A biochemical evalution of human alkaline phosphatase is postponed until the above considerations have been presented. In our view, the most reasonable analytical approach is based on the measurement of L-phenyl-alanine-sensitive and -insensitive moieties along with their respective heat stabilities. To this may be added information gathered from starch-gel electrophoresis with native and heated serum and from the presence of L-phenylalanine-sensitive bands on the gels following electrophoresis. Experiments of a different type can be included, in which the serum is incubated with neuraminidase and susceptibility of the glycoprotein is established following electrophoresis. Finally, the data on L-phenyl-alanine inhibition of heat-sensitive and -insensitive moieties appear to make sense, if the population of normal subjects is divided into one with the slow-moving intestinal band and one without it. It is from this consideration and other indirect and direct inferences that the intestine is... 

The greater heat sensitivity of bone alkaline phosphatase as compared to that of liver, kidney, and intestine was first reported without comnaent by Moss and King (M36). The incubation periods (at 55°C and pH 7.0)... 

Our data support the statement that the heat sensitivity of each enzyme source remains characteristic and independent of the influence of the others in the mixture, and that the resultant heat inactivation is an additive function of the heat-sensitivities of members of the mixture. Bone enzyme from different sources is very consistently heat-sensitive (85-90%), unlike intestinal (50-65%), and liver enzyme (50-75%). However, the heat sensitivity of the LPSAP of normal serum can vary from 33 to 85% and of the non-LPSAP fraction from 50 to 95%. Therefore one cannot determine the identity of the organ sources of serum alkaline phosphatase with a knowledge of only the heat sensitivity and the total alkaline phosphatase. However, by correcting the heat-inactivation of serum by that contributed by intestinal component, one obtains the heat-inactivation of non-intestinal sources of alkaline phosphatase. If this value is 90% or more, the non-intestinal component could be presumed to be of osseous origin if 60% or less, of hepatic origin. 

The L-phenylalanine-sensitive component of serum alkaline phosphatase exhibits heat sensitivity that on occasion is far below or far above the expected heat sensitivity for intestine. For this reason, it is... 

The tissue preparations were diluted (1 12) in heat-inactivated sera (1 hour at 55°C), and incubated in 0.02 M Veronal buffer (pH 9.8) containing 0.018 M disodium phenyl phosphate for 2 hours at 37°C. Phenol was measured via a diazo coupling procedure. Conditions for heat-inactivating the tissue enzymes were 16 minutes at 55°C. By subtracting from the total activity the intestinal component, which is measured by n-phenylalanine sensitivity, one obtains the sum of the activities of liver and bone. The ratio of the two was computed from the heat inactivation minus that attributed to intestine, employing 91.2% heat inactivation to represent 100% bone and 51.4% heat inactivation indicating all liver. In this way one arrives at values for bone, liver, and intestinal alkaline phosphatase. 

We assume that normal individuals have the same organ sources of serum alkaline phosphatase whether or not the slow intestinal band is present. Thus, persons with the band have more total and heat-stable LPSAP and those lacking the band, more heat-stable non-LPSAP. This can be explained if some of the intestinal alkaline phosphatase, before, during, or after absorption loses its sensitivity to L-phenylalanine but... 

Since there is no difference in the amount of alkaline phosphatase in the intestinal mucosa of subjects of various blood types and secretor status (L2) and since this enzyme is presumed to be L-phenylalanine-sensitive (L14), then the difference in the relative amount of heat-stable LPSAP in persons with or without the slow band may relate to events... 

The alkaline phosphatase of both human intestine and placenta are L-phenyl-alanine-sensitive and undergo uncompetitive inhibition to the same extent (nearly 80%) by 0.005 M L-phenylalanine. However, we have been able to find several distinguishing biochemical characteristics of the two enzymes (1) the anodic mobility of intestinal alkaline phosphatase remains unchanged after neuraminidase treatment, whereas the placental enzyme is sialidase-seusitive and hence the electrophoretic mobility on starch gel is considerably reduced by such treatment, (2) the Michaelis constant of placental alkaline phosphatase at a definite pH is appreciably higher than that of the intestinal enzyme (at pH 9.3 the Km values of placenta and intestine are 316 and 160 ixM, respectively), and (3) the pH optima (with 0.018 Af phenyl phosphate as substrate) of the two enzymes are different the values for intestinal and placental enzymes with 0.006 Af n-phenylalanine are 9.9 and 10.6, respectively, and the respective values in the presence of 0.005 Af L-phenylalanine are 10.2 and 11.1. Finally, contrary to the behavior of intestinal alkaline phosphatase, placental enzyme is completely heat stable (P19). 

Case A (Fig. 36). This female patient (blood type B) with cancer of the endometrium metastatic to liver and lung showed no change in alkaline phosphatase on the first 60 days of Delalutin (progesterone) therapy, and then showed a rise for the next 80 days. The elevated values over this period showed a reasonably close proportionate rise in the LPSAP and non-LPSAP. This circumstance applies also to the heat-sensitive and -insensitive fractions. The percent heat inactivation remains constant for both LPSAP and non-LPSAP moieties at 62 and 50%... 

Case B (Fig. 37). This female patient (blood type A) with cancer of the breast metastatic to bone showed a rise in alkaline phosphatase while on androgen (Halotestin) therapy. There was a proportionate increase in both LPSAP and non-LPSAP moieties, which could be attributed to the alteration in the heat-sensitive portions the heat-insensitive fractions showed no appreciable alteration. With the increase, the per-... 

With the aid of photomicrographs of the L-phenylalanine-sensitive placental alkaline phosphatase, one can picture the placental villi in contact with the maternal circulation (H3) and so enriching it with alkaline phosphatase. In support of this view, we have demonstrated a progressive rise in pregnancy of heat-stable LPSAP with an optimum pH of 10.7. 

The portal blood-borne alkaline phosphatase from the intestine undergoes mixing in the liver with the enzyme that has traveled the lymphatic route. The proportions of these two populations of enzyme molecules, and their heat lability and sensitivity to L-phenylalanine are under the control of a number of physiological variables already discussed. 

The biochemical fractions of the serum alkaline phosphatase in cirrhosis are listed in Table 13 according to blood types A and 0. It may be advantageous to compare the various moieties in these patients with the data on normal subjects of corresponding blood type (Table 10). In some cases (4, 5, 14) the elevation is proportionate in the LPSAP and non-LPSAP moieties, whereas in others (1, 2, 3, 9, 10, 12, 13, 15) there is a disproportionate elevation in the LPSAP fraction. Subject 7 exhibits the largest contribution of LPSAP (60%). There appears to be no similarity of the heat sensitivities of LPSAP and non-LPSAP in subjects 4, 5, 7, 14, and 15 with total LPSAP hyperphosphatasemia. In fact, the relatively high heat inactivation of sera 5, 6, 7, and 12 would register negatively for liver in a heat-inactivation test of total alkaline phosphatase (P19). 

Starch-gel migration of bone alkaline phosphatase yields bands in locations that can be occupied by gastrointestinal juice alkaline phosphatase as well as by the enzyme of kidney, lung, and spleen. High heat sensitivity is a property of the alkaline phosphatase from these various sources. Hence findings based on starch-gel electrophoresis and heat sensitivity in themselves are not diagnostic of a bone source. 

In general, it would seem that osteoblasts could not compare as a source of alkaline phosphatase with intestine or placenta. In pregnancy the level of alkaline phosphatase is rarely increased above 10 Bodansky or Shinowara units, and yet the placenta s microvilli, which are extremely rich in alkaline phosphatase, are directly immersed in the ample and efficient maternal blood supply (H3). In the nonpregnant individual, therefore, before hyperphosphatasemia can be attributed to bone it would appear necessary to evaluate the intestinal contribution that is evident as heat-sensitive non-LPSAP protein. 

The application of the heat-sensitive measurement by Posen et al. (P19, P20) has been restricted to sera of patients with uncomplicated liver or bone disease. Here, sera with high inactivation of alkaline phosphatase are interpreted as bone and those that are relatively heat-stable as liver. Particularly disturbing to us were sera from patients with cirrhosis of the liver (patients 5, 6, and 12, Table 13) exhibiting heat sensitivity of non-LPSAP in the bone range. The measurement of heat sensitivity of serum alkaline phosphatase alone cannot be used for the certain identification of liver or bone sources or their mixtures. 

The enzyme alkaline phosphatase is naturally present in milk. As it is sensitive to heat it is used to measure the effectiveness of pasteurization. Milk is diluted with a buffer (pH 10.6) containing disodium phenol phosphate which is hydrolyzed by the enzyme releasing phenol. The phenol is reacted with 2,6-di-bromoquinonechloroimide producing a color that is measured spectrophotometrically at 610 nm. An earlier version of this test uses p-nitrophenyl phosphate and the amount of yellow p-nitrophenyl which is released is compared with standard comparator disks. 

When ligating an insert, always perform a negative control with the same plasmid but no insert DNA. Ideally, you would see more colonies on the plate with the insert than on the negative control. However, we have found that when the negative control plate has more colonies, the smaller number of colonies on the experimental plate often contain the correct ligation product. If the plasmid recircularizes frequently, you can use a heat-sensitive alkaline phosphatase to remove the 5 phosphates from your vector. 

---