- Email: [email protected]
Protein kinase C subspecies
1594
SELECTED SUMMARIES
evening meal. After correction for the endogenous bile acid isotopes, bile acid kinetics were determined from the decay curves, which were found to be first order and monoexponential. Individual bile acid pools as well as synthesis rates were calculated. By determining the ratio of [13C]DCA, which was derived from administered [13C]CA, to [‘H]DCA, it was possible to calculate the percentage of conversion from the primary to the secondary bile acid. Gallbladder motor function was also studied preoperatively, using ultrasound to assess contraction during cholecystokinin infusion. Duodenal bile was sampled and assayed for bilirubin, cholesterol, and lipids. At surgery, gallbladder bile was collected by direct aspiration. All bile samples were analyzed for individual bile acids, using gas-liquid chromatography. Six of the 9 subjects had cholesterol stones [group A) and the other 3 had mixed stones or no stones (group B). Two of the group A patients had impaired gallbladder emptying, whereas the fraction of the bile acid pool stored in the gallbladder during fasting was normal in all the patients. The ratio of bile acids to bilirubin (which was considered a constant indicator of biliary excretion] increased threefold after cholecystectomy. This significantly increased cholesterol solubility. The major (statistically significant) changes in bile acid kinetics at 3 mo were a 37% decrease in the synthesis rate of CA, a 16% decrease in total bile acid pool, and a 30% increase in the fraction of CA that was changed to DCA. This shift of CA to DCA could be readily demonstrated by noting a more rapid appearance of a 13C peak in the DCA pool after surgery. Chenodeoxycholic acid kinetics were not altered by surgery. The DCA pool, which represents most of the secondary bile acids, did not increase significantly in spite of the increased shift of CA to DCA. This was presumably due to the associated decrease in the CA pool. Comment. This study, on a narrowly restricted group of patients, confirmed the previous findings of an increased total bile acid concentration in duodenal bile after cholecystectomy [Dig Dis Sci 1973;18:445-55). The overall bile acid pool reduction of 16% (p > 0.05) was due almost exclusively to a decrease in CA. Shaffer and Small [J Clin Invest 1977;59:828-l0) had found a similar reduction in the total bile acid pool but had attributed it to an enhanced loss from the enterohepatic circulation. The present study indicates there is an actual decrease in CA synthesis. In a similar study by Almond et al. (N Engl J Med 1973;289:1213-6), 5 of 10 subjects had low preoperative CA synthesis rates, and this group actually increased the CA synthesis postoperatively. The remaining subjects, with normal preoperative CA synthesis rates, decreased this rate after cholecystectomy, as did the patients in the present study. This discrepancy raises the serious question of whether such small studies can yield valid conclusions, particularly when subgroups that are identified on the basis of the results are then compared. The exclusion of males, therefore, seems unfortunate as there is some evidence that bile acid kinetics may differ between males and females. Singletary (Hepatology 1986; 6:574-98) demonstrated receptor sites for progesterone on the gallbladder in humans, both males and females, and animal studies by Ryan and Pellechia (Gastroenterology 1982;87:674-8) showed that progesterone decreased the responsiveness of gallbladder muscle to acetylcholine and cholecystokinin. As all of the women in this study were under 50 yr old, and presumably premenopausal, a sluggish response of the gallbladder (or possi-
GASTROENTEROLOGY Vol. 97, No. 6
bly of the biliary tree) to cholecystokinin infusion during the luteal phase of the menstrual cycle might alter bile acid concentration in the duodenum. Almost nothing is known concerning possible effects of estrogen and progesterone on postcholecystectomy bile acid output. The authors think that the most likely explanation for the enhanced conversion of CA to DCA is an increase in the 7dehydroxylation of CA in the ileum. The increased DCA would then be reabsorbed by the efficient absorptive sites in the ileum, leading to an enhanced transhepatic flux of bile acids with a consequent decrease in CA synthesis. This is a reasonable hypothesis but it depends in part on the assumption that there is a comparable colonization of the distal ileum with the appropriate deconjugating bacteria in all patients. The fact that some postcholecystectomy patients develop diarrhea that is helped by bile chelating agents suggests that this increased deconjugation and enhanced absorption is not universal. Presumably the type, location, and concentration of the gut flora are additional variables to be considered in studying bile acid kinetics. The data from this study are important and not easy to acquire. Because of the numerous variables that can affect bile acid kinetics, however, both before and after cholecystectomy, many additional studies will be required to provide a truly representative picture. J. SWEETING,M.D.
PROTEIN KINASE C SUBSPECIES Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y [Biotechnology Laboratories, Central Research Division, Takeda Chemical Industries, Osaka, Japan; and Department of Biochemistry, Kobe University School of Medicine, Kobe, Japan) Protein kinase C &’ subspecies from rat brain: its structure, expression, and properties. Proc Nat1 Acad Sci USA 1989;86:3099-103. This paper, by the original discoverer of protein kinase C and his coworkers, describes a new protein kinase C subspecies, designated 5, that is present in rat brain. Utilizing a molecular biology approach, the authors isolated two complementary deoxyribonucleic acid (cDNA) clones that encode the 5 subspecies of protein kinase C from a rat brain cDNA library. The nucleotide sequence of the cDNA inserts were determined and amino acid sequence of the 5 subspecies deduced. It was found that the open reading frame encodes 592 amino acids giving a calculated molecular mass of 67,740 daltons. Of note is that (a) the 5 subspecies of protein kinase C lacks the C, region, which in other protein kinase C species binds Ca’+; and (b) the 5 subspecies contains only one set of the characteristic cysteine-rich zinc fingerlike sequence in the region C,, whereas all other subspecies identified to date contain a tandem repeat of the sequence. Northern blot hybridization analysis indicated that RNA transcripts for the protein kinase C subspecies are present in rat brain, kidney, and lung. The cDNA for this 6 subspecies was expressed in mammalian COS-7 cells, using a plasmid expression vector. Three days after transfection, proteins from cell homogenates were separated using high-performance liquid chromatography. Protein kinase from the transfected cells eluted in two major peaks, whereas that from nontransfected, control cells showed only one major
December 1989
peak (previously identified as the LYsubspecies of protein kinase C). The additional protein kinase C obtained with the transfected cells contained two proteins that reacted with an antibody raised against the 5 subspecies. These proteins had estimated molecular masses of 64 and 30 kilodaltons, with the latter possibly being a proteolytic fragment of the larger molecule. The authors then studied the catalytic properties of the C protein kinase C subspecies. Key findings, in marked contrast to the (Y, PI, @I, or y subspecies, include the following. (a) Using calf thymus H, histone as substrate, kinase activity of the 5 subspecies was independent of the presence of Ca’+, phospholipid, or diacylglycerol (although the activity was enhanced by phospholipid). (b) The phorbol ester, phorbol dibutyrate, did not bind to the 5 subspecies. In keeping with this observation, COS-7 cells expressing this protein kinase C species displayed levels of [3H]phorbol dibutyrate binding similar to those of nontransfected control cells. Comment. It has recently become apparent that many protein kinase enzymes exist as families of related subspecies. The members of these families were most often identified as putative protein kinases based on their amino acid sequences, deduced from the nucleotide sequences of cloned genes or cDNAs. Protein kinase C, originally discovered by the authors of the current article in 1977, is now known to be ubiquitous in tissues and organs. The physiologic importance of this enzyme is well documented. It was first discovered by the traditional biochemical approaches, such as protein purification and enzymatic analysis. More recently, molecular biological approaches have been linked to biochemical analyses and have identified multiple, distinct forms of protein kinase C with closely related structures. Initially, four cDNA clones, a, PI, PII, and y, were isolated. These four protein kinase C species all consist of a single poly-
SELECTED SUMMARIES 1595
peptide chain with four conserved (C,-C,) and five variable [V,-V,) regions. The amino terminal portion, containing the C, and C, regions, appears to be the regulatory domain responsible for binding Ca*+, phospholipid, and diacylglycerol [or phorbol ester). The carboxy-terminal portion, containing the C, and C, regions, appears to be the protein kinase domain; it contains large clusters of amino acid sequences found in other protein kinases. Recently, additional cDNA clones were isolated from a rat brain library using a mixture of a, PII, and y cDNAs as probes under low-stringency conditions. At least three additional clones designated 6, E, and 5 were isolated, with the { subspecies being the subject of this report. The (I, PI, @I, and y subspecies of protein kinase C are activated by diacylglycerol (or phorbol ester] in the presence of phospholipid and Ca’+. Only subtle differences in their kinetic properties exist and they have closely related structures. In contrast, the structure of the &’subspecies reported in this paper is closely related to the S and E subspecies, but distinct from those of a, PI, PII, and y subspecies. Structurally, the 6, E, and i subspecies lack the Ca’+ binding region C,. Functionally, the enzymatic activity of these subspecies is independent of the presence of Ca2+, and also does not show an absolute requirement for diacylglycerol. The tools of molecular biology will doubtless enable the identification of other protein kinase species in the near future. However, our acquisition of knowledge regarding the physiologic significance of these enzymes is unlikely to keep pace with the structural and biochemical discoveries. One major hurdle will be the known intracellular compartmentalization of protein kinase C subspecies. Although these enzymes may phosphorylate multiple substrates in broken cell preparations, their access to target proteins in vivo may be highly restricted. Only when we are able to identify potential target substrates will we be able to begin to form a clearer understanding of the significance of multiple protein kinase subspecies. K. DHARMSATHAPHORN,M.D. K. E. BARRETT,PH.D.
SELECTED SUMMARIES
evening meal. After correction for the endogenous bile acid isotopes, bile acid kinetics were determined from the decay curves, which were found to be first order and monoexponential. Individual bile acid pools as well as synthesis rates were calculated. By determining the ratio of [13C]DCA, which was derived from administered [13C]CA, to [‘H]DCA, it was possible to calculate the percentage of conversion from the primary to the secondary bile acid. Gallbladder motor function was also studied preoperatively, using ultrasound to assess contraction during cholecystokinin infusion. Duodenal bile was sampled and assayed for bilirubin, cholesterol, and lipids. At surgery, gallbladder bile was collected by direct aspiration. All bile samples were analyzed for individual bile acids, using gas-liquid chromatography. Six of the 9 subjects had cholesterol stones [group A) and the other 3 had mixed stones or no stones (group B). Two of the group A patients had impaired gallbladder emptying, whereas the fraction of the bile acid pool stored in the gallbladder during fasting was normal in all the patients. The ratio of bile acids to bilirubin (which was considered a constant indicator of biliary excretion] increased threefold after cholecystectomy. This significantly increased cholesterol solubility. The major (statistically significant) changes in bile acid kinetics at 3 mo were a 37% decrease in the synthesis rate of CA, a 16% decrease in total bile acid pool, and a 30% increase in the fraction of CA that was changed to DCA. This shift of CA to DCA could be readily demonstrated by noting a more rapid appearance of a 13C peak in the DCA pool after surgery. Chenodeoxycholic acid kinetics were not altered by surgery. The DCA pool, which represents most of the secondary bile acids, did not increase significantly in spite of the increased shift of CA to DCA. This was presumably due to the associated decrease in the CA pool. Comment. This study, on a narrowly restricted group of patients, confirmed the previous findings of an increased total bile acid concentration in duodenal bile after cholecystectomy [Dig Dis Sci 1973;18:445-55). The overall bile acid pool reduction of 16% (p > 0.05) was due almost exclusively to a decrease in CA. Shaffer and Small [J Clin Invest 1977;59:828-l0) had found a similar reduction in the total bile acid pool but had attributed it to an enhanced loss from the enterohepatic circulation. The present study indicates there is an actual decrease in CA synthesis. In a similar study by Almond et al. (N Engl J Med 1973;289:1213-6), 5 of 10 subjects had low preoperative CA synthesis rates, and this group actually increased the CA synthesis postoperatively. The remaining subjects, with normal preoperative CA synthesis rates, decreased this rate after cholecystectomy, as did the patients in the present study. This discrepancy raises the serious question of whether such small studies can yield valid conclusions, particularly when subgroups that are identified on the basis of the results are then compared. The exclusion of males, therefore, seems unfortunate as there is some evidence that bile acid kinetics may differ between males and females. Singletary (Hepatology 1986; 6:574-98) demonstrated receptor sites for progesterone on the gallbladder in humans, both males and females, and animal studies by Ryan and Pellechia (Gastroenterology 1982;87:674-8) showed that progesterone decreased the responsiveness of gallbladder muscle to acetylcholine and cholecystokinin. As all of the women in this study were under 50 yr old, and presumably premenopausal, a sluggish response of the gallbladder (or possi-
GASTROENTEROLOGY Vol. 97, No. 6
bly of the biliary tree) to cholecystokinin infusion during the luteal phase of the menstrual cycle might alter bile acid concentration in the duodenum. Almost nothing is known concerning possible effects of estrogen and progesterone on postcholecystectomy bile acid output. The authors think that the most likely explanation for the enhanced conversion of CA to DCA is an increase in the 7dehydroxylation of CA in the ileum. The increased DCA would then be reabsorbed by the efficient absorptive sites in the ileum, leading to an enhanced transhepatic flux of bile acids with a consequent decrease in CA synthesis. This is a reasonable hypothesis but it depends in part on the assumption that there is a comparable colonization of the distal ileum with the appropriate deconjugating bacteria in all patients. The fact that some postcholecystectomy patients develop diarrhea that is helped by bile chelating agents suggests that this increased deconjugation and enhanced absorption is not universal. Presumably the type, location, and concentration of the gut flora are additional variables to be considered in studying bile acid kinetics. The data from this study are important and not easy to acquire. Because of the numerous variables that can affect bile acid kinetics, however, both before and after cholecystectomy, many additional studies will be required to provide a truly representative picture. J. SWEETING,M.D.
PROTEIN KINASE C SUBSPECIES Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y [Biotechnology Laboratories, Central Research Division, Takeda Chemical Industries, Osaka, Japan; and Department of Biochemistry, Kobe University School of Medicine, Kobe, Japan) Protein kinase C &’ subspecies from rat brain: its structure, expression, and properties. Proc Nat1 Acad Sci USA 1989;86:3099-103. This paper, by the original discoverer of protein kinase C and his coworkers, describes a new protein kinase C subspecies, designated 5, that is present in rat brain. Utilizing a molecular biology approach, the authors isolated two complementary deoxyribonucleic acid (cDNA) clones that encode the 5 subspecies of protein kinase C from a rat brain cDNA library. The nucleotide sequence of the cDNA inserts were determined and amino acid sequence of the 5 subspecies deduced. It was found that the open reading frame encodes 592 amino acids giving a calculated molecular mass of 67,740 daltons. Of note is that (a) the 5 subspecies of protein kinase C lacks the C, region, which in other protein kinase C species binds Ca’+; and (b) the 5 subspecies contains only one set of the characteristic cysteine-rich zinc fingerlike sequence in the region C,, whereas all other subspecies identified to date contain a tandem repeat of the sequence. Northern blot hybridization analysis indicated that RNA transcripts for the protein kinase C subspecies are present in rat brain, kidney, and lung. The cDNA for this 6 subspecies was expressed in mammalian COS-7 cells, using a plasmid expression vector. Three days after transfection, proteins from cell homogenates were separated using high-performance liquid chromatography. Protein kinase from the transfected cells eluted in two major peaks, whereas that from nontransfected, control cells showed only one major
December 1989
peak (previously identified as the LYsubspecies of protein kinase C). The additional protein kinase C obtained with the transfected cells contained two proteins that reacted with an antibody raised against the 5 subspecies. These proteins had estimated molecular masses of 64 and 30 kilodaltons, with the latter possibly being a proteolytic fragment of the larger molecule. The authors then studied the catalytic properties of the C protein kinase C subspecies. Key findings, in marked contrast to the (Y, PI, @I, or y subspecies, include the following. (a) Using calf thymus H, histone as substrate, kinase activity of the 5 subspecies was independent of the presence of Ca’+, phospholipid, or diacylglycerol (although the activity was enhanced by phospholipid). (b) The phorbol ester, phorbol dibutyrate, did not bind to the 5 subspecies. In keeping with this observation, COS-7 cells expressing this protein kinase C species displayed levels of [3H]phorbol dibutyrate binding similar to those of nontransfected control cells. Comment. It has recently become apparent that many protein kinase enzymes exist as families of related subspecies. The members of these families were most often identified as putative protein kinases based on their amino acid sequences, deduced from the nucleotide sequences of cloned genes or cDNAs. Protein kinase C, originally discovered by the authors of the current article in 1977, is now known to be ubiquitous in tissues and organs. The physiologic importance of this enzyme is well documented. It was first discovered by the traditional biochemical approaches, such as protein purification and enzymatic analysis. More recently, molecular biological approaches have been linked to biochemical analyses and have identified multiple, distinct forms of protein kinase C with closely related structures. Initially, four cDNA clones, a, PI, PII, and y, were isolated. These four protein kinase C species all consist of a single poly-
SELECTED SUMMARIES 1595
peptide chain with four conserved (C,-C,) and five variable [V,-V,) regions. The amino terminal portion, containing the C, and C, regions, appears to be the regulatory domain responsible for binding Ca*+, phospholipid, and diacylglycerol [or phorbol ester). The carboxy-terminal portion, containing the C, and C, regions, appears to be the protein kinase domain; it contains large clusters of amino acid sequences found in other protein kinases. Recently, additional cDNA clones were isolated from a rat brain library using a mixture of a, PII, and y cDNAs as probes under low-stringency conditions. At least three additional clones designated 6, E, and 5 were isolated, with the { subspecies being the subject of this report. The (I, PI, @I, and y subspecies of protein kinase C are activated by diacylglycerol (or phorbol ester] in the presence of phospholipid and Ca’+. Only subtle differences in their kinetic properties exist and they have closely related structures. In contrast, the structure of the &’subspecies reported in this paper is closely related to the S and E subspecies, but distinct from those of a, PI, PII, and y subspecies. Structurally, the 6, E, and i subspecies lack the Ca’+ binding region C,. Functionally, the enzymatic activity of these subspecies is independent of the presence of Ca2+, and also does not show an absolute requirement for diacylglycerol. The tools of molecular biology will doubtless enable the identification of other protein kinase species in the near future. However, our acquisition of knowledge regarding the physiologic significance of these enzymes is unlikely to keep pace with the structural and biochemical discoveries. One major hurdle will be the known intracellular compartmentalization of protein kinase C subspecies. Although these enzymes may phosphorylate multiple substrates in broken cell preparations, their access to target proteins in vivo may be highly restricted. Only when we are able to identify potential target substrates will we be able to begin to form a clearer understanding of the significance of multiple protein kinase subspecies. K. DHARMSATHAPHORN,M.D. K. E. BARRETT,PH.D.