Function of surfactants in hair dyeing by oxidation dyes 2. Effect on formation of oxidation dyes by p-aminophenol and 5-amino-o-cresol in dye bath(1).

The effect of surfactants on an oxidation-hair-dye-formation reaction in a dye bath was studied in order to learn the mechanism of the effect of surfactants on the dyeability of hair by the oxidation dye. The dye-formation behaviours for the p-aminophenol and 5-amino-o-cresol system with the surfactants, of which the hydrophilic parts have different charges, were compared changing the concentration of surfactants. It was found that the same dyes are produced, regardless of the charge of surfactants added, and the rate of dye produced in the dyebath is increased in the presence of surfactants. The order of the production rate is, with an anionic surfactant > with non-ionic surfactant > with cationic surfactant > without surfactant. The relation between the dyeability of hair and the rate of dye produced in the dyebath in the presence of surfactants is not found. The major factor governing the dyeability of hair is different from the mechanism of the increased dye in the solution. It was also found that the dye-formation rate is increased by immersing hair into the reaction solution, and hair works as an accelerator for the dye-formation reaction.

The function of carbohydrate moiety and alteration of carbohydrate composition in human alkaline phosphatase isoenzymes.

The relationship between the structure and function of alkaline phosphatase (orthoposphoric monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) isoenzymes is under investigation in a number of laboratories. The present study deals with the effects of glycosidase digestion on the alkaline phosphatase isoenzymes. Changes in physicochemcial properties, activity, affinity for various lectins and blood group antisera, carbohydrate composition and biological half-life were investigated. The desialylated hepatic enzyme was shown to be more heat labile and more sensitive to protease digestion in the presence of 0.5% sodium dodecyl sulfate than native hepatic enzyme. Helix contents of the native and desialated hepatic enzyme were calculated to be 39.0 and 30.8%, respectively, and apparent molecular weights 175,000 and 167,000, respectively. Intestinal enzyme preparations treated with alpha-mannosidase, exo-N-acetyl-Dglucosaminidase and endo-N-acetyl-D-glucosaminidase-D displayed a decrease in enzyme activity. Among these, the alpha-mannosidase-treated enzyme activity was the most clearly reduction. The maximum activity of the alpha-mannosidase-treated intestinal enzyme was observed to change from 40 mM Mg2+ to 5--10 mM Mg2+.

Partial purification of human intestinal alkaline phosphatase with affinity chromotography. Some properties and interaction of concanavalin A with alkaline phosphatase.

1. Alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from human intestine was purified with concanavalin A-Sepharose and tyraminyl derivative-Sepharose affinity chromatography. The enzyme obtained with these techniques had a specific activity of approx. 513.2 mumol p-nitrophenylphosphate hydrolyzed per min per mg of protein at pH 10.0. 2. The highly purified enzyme showed one major enzymatically active band and a possible minor enzymatically active band on acrylamide gel and cellogel electrophoresis, and the two fraction types showed identical antigenicity. 3. The highly purified intestinal enzyme was compared with the purified hepatic enzyme: the saccharide content of each showed a marked difference. 4. The interaction of alkaline phosphatase with concanavalin A, a carbohydrate-binding protein, was studied. Concanavalin A showed an organ-specific behavior to alkaline phosphatase isoenzyme, i.e., the effect on the enzyme activity, and the optimum pH of the activity. 5. The concanavalin A and alkaline phosphatase complex showed a protective effect against heat denaturation and inactivation of proteinase digestion. There was no difference in stability between the intestinal enzyme and the hepatic enzyme. 6. Alkaline phosphatase preparations from human intestine and human liver can bind with concanavalin A; these interactions of concanavalin A; these interactions of concanavalin A with the enzyme occurred reversibly when alpha-methyl-D-mannoside was added. 7. The double reciprocal plots of 1/v vs. 1/s at higher concentrations of concanavalin A showed that the mechanism of inhibition was "mixed type". From the results of Dixon plots, the inhibition constant (Ki) was calculated to the 0.025 muM for human intestinal enzyme. 8. The effect of concanavalin A on L-phenylalanine inhibition of the intestinal alkaline phosphatase indicates that concanavalin A does not interfere with L-phenylalanine binding, but its effect on L-homoarginine inhibition of the hepatic enzyme seems to show that concanavalin A interfered with L-homoarginine binding.

Partial purification and some properties of human liver alkaline phosphatase.

1. Alkaline phosphatase (EC 3.1.3.1) from human liver was solubilized from the homogenate using 0.2% Triton X-100 containing 0.2 M lithium 3,5-diiodosalicylate, and the pellet obtained was resolubilized with 20% n-butanol. The procedure resulting in 3842-fold purification included acetone fractionation, ammonium sulfate precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration, hydroxyapatite gel chromatography and further concanavalin A/Sepharose 4B affinity chromatography. 2. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 8.6, and exhibited one-seventh of the alkaline p-nitrophenylphosphatase activity in the hepatic enzyme preparation contains of the alkaline pyrophosphatase activity. 3. The highly purified enzyme was a sialic-acid containing glycoprotein. 4. Sialidase-treated hepatic enzyme clearly presented the phenomenon of delayed mobility, and the delayed enzyme fraction stained more strongly than that of non-treated hepatic alkaline phosphatase. 5. In order to investigate the role of the carbohydrate region(s) of the hepatic alkaline phosphatase molecule on substrate binding, the effect of sialidase treatment on the rate of substrate inhibition of alkaline phosphatase was studied. In the case of hepatic enzyme without sialidase, substrate inhibition of alkaline phosphatase activity was clearly shown, while in the case of the hepatic enzyme with sialidase, there was hardly any substrate inhibition in the range of 1-8 mM p-nitrophenylphosphate.

Inhibition of alkaline phosphatase by sialic acid.

The interaction of human organ alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) with sugars was studied. Hexosamines, N-acetylneuraminic acid (NANA or sialic acid), N-acetylmuramic acid and N-acetylglycolylneuraminic acid inhibited human organ alkaline phosphatase activities. Of these, sialic acid was the most effective inhibitor. The pH profiles for the enzymes in the absence and presence of sialic acid were similar. The sialic acid - enzyme complex was more heat stable than the free enzyme between 20 and 45 degrees C. Lineweaver-Burk plots of 1/v versus 1/S at various concentrations of sialic acid showed intersecting straight lines indicating that the mechanism of inhibition was a mixed type. The Ki value obtained from the plots of 1/v versus the square of sialic acid concentration was 0.07 mM for the hepatic, sialidase-treated hepatic, and intestinal alkaline phosphatases. The respective Hill coefficients varied somewhat with the alkaline phosphatase isoenzyme. Hyperbolic curves were obtained when the percentage of remaining activity was plotted against the substrate concentration at different concentrations of sialic acid. The Hill coefficient was lowered in the presence of sialic acid. The sialidase-treated hepatic enzymes used gave the most effective conversion. Partial denaturation of the enzyme with urea, or pronase digestion had a little if any effect on the sialic acid inhibition with constant time.

A possible mechanism for the changes in hepatic and intestinal alkaline phosphatase activities in bile-duct-ligated rats or guinea pigs.

The effects of bile-duct ligation on hepatic and intestinal (jejunum) alkaline phosphatase activities were studied using rats and guinea pigs. In ligated rats, the enzyme activity was increased 4.1-fold in the liver after 24 h and 2.8-fold in the intestine after 12 h. In guinea pigs, the hepatic and intestinal enzyme activities were increased 2.3-fold and 1.5-fold after 100 and 24 h, respectively. The intestinal activity was induced sooner after ligation than hepatic activity. The induction of alkaline phosphatase was inhibited by prior treatment of animals with amanitin, an inhibitor of RNA polymerase activity. This result indicates that the induction is associated with de novo enzyme synthesis. The content of cyclic AMP in liver and intestine increased immediately after ligation. The increase in alkaline phosphatase activities was also inhibited by pretreatment with chlorpromazine, an inhibitor of adenylate cyclase activity. Hence, cellular cyclic AMP may be implicated in playing a role in the induction of alkaline phosphatase by bile-duct ligation.

A precursor form of human kidney gamma-glutamyl transferase in normal and cancerous tissues, and its possible post-translational modification.

We have found three molecular forms of human gamma-glutamyl transferase (GGT) in normal and renal cell carcinomatous tissues, and also have reported the marked differences in the sugar chains of GGTs between normal and cancerous tissues by serial lectin affinity chromatographies. In this study, the peptide maps of three purified GGTs (79 kDa, 50 kDa and 25 kDa) obtained by lysylendopeptidase digestion, the subcellular localization of GGTs, and the sugar chains of GGTs were compared between normal and cancerous tissues. According to the results, the total peptide bands of the digested 79 kDa component represented the sum of those of the digested 50 and 25 kDa components on 12.5% SDS-PAGE. In addition, C-terminal and N-terminal amino-acid sequences of the 79 kDa protein were the same as the sequences of light and heavy subunits, respectively, suggesting that the 79 kDa component is of the precursor form of the 50 kDa mature heavy and 25 kDa light subunits, respectively. On the other hand, the GGT activity in renal cell carcinomatous tissues was significantly increased in the microsomal fraction and decreased in the soluble fraction compared with that of normal tissues. Meanwhile, the sugar moiety of GGTs in the respective subcellular fractions was obviously different between normal and cancerous tissues. In particular, a reduced multiantennary complex type sugar chain and an elevated high-mannose or hybrid-type sugar chain in the microsomal fraction were observed in the GGT in cancerous tissues.

Rat ileal alkaline phosphatase activity and secretion is stimulated by alterations in calcium metabolism.

The mechanism of calmodulin-stimulated alkaline phosphatase activity was studied in the rat. In calmodulin-treated rats (2.5 micrograms/animal, intraperitoneally) alkaline phosphatase (ALP) activity was elevated 11-fold in the ileum, 1.5-fold in the duodenum and calvarium, 3-fold in serum, and not at all in liver. The elevated ALP activity was prevented by prior treatment with flunarizine, a calcium channel blocker, and by W-7, a calmodulin antagonist. cAMP content in ileum paralleled the timing and changes in ALP activity, but was not elevated in the duodenum or calvarium. Calcium ionophore A23187 and calcitonin treatment also increased ileal, duodenal, and calvarial ALP activity, but by less than the response to calmodulin. All of these treatments caused a 2-fold elevation in serum 1,25-dihydroxyvitamin D-3 (1,25(OH)2D3) levels. Pretreatment of the animals with parathyroid hormone prevented the rise of both ALP activity and of 1,25(OH)2D3. Administration of 1,25(OH)2D3 alone stimulated a different pattern of increased ALP activity, greater in duodenum than ileum. The uptake of 45Ca by calmodulin was also elevated in ileum and calvarium. These data suggest that shifts in calcium movement, perhaps mediated by vitamin D, can alter ALP activity, and may provide a mechanism for rapid control of the secretion of this enzyme.

Liver-like alkaline phosphatase in the tissue-unspecific type enzyme found in rabbit organs.

Rabbit liver and kidney tissues are known to produce an intestinal-like alkaline phosphatase (IAP-like enzyme) as a dominant isozyme, with a minor isozyme of tissue-unspecific type (UAP), unlike humans and other mammalians. We investigated immunohistochemically and biochemically these unique isozymes in the rabbit liver and bone, and compared them with the human isozyme. In rabbit liver, UAP was found to be localized only in the apical part of the membrane of cells lining the bile duct, whereas IAP-like enzyme was found in the sinusoidal membrane of hepatocytes. Rabbit liver UAP was separated from IAP-like enzyme by DEAE-cellulose column chromatography. Rabbit bone tissue contained only one UAP isozyme. The two UAPs were biochemically and physicochemically compared with human liver AP. Both UAPs reacted with an anti-human liver AP monoclonal antibody, not with an anti-human bone AP monoclonal antibody, indicating that both enzymes have the same antigenicity as human liver AP. Rabbit liver and bone UAPs had similar N-linked sugar-chain heterogeneities to the respective human enzymes. In addition, rabbit bone AP also had an O-linked sugar chain, as did human bone AP, unlike rabbit and human liver APs.

Inhibitory effect of phenothiazine derivatives on bone in vivo and osteoblastic cells in vitro.

We tested the inhibitory effects of phenothiazine derivatives on bone in vivo and osteoblastic cells in vitro. Chlorpromazine (CPZ) and trifluoperazine (TFPZ) dose-dependently decreased alkaline phosphatase activity in calvariae of rats: half-maximal inhibitory effects of CPZ and TFPZ were at 2.0 and 4.0 mg/kg, respectively. These effects were more specific for calvaria and ileum than for liver and duodenum. CPZ inhibited the proliferation of osteoblastic clone MC3T3-E1 cells to a greater extent than that of liver epithelial clone RLC-18(4) cells in vitro. CPZ, TFPZ and perphenazine (PNZ) also affected rather specifically alkaline phosphatase activity and collagen synthesis and were not cytotoxic. These in vivo and in vitro findings suggest inhibitory effects on osteoblastic cell function(s). However, promethazine (PMZ) had little effect in vivo and in vitro. In addition, increases in serum calcium and phosphate induced by CPZ indicate its possible involvement in bone resorption.

Modulation of reactive oxygen species in endothelial cells by peroxynitrite-treated lipoproteins.

Peroxynitrite has been implicated in the oxidative modification of low-density lipoprotein (LDL) particles, and nitrotyrosine residues in the LDL have been detected in atherosclerotic plaques. Studies have suggested that lipoproteins modified by peroxynitrite lead to the onset of atherosclerotic vascular disease. We therefore prepared in vitro lipoproteins oxidatively modified by peroxynitrite (NO(2)-lipoprotein) and investigated the effect of NO(2)-lipoprotein on the viability of cultured endothelial cells. After exposure of a high-density lipoprotein (HDL) to peroxynitrite, some intermolecular complexes of apolipoproteins in HDL were detected on immunoblotting with monoclonal antibodies against apolipoprotein AI and AII, suggesting that nitration of HDL by peroxynitrite causes intermolecular cross-linking of the apolipoproteins in the particles. Treatment with 1 mM peroxynitrite increased the 3-nitrotyrosine level to 28.5 mmol/mol of tyrosine residues in the prepared NO(2)-HDL, as quantitated by HPLC, and the amount in NO(2)-lipoprotein depended on the peroxynitrite concentration. HDL exhibited a shorter lag phase and the reaction plateaued more rapidly than that with LDL. To clarify whether or not NO(2)-lipoproteins affect the function of endothelial cells, we first examined the viability of cultured human aortic endothelial cells (HAECs) exposed to NO(2)-lipoproteins. Incubation with either NO(2)-HDL or NO(2)-LDL significantly reduced the HAEC viability at 72 h. The results of RT-PCR and Western blotting showed that NO(2)-HDL markedly suppressed at 48 h not only the expressed levels of mRNA and protein but also the activity of catalase in HAECs. In contrast, NO(2)-LDL significantly reduced the expression and activity of Cu(2+),Zn(2+)-superoxide dismutase (CuZn-SOD) in the cells. Neither NO(2)-HDL nor NO(2)-LDL interfered with nitric oxide production or expression of cyclooxygenases and NADPH oxidase in HAECs. Increased radical production in NO(2)-lipoprotein-treated HAECs implied that reactive oxygen species such as superoxide anions and hydroxyl radicals may contribute to the mechanism of the toxic effect induced in endothelial cells by NO(2)-lipoprotein. Overall, NO(2)-lipoprotein may lead to deterioration of the vascular function through these endothelial cell responses.

Abnormal alkaline phosphatase of hepatic type in cerebrospinal fluid of a patient with intracranial metastasis from lung cancer.

High alkaline phosphatase (ALP) activity was found in the cerebrospinal fluid of a patient with intracranial metastases from adenocarcinoma of the lung. On agarose gel electrophoresis of the major ALP isoenzyme found in the cerebrospinal fluid, its mobility was different from those of the usual serum ALP isoenzymes. This abnormal mobility might be due to the linked glycan phosphatidylinositol anchor in the ALP molecule, as the mobility became the same as that of the common liver type ALP after treatment with phosphatidylinositol specific phospholipase. The immunochemical antigenicity of the cerebrospinal fluid ALP was identical with that of the common serum liver type ALP, but its sugar moiety was similar to the membranous liver-type ALP rather than the serum liver type ALP. The molecular size of the cerebrospinal fluid ALP was 140 kilodaltons, 12 less than the common serum liver type ALP, suggesting that the ALP in the patient's cerebrospinal fluid was derived from the intracranial metastatic carcinoma.

The ligands/activators for peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARgamma increase Cu2+,Zn2+-superoxide dismutase and decrease p22phox message expressions in primary endothelial cells.

We examined the effects of a variety of ligands/activators of the peroxisome proliferator-activated receptor (PPAR) on the expression of the superoxide scavenger enzyme, Cu2+,Zn2+-superoxide dismutase (CuZn-SOD), and the superoxide generating enzyme nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase in primary cultures of human umbilical vein endothelial cells (HUVEC) and human aorta endothelial cells (HAEC). Our data show that 3 types of PPARs, PPARalpha, PPARbeta/delta/Nuc1, and PPARgamma are expressed in endothelial cells. Bezafibrate, which is a ligand/activator for PPARalpha, increased the CuZn-SOD gene expression and protein levels in endothelial cells. Troglitazone and pioglitazone, which are ligands/activators for PPARgamma, also induced PPARalpha gene and protein expression and increased CuZn-SOD gene expression and protein levels in addition to increasing PPARgamma gene and protein expression in endothelial cells. Moreover, with treatment of monounsaturated and polyunsaturated fatty acids (PUFA), the CuZn-SOD mRNA levels were positively correlated with PPARalpha mRNA levels (r = .872, P < .0001) in primary endothelial cells. In addition, the phorbol myristate acetate (PMA)-stimulated or PMA-nonstimulated 22-kd a-subunit (p22phox) mRNA levels and 47-kd a-subunit (p47phox) protein levels in NADPH oxidase were decreased by treatment with PPARalpha and PPARgamma ligands/activators. These results suggest that PPARalpha and PPARgamma gene and protein expression in endothelial cells may play a physiologic role as radical scavengers, although the details of these mechanisms remain to be established.

Intestinal alkaline phosphatase isoforms in rabbit tissues differ in glycosylation patterns.

OBJECTIVES: In patients with advanced liver cirrhosis or chronic nephritis, an intestinal alkaline phosphatase (IAP)-like enzyme is ectopically expressed in the liver or kidney. In this study, we used rabbit organs as a human pathological model, because the rabbit liver or kidney expresses an IAP-like enzyme as the predominant isozyme, unlike humans. METHODS: IAP and the IAP-like enzyme were purified from rabbit intestine and kidney, respectively, by immunoaffinity chromatography using a monoclonal antihuman IAP antibody. Some properties of the IAP and IAP-like enzyme expressed in rabbit organs are compared. RESULTS: Some of their catalytic and physicochemical properties differed. In particular, the net charge, molecular mass, and hydrophobicity of IAP from rabbit intestine was slightly different from the IAP-like enzyme from rabbit kidney. There was a difference in the sugar chain structure between the two enzymes according to the results of lectin affinity chromatography, and part of the peptide maps differed slightly. However, there was no difference in the peptide maps after treatment with endo-N-acetylglucosaminidase F. CONCLUSIONS: The primary structures of IAP and the IAP-like enzyme are basically similar, except for the glycosylation process of the respective AP isozymes.

Comparative studies on the properties of purified gamma-glutamyl transferase from human reproductive system and the kidney.

Gamma-glutamyl transferase (GGT) of the human seminal plasma and reproductive tissues was purified and its properties were compared to those of the enzyme from kidney. A single band of GGT was obtained by polyacrylamide gel electrophoresis. Purification was 1080-fold for seminal plasma, 206-fold for prostate, 608-fold for testis and 382-fold for kidney. Similar Km value (0.87-1.06 mM) and optimum pH (8.2-8.5) were obtained for the enzymes of the four different sources. Their thermal stabilities were identical. However, inhibitions by Zn2+ and Cu2+ were different between kidney and reproductive system GGT. Molecular mass of the native enzyme was 78 kDa for seminal plasma, prostate and testis and 79 kDa and 105 kDa for kidney. The subunit molecular masses of the enzymes from seminal plasma, prostate and kidney consisted of three proteins, suggesting the precursor form, and the heavy and light subunits of the mature form.

A case of benign familial hyperphosphatasemia of intestinal origin.

We recently encountered a case of hyperphosphatasemia, in which > 90% of serum alkaline phosphatase (ALP) was of intestinal origin. The patient, a 51-year-old man, was found to have hyperphosphatasemia (2,341 U/L) during a routine medical check-up. All other laboratory tests and physical findings were normal. The agarose gel electrophoresis pattern of the patient's serum ALP was identical to that of common intestinal ALP from healthy adults, and only a single band of intestinal ALP was detected by immunoaffinity electrophoresis. In addition, 89% of total ALP was defined as intestinal ALP by an immunoprecipitation method. The molecular mass of the ALP was 154 kDa, almost identical with that of adult duodenal ALP. Analysis of the sugar chain structure showed an increased la fraction (74%) compared with adult duodenal ALP. Genealogical study revealed that two persons in the 5 members of the proband's family had hyperphosphatasemia of intestinal origin, indicating possible autosomal dominant inheritance.

Multiple forms of human intestinal alkaline phosphatase: chemical and enzymatic properties, and circulating clearances of the fast- and slow-moving enzymes.

Two forms of alkaline phosphatase (orthophosphoric monoester phosphohydrolase (alkaline optimum, EC 3.1.3.1) have been purified from human small intestine by column chromatography on DEAE-cellulose and tyraminyl derivative affinity gel, and by preparative disc gel electrophoresis. Intestinal phosphatases were electrophoretically separated into two components, fast- and slow-moving enzymes, with apparent molecular weights of 140000 and 168000 and with subunit weights of 68000 and 80000, respectively. Analyses of carbohydrate and amino acid revealed marked differences in the two enzymes. Enzymatic properties and affinities for an anti-blood group antibody were also found to differ. Papain digestion released a hydrophobic small peptide from the slow-moving enzyme and its enzymatic properties resembled those of the fast-moving enzyme. Circulating clearance (T1/2) of the slow- and fast-moving enzymes from adult intestine was found to be 7.5 h and 1.3 h, respectively; that of fetal intestinal enzyme was 2.8 h. Sialidase, sialidase/beta-galactosidase, or sialidase/beta-galactosidase/N-acetyl-beta-glucosaminidase treatment of the fetal enzyme reduced the value to about 40 min. Further, digestion with alpha-fucosidase, alpha-mannosidase or both restored it to nearly the original level. Organ distribution of injected 125I-labelled enzymes indicates that the desialylated hepatic enzyme was selectively distributed in liver, while the degalactosylated intestinal enzyme was incorporated into liver lymph fluid, and small intestine. These results suggest that the pathway of circulating clearance of alkaline phosphatase has several routes.

Determination of isoenzyme contents of lactic dehydrogenase activity and 2-hydroxybutyric dehydrogenase activity in lactic dehydrogenase preparations.

In this inestigation, a determination of the isoenzyme contents of LDH and HBD activities in lactate dehydrogenase preparations and the differences in the interaction of these preparations with NAD analogues were examined. The results obtained were as follows. 1. The activity ratio between oxidation and reduction in LDH reaction is shown to be similar in both H4 and M4 preparations, whereas for HBD activity, the ratio seems to be lower in the M4 preparation than in H4(H4/M = 1/2). 2. NAD and its analogues (NXD, TNAD, and TNXD) are shown to be useful coenzymes for the LDH reaction, while 3-acetyl derivatives appear to be unsuitable for this purpose because of their lower activity. HBD activity with 3-acetyl NXD is shown to be higher than with TNAD or TNXD. among these NAD analogues, 3-acetyl NXD gives the highest HBD activity, especially in the M4 preparation. 3. The LDH activity of H4 relative to M4 preparations has been shown to be maximal when 450 mM lactic acid with NAD or 15 mM lactic acid with TNXD are used. Under these conditions, the contents of LDH subunits can be estimated with considerable reliability. As to HBD activity, the content of LDH subunit having HBD activity has been estimated by determing the enzyme activity under conditions in which either 300 mM 2-hydroxybutyrate with 3-acetyl NXD or 15 mM 2-hydroxybutyrate with NAD are employed.

Partial characterization of human ileal alkaline phosphatase: differences between human ileal and duodenal enzymes.

Properties of human ileal and duodenal alkaline phosphatase (ALP) were compared. The pH optimum, Km values, heat stability, inhibition of activity by amino acids, and antigenicity of ileal and duodenal ALPs were similar. Affinity for DEAE and Tyraminyl derivatives/Sepharose chromatographies, substrate specificity, molecular mass, isoelectric point, and sugar chain structure differed, suggesting two forms of intestinal enzyme. The N-terminal amino acid sequence, or peptide mapping or both suggest that the two major intestinal ALPs are identical, but the minor ALP may be differed from the sequence of major one.

Determination of proteins by a reverse biuret method combined with the copper-bathocuproine chelate reaction.

A method of protein determination has been developed which combines the biuret reaction and the copper(I)-bathocuproine chelate reaction. Protein in the specimen forms a Cu(2+)-protein chelate complex (biuret reaction) during the first step. Excess Cu2+ is reduced to Cu+ by ascrobic acid, allowing the Cu+ to form a Cu(+)-bathocuproine chelate complex during the second step. The amount of Cu(+)-bathocuproine chelate complex formed is inversely proportional to the protein concentration. The sensitivity (epsilon = 1.4 x 10(6) 1.mol-1.cm-1 against human albumin) of this method was higher than that of the original Lowry (9.8 x 10(5)), pyrogallol red (1.0 x 10(6)) and commercially available Coomassie Brilliant Blue G.250 methods (6.7 x 10(5)). The color intensities of human gamma-globulin, human globulin (fractions IV-1 and IV-4), bovine albumin, egg albumin and horse gamma-globulin against human albumin (100%) ranged from 92 to 101%. The results obtained with the present method (y) correlated well with those determined by the biuret method (r = 0.998, y = 0.98 chi - 0.002, x = 1.31, y = 1.29 g/l) in 30 diluted sera. These results confirm that this assay is similar in sensitivity to the original Lowry method, is rapid and has similar reactivity to each of the various proteins in biological fluids.