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On the importance of being (stereo) specific
CIinicu Chin&u Act4 Elsevier
129
186 (1989) 129-132
CCA 04558
His torical Perspective
On the importance of being (stereo) specific * William H. Fishman LQJolla
Cancer Research Foundation, Lu Jolla, CA (USA)
(Received
14 June 1989; accepted 19 June 1989)
The current era of exciting advances in the molecular and cell biology of the alkaline phosphatase isozymes represents to me ‘the promised land’ towards which I and others have been struggling to reach over forty years. The papers including in the Symposium have given us a picture which can be the platform of knowledge upon which to build future progress. My purpose is to illuminate a key signpost which has guided me along the route of my scientific travels in clinical enzymology. It is both the stereo specificity and organ specificity of amino acid inhibitors of enzymes. Organ specific inhibition of phosphatases
[l]
Long before isozymes were defined on the basis of differences in their electrophoretic migration in starch gel, there were indications that different forms of the same enzyme existed. For example, Oscar Bodansky raised the question in 1937 ‘ . . .Are the phosphatases of bone, kidney, intestine and serum identical? The use of bile aids in their differentiation.. . ’ And in 1949, Abdul-Fadl and King distinguished the acid phosphatase in erythrocytes and in prostate with L-tartrate and sodium fluoride. The practical significance of the L-tartrate inhibition of prostatic and not of erythrocytic acid phosphatase was the opportunity to eliminate a major contaminant (red cells) from serum which could otherwise greatly inflate acid phosphatase determinations and make accurate interpretation of prostate status impossible. Fishman and Lemer (1953) found the optimal conditions for measuring ‘prostatic’ acid phosphatase in patient’s sera. This method was utilized in many hospitals until it was superceded by radioimmunoassay.
* Remarks following the Tribute Dinner in honor of Dr. Fishman’s 75th birthday and his appointment as Resident emeritus of the La Jolla Cancer Research Foundation. Correspondence to: William H. Fishman, La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA.
0009-8981/89/$03.50
0 1989 Elsevier Science Publishers B.V. (Biomedical
Division)
130
Attempts to distinguish bone from liver alkaline phosphataw [l] In 1954, the late Dr. Daniel Lazlo at Montifiore Hospital challenged me to find a way to measure separately liver and bone alkaline phosphatases in the sera of breast cancer patients undergoing hormonal therapy. Oncologists were simply unable to interpret the significance of an elevation in the serum alkaline phosphatase as it could reflect either exacerbation or remission of the lesions. Based on the success with L-tartrate inhibition, I assembled 139 chemicals including bile acids, metals, -SH compounds and amino acids, all of which had some history as enzyme inhibitors. The rat was chosen to supply various tissues and serum. After two years of intensive work, we failed to find a substance which added to enzyme digests would inhibit liver or bone specifically. As Dr. Riggs at the Beckman Research Institute of the City of Hope would describe it, this was a great failure which nevertheless had an unexpected benefit. The benefit was that an intestine specific inhibitor, L-phenylalanine, was recognized which was used to identify this alkaline phospbatase in the mixture found in rat serum. D-Phenylahurine was not an inhibitor. Intestinal alkaline phosphatase was the dominant component in rat serum. When the L-phenylalanine inhibition was applied to human serum, there was good news and bad news. The good news was that the L-phenylalanine was a much better inhibitor of human as compared to rat intestinal alkaline phosphatase. The bad news was that the intestinal enzyme usually represented a relatively small portion of the total serum alkaline phosphatase. L-Phenylalanine di~shes isozymes Ill.
in~tinal and placental isozymes from liver and bonfz
Human placental alkahne phosphatase undergoes the same inhibition by L-phenylalanine as human intestinal isozyme while the liver and bone enzymes are unaffected. L-Homoarginine has just the opposite organ specificity. Both L-phenylalanine and L-homoarginine exhibit the rare uncompetitive type of inhibition, D-phenylalanine does not. Siiicance
of D-m*
in the control [l]
D-Phenylidanine is employed in controls in equimolar amounts to L-phenylalanine in a variety of contexts; in enzyme assays; in electrophoretic studies; in hi&chemical studies and in investigations of the physiology of fat absorption. The only difference between the test and control solutions then is the difference in the arrangement of the a-carbon amino and hydrogen groups. It is this configuration in the L-enantiomorph which accounts for the inhibition, which has been classified as uncompetitive. L-Tryptophan exhibits the same stereospecific, organ specific inhibition.
131
Discovery of the Regan Isozyme, ‘PLAP’ [1,2]
It was the r_-phenylalanine inhibition, coupled with heat inactivation and starch gel electrophoresis, which led us to identify the Regan Isozyme in the serum and tumor tissue of a patient with terminal lung cancer. The Regan isozymes’ properties were indistinguishable from those of human placental alkaline phosphatase. The apparent reexpression of a placental gene in a cancer resident in a male has been an observation which has challenged us to find the explanation. In truth, from this has grown my conviction that the understanding of cancer lies at the interface of oncology and developmental biology. This led, in 1976, to the creation of the La Jolla Cancer Research Foundation and to its present direction. Origin of my regard for the importance of stereospecificity
Where did the idea come to me to evaluate the D- and L-forms of amino acids? In 1941 as an instructor in biochemistry at the then newly opened Bowman-Gray School of Medicine in Winston-Salem, I was working with Dr. Camillo Artom on the changes in liver phospholipids induced by changes in diets fed to rats. Groups of rats received either choline, ethanolamine or DL-serine by stomach tube. All the animals receiving DL-serine died [3]. Dr. Artom exclaimed, ‘these rats did not die because they were tired of living’. We were able to prove that it was the D-serine enantiomorph which was toxic. It produced necrosis of the proximal tubules of the kidney. Here was my first experience where the configuration on the a-carbon of an amino acid could have such profound biological effects [4]. Looking back to this experience, I can now identify fairly accurately why I was motivated to investigate the behavior of enantiomorphs of amino acids separately rather than to assume that the D- and L-forms were equal in their biological and biochemical significance. The L-phenylalanine inhibition has been a key signpost in my career and I am honored to have an opportunity to share my thoughts on the importance of being stereo specific. References 1 Fishman WH. ‘Oncodevelopmental enzymes’, The clinical biochemistry of cancer. Proceedings of the Second Arnold 0. Beckman Conference in Clinical Chemistry. Fleisher M, ed. AACC publication, 1978;132-154. 2 Fisbman WH. Oncotrophoblast gene expression-placental alkaline phosphatase. Adv Cancer Res 1986;46:1-36. 3 Fishman WH, Artom C. Serine injury. J Biol Chem 1942;45:345-346. 4 Artom C, Morehead RP, Fishman WH. The relative toxicity of L- and DL-serinein rats. Proc Sot Exp Med Biol 1945;60:284.
129
186 (1989) 129-132
CCA 04558
His torical Perspective
On the importance of being (stereo) specific * William H. Fishman LQJolla
Cancer Research Foundation, Lu Jolla, CA (USA)
(Received
14 June 1989; accepted 19 June 1989)
The current era of exciting advances in the molecular and cell biology of the alkaline phosphatase isozymes represents to me ‘the promised land’ towards which I and others have been struggling to reach over forty years. The papers including in the Symposium have given us a picture which can be the platform of knowledge upon which to build future progress. My purpose is to illuminate a key signpost which has guided me along the route of my scientific travels in clinical enzymology. It is both the stereo specificity and organ specificity of amino acid inhibitors of enzymes. Organ specific inhibition of phosphatases
[l]
Long before isozymes were defined on the basis of differences in their electrophoretic migration in starch gel, there were indications that different forms of the same enzyme existed. For example, Oscar Bodansky raised the question in 1937 ‘ . . .Are the phosphatases of bone, kidney, intestine and serum identical? The use of bile aids in their differentiation.. . ’ And in 1949, Abdul-Fadl and King distinguished the acid phosphatase in erythrocytes and in prostate with L-tartrate and sodium fluoride. The practical significance of the L-tartrate inhibition of prostatic and not of erythrocytic acid phosphatase was the opportunity to eliminate a major contaminant (red cells) from serum which could otherwise greatly inflate acid phosphatase determinations and make accurate interpretation of prostate status impossible. Fishman and Lemer (1953) found the optimal conditions for measuring ‘prostatic’ acid phosphatase in patient’s sera. This method was utilized in many hospitals until it was superceded by radioimmunoassay.
* Remarks following the Tribute Dinner in honor of Dr. Fishman’s 75th birthday and his appointment as Resident emeritus of the La Jolla Cancer Research Foundation. Correspondence to: William H. Fishman, La Jolla Cancer Research Foundation, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA.
0009-8981/89/$03.50
0 1989 Elsevier Science Publishers B.V. (Biomedical
Division)
130
Attempts to distinguish bone from liver alkaline phosphataw [l] In 1954, the late Dr. Daniel Lazlo at Montifiore Hospital challenged me to find a way to measure separately liver and bone alkaline phosphatases in the sera of breast cancer patients undergoing hormonal therapy. Oncologists were simply unable to interpret the significance of an elevation in the serum alkaline phosphatase as it could reflect either exacerbation or remission of the lesions. Based on the success with L-tartrate inhibition, I assembled 139 chemicals including bile acids, metals, -SH compounds and amino acids, all of which had some history as enzyme inhibitors. The rat was chosen to supply various tissues and serum. After two years of intensive work, we failed to find a substance which added to enzyme digests would inhibit liver or bone specifically. As Dr. Riggs at the Beckman Research Institute of the City of Hope would describe it, this was a great failure which nevertheless had an unexpected benefit. The benefit was that an intestine specific inhibitor, L-phenylalanine, was recognized which was used to identify this alkaline phospbatase in the mixture found in rat serum. D-Phenylahurine was not an inhibitor. Intestinal alkaline phosphatase was the dominant component in rat serum. When the L-phenylalanine inhibition was applied to human serum, there was good news and bad news. The good news was that the L-phenylalanine was a much better inhibitor of human as compared to rat intestinal alkaline phosphatase. The bad news was that the intestinal enzyme usually represented a relatively small portion of the total serum alkaline phosphatase. L-Phenylalanine di~shes isozymes Ill.
in~tinal and placental isozymes from liver and bonfz
Human placental alkahne phosphatase undergoes the same inhibition by L-phenylalanine as human intestinal isozyme while the liver and bone enzymes are unaffected. L-Homoarginine has just the opposite organ specificity. Both L-phenylalanine and L-homoarginine exhibit the rare uncompetitive type of inhibition, D-phenylalanine does not. Siiicance
of D-m*
in the control [l]
D-Phenylidanine is employed in controls in equimolar amounts to L-phenylalanine in a variety of contexts; in enzyme assays; in electrophoretic studies; in hi&chemical studies and in investigations of the physiology of fat absorption. The only difference between the test and control solutions then is the difference in the arrangement of the a-carbon amino and hydrogen groups. It is this configuration in the L-enantiomorph which accounts for the inhibition, which has been classified as uncompetitive. L-Tryptophan exhibits the same stereospecific, organ specific inhibition.
131
Discovery of the Regan Isozyme, ‘PLAP’ [1,2]
It was the r_-phenylalanine inhibition, coupled with heat inactivation and starch gel electrophoresis, which led us to identify the Regan Isozyme in the serum and tumor tissue of a patient with terminal lung cancer. The Regan isozymes’ properties were indistinguishable from those of human placental alkaline phosphatase. The apparent reexpression of a placental gene in a cancer resident in a male has been an observation which has challenged us to find the explanation. In truth, from this has grown my conviction that the understanding of cancer lies at the interface of oncology and developmental biology. This led, in 1976, to the creation of the La Jolla Cancer Research Foundation and to its present direction. Origin of my regard for the importance of stereospecificity
Where did the idea come to me to evaluate the D- and L-forms of amino acids? In 1941 as an instructor in biochemistry at the then newly opened Bowman-Gray School of Medicine in Winston-Salem, I was working with Dr. Camillo Artom on the changes in liver phospholipids induced by changes in diets fed to rats. Groups of rats received either choline, ethanolamine or DL-serine by stomach tube. All the animals receiving DL-serine died [3]. Dr. Artom exclaimed, ‘these rats did not die because they were tired of living’. We were able to prove that it was the D-serine enantiomorph which was toxic. It produced necrosis of the proximal tubules of the kidney. Here was my first experience where the configuration on the a-carbon of an amino acid could have such profound biological effects [4]. Looking back to this experience, I can now identify fairly accurately why I was motivated to investigate the behavior of enantiomorphs of amino acids separately rather than to assume that the D- and L-forms were equal in their biological and biochemical significance. The L-phenylalanine inhibition has been a key signpost in my career and I am honored to have an opportunity to share my thoughts on the importance of being stereo specific. References 1 Fishman WH. ‘Oncodevelopmental enzymes’, The clinical biochemistry of cancer. Proceedings of the Second Arnold 0. Beckman Conference in Clinical Chemistry. Fleisher M, ed. AACC publication, 1978;132-154. 2 Fisbman WH. Oncotrophoblast gene expression-placental alkaline phosphatase. Adv Cancer Res 1986;46:1-36. 3 Fishman WH, Artom C. Serine injury. J Biol Chem 1942;45:345-346. 4 Artom C, Morehead RP, Fishman WH. The relative toxicity of L- and DL-serinein rats. Proc Sot Exp Med Biol 1945;60:284.