Separation of alkaline phosphatase isoenzymes from human kidney and comparison with alkaline phosphatases from other human tissues, urine, and Escherichia coli.

1983 ◽  
Vol 29 (1) ◽  
pp. 100-106 ◽  
Author(s):  
A W Hodson
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Human Kidney ◽  
Human Tissues ◽  
Ammonium Sulfate Precipitation ◽  
Alkaline Phosphatases ◽  
Alkaline Phosphatase Isoenzymes ◽  
Starch Gel

Abstract Human kidney isoenzymes of alkaline phosphatase (EC 3.1.3.1) after extraction with butan-1-ol were separated by ammonium sulfate precipitation, gel filtration, and chromatofocusing fractionation methods. The separation at each fractionation step was monitored by starch gel and equilibrium-gradient-pore electrophoresis, the latter technique also being used to determine molecular mass. The determined molecular mass (daltons) of alkaline phosphatase from human placenta was 132 000, from urine 95 000, and three isoenzymes from kidney were 195 000, 140 000, and 95 000, respectively. The mass of Escherichia coli alkaline phosphatase was 80 000 daltons, and that of human liver alkaline phosphatase was assumed to be 160 000 daltons. The urinary isoenzyme and the electrophoretically fastest migrating kidney isoenzyme were similar with regard to pH optima, charge, and molecular mass as well as response to L-phenylalanine, L-homoarginine, heat, and urea. Bacterial alkaline phosphatase could be distinguished from the alkaline phosphatases in human tissues and urine by differences in the response to changes in pH and several other physicochemical properties.

Properties of High-Molecular-Mass Placental Alkaline Phosphatases in Normal Pregnancy Sera

1998 ◽  
Vol 35 (4) ◽  
pp. 515-521 ◽  
Author(s):  
Makoto Matsushita ◽  
Tsutomu Irino ◽  
Masakazu Minowa ◽  
Tsugikazu Komoda ◽  
Torgny Stigbrand
Keyword(s):  
Alkaline Phosphatase ◽  
Pregnant Women ◽  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
High Molecular Mass ◽  
Placental Alkaline Phosphatase ◽  
Alkaline Phosphatases ◽  
Serum Samples ◽  
Triton X

We examined the appearance of high-molecular-mass placental alkaline phosphatases (HPLAPs) in the serum of normal pregnant women by means of polyacrylamide gel electrophoresis (PAGE) in the presence of Triton X-100. The HPLAPs were undetectable or only slightly detectable by PAGE in the absence of Triton X-100. The HPLAPs were detected in all sera sampled during the last trimester of pregnancy. The catalytic activities of total placental alkaline phosphatase (TPLAP) and HPLAPs were correlated (r = 0.96) and the ratio of HPLAPs/TPLAP catalytic activity was 0.20 (0.06) (mean and SD) in 40 serum samples from pregnant women. The HPLAPs appear to be formed from a common dimeric placental alkaline phosphatase (PLAP) (common-PLAP), as judged by the fact that they were formed again after removal of HPLAPs from serum by gel filtration. The formation of HPLAPs was more prominently observed with the faster fractions of gel filtration. The apparent molecular mass of the HPLAPs in pregnancy serum was 720 K Da by gel filtration. HPLAPs were not converted to common-PLAP by phosphatidylinositol-specific phospholipase (PIPL) C and PIPL-D treatments. The HPLAPs were selectively incorporated into liposomes consisting of phosphatidylcholine/cholesterol, and most of the PIPL-D-treated PLAP could form HPLAPs, while a small amount of PLAP could not form HPLAPs. On the other hand, HPLAPs in pregnant women's sera and HPLAPs prepared from partially purified PLAP in vitro could be converted to common-PLAP by brief treatment with subtilisin. However, the highly purified PLAP could not form HPLAPs in the presence of Triton X-100. These results suggest that PIPL-D-resistant and PLAP-associated serum protein may regulate the conversion of PLAP to HPLAP in the presence of Triton X-100.

Alkaline phosphatase. I. Kinetics and inhibition by levamisole of purified isoenzymes from humans.

1976 ◽  
Vol 22 (7) ◽  
pp. 972-976 ◽  
Author(s):  
H Van Belle
Keyword(s):  
Alkaline Phosphatase ◽  
Substrate Concentration ◽  
Human Liver ◽  
Alkaline Phosphatases ◽  
Mechanism Of Inhibition ◽  
Optimum Ph ◽  
Alkaline Phosphatase Isoenzymes

Abstract I studied the kinetics and sensitivity toward inhibition by levamisole and R 8231 of the most important human alkaline phosphatase isoenzymes. N-Ethylaminoethanol proved superior to the now widely used diethanolamine buffer, especially for the enzymes from the intestine and placenta, behaving as an uncompetitive activator. The optimum pH largely depends on the substrate concentration. The addition of Mg2+ has no effect on the activities. The meaning of Km-values for alkaline phosphatases is questioned. Isoenzymes from human liver, bone, kidney, and spleen are strongly inhibited by levamisole or R 8231 at concentrations that barely affect the enzymes from intestine or placenta. The inhibition is stereospecific, uncompetitive, and not changed by Mg2+. Inhibition is counteracted by increasing concentrations of N-ethylaminoethanol. The mechanism of inhibition is suggested to be formation of a complex with the phosphoenzyme.

Alkaline phosphatase isoenzymes.

1982 ◽  
Vol 28 (10) ◽  
pp. 2007-2016 ◽  
Author(s):  
D W Moss
Keyword(s):  
Alkaline Phosphatase ◽  
Quantitative Analysis ◽  
Posttranslational Modifications ◽  
Quantitative Methods ◽  
Molecular Forms ◽  
Future Research ◽  
Alkaline Phosphatases ◽  
Isoenzyme Analysis ◽  
Alkaline Phosphatase Isoenzymes ◽  
Alkaline Phosphatase Isoenzyme

Abstract The human alkaline phosphatases constitute a system of multiple molecular forms of enzymes in which heterogeneity is partly due to genetic factors and partly to posttranslational modifications. Recognition of the nature and occurrence of these multiple forms has made a significant contribution both to the understanding of changes in alkaline phosphatase values for serum in disease and to the use of alkaline phosphatase measurements in diagnosis. Many of the diagnostic advantages of alkaline phosphatase isoenzyme analysis can be obtained with the aid of qualitative methods such as zone electrophoresis. However, quantitative methods are needed to take full advantage of the potential benefits of isoenzyme analysis. Selective inactivation methods can be applied successfully to the quantitative analysis of bone and liver alkaline phosphatases in serum. However, the aim of future research should be to remove the limitations at present imposed on quantitative analysis by the close similarities of bone and liver alkaline phosphatases.

PROPERTIES AND CLASSIFICATION OF THE SOLUBLE ESTERASES OF HUMAN BRAIN

1966 ◽  
Vol 44 (2) ◽  
pp. 225-232 ◽  
Author(s):  
D. J. Ecobichon
Keyword(s):  
Alkaline Phosphatase ◽  
Human Brain ◽  
Esterase Activity ◽  
Water Soluble ◽  
Human Tissues ◽  
Esterase Pattern ◽  
Vertical Zone ◽  
Zone Electrophoresis ◽  
Starch Gel

Water-soluble proteins and enzymes of human brain were separated by vertical zone electrophoresis in starch gel. Fifteen bands of esterase activity were detected in brain. Various substrates and inhibitors were used in efforts to identify enzymes in addition to a comparison of the esterase pattern with patterns obtained from other human tissues. One zone, composed of four bands of acetylesterase activity, was found to be common to all the tissues investigated with the exception of serum. Two bands of cholinesterase and two bands of A-esterase activity were identified. The remaining bands, which were aliesterases possessing broad overlapping substrate specificity and inhibitor sensitivity, were electrophoretically different from those of other tissues. Observations on alkaline phosphatase, acid phosphatase, and lactate dehydrogenase were recorded for comparison with the data on esterases.

Purification of an alkaline phosphatase-lipoprotein-X complex.

1980 ◽  
Vol 26 (5) ◽  
pp. 604-608 ◽  
Author(s):  
R N Weijers ◽  
H Kruijswijk ◽  
J M Baak
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Gel Filtration ◽  
Relative Molecular Mass ◽  
Agarose Gel ◽  
Extrahepatic Cholestasis ◽  
Abnormal Position ◽  
Alkaline Phosphatase Isoenzyme ◽  
Isoenzyme Patterns

Abstract An alkaline phosphatase isoenzyme was observed in an abnormal position in the agar-agarose gel pattern of sera from several patients suffering from intrahepatic and extrahepatic cholestasis. We purified this isoenzyme, by gel filtration and affinity chromatography, from the serum of a patient suffering from extrahepatic cholestasis. Analysis demonstrated an alkaline phosphatase-lipoprotein-X complex with a relative molecular mass of at least 669,000. We discuss the interpretation of alkaline phosphatase isoenzyme patterns produced by different techniques.

scholarly journals Synthesis and secretion of alkaline phosphatase in vitro from first-trimester and term human placentas

1981 ◽  
Vol 194 (3) ◽  
pp. 857-866 ◽  
Author(s):  
H Galski ◽  
S E Fridovich ◽  
D Weinstein ◽  
N De Groot ◽  
S Segal ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Time Course ◽  
De Novo ◽  
Placental Tissue ◽  
First Trimester ◽  
Alkaline Phosphatases ◽  
Heat Stable ◽  
Alkaline Phosphatase Isoenzymes ◽  
Specific Activities

The synthesis and secretion of alkaline phosphatases in vitro by human placental tissue incubated in organ culture were studied. First-trimester placenta synthesizes and secretes two different alkaline phosphatase isoenzymes (heat-labile and heat-stable), whereas in term placenta nearly all the alkaline phosphatase synthesized and secreted is heat-stable. The specific activities of alkaline phosphatases in first-trimester and term placental tissue remain constant throughout the time course of incubation. In the media, specific activities increase with time. Hence, alkaline phosphatase synthesis seems to be the driving force for its own secretion. The rates of synthesis de novo and of alkaline phosphatases were measured. The specific radioactivities of the secreted alkaline phosphatases were higher than the corresponding specific radioactivities in the tissue throughout the entire incubation period. The intracellular distribution of the alkaline phosphatase isoenzymes was compared.

scholarly journals Purification and properties of molecular-weight variants of human placental alkaline phosphatase

1968 ◽  
Vol 108 (5) ◽  
pp. 779-792 ◽  
Author(s):  
Nimai K. Ghosh ◽  
William H. Fishman
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Large Scale ◽  
Equilibrium Dialysis ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Exchange Chromatography ◽  
Placental Alkaline Phosphatase ◽  
Ph Optimum ◽  
Starch Gel

1. Alkaline phosphatase of human placenta was purified by a procedure involving homogenization with tris buffer, pH8·6, extraction with butanol, ammonium sulphate fractionation, exposure to heat, ethanol fractionation, gel filtration, triethylaminoethylcellulose anion-exchange chromatography, continuous curtain electrophoresis on paper and equilibrium dialysis. Methods for both laboratory-scale and large-scale preparation were devised. 2. Two major molecular-weight variants designated A and B were separated by molecular sieving with Sephadex G-200 and variant A was purified 4000-fold. 3. Variant B, which comes off the Sephadex G-200 column before variant A, is the electrophoretically slower-moving species on starch gel and is quite heterogeneous. 4. Purified variant A was fairly homogeneous on the basis of electrophoretic studies on starch gel and Sephadex gel, ultracentrifugation and immunodiffusion. 5. The respective molecular weights for variants A and B were 70000 and over 200000 on the basis of sucrose-density-gradient ultracentrifugation. Variant A exhibited a sedimentation coefficient of 4·2s. 6. Crystalline variant B could be converted into fast-moving variant A and vice versa. 7. Kinetic studies indicated no difference between the two variants. These include linear rates of hydrolysis, pH optimum, Michaelis constants and uncompetitive stereospecific l-phenylalanine inhibition. 8. The amino acid compositions of variants A and B and of placental albumin were determined.

Alkaline phosphatase isoenzymes of human kidney and urine

1975 ◽  
Vol 61 (1) ◽  
pp. 53-62 ◽  
Author(s):  
A.W. Hodson ◽  
A.L. Latner
Keyword(s):  
Alkaline Phosphatase ◽  
Human Kidney ◽  
Alkaline Phosphatase Isoenzymes

scholarly journals Production of the HBc Protein from Different HBV Genotypes in E. coli. Use of Reassociated HBc VLPs for Packaging of ss- and dsRNA

2021 ◽  
Vol 9 (2) ◽  
pp. 283
Author(s):  
Ivars Petrovskis ◽  
Ilva Lieknina ◽  
Andris Dislers ◽  
Juris Jansons ◽  
Janis Bogans ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
E Coli ◽  
The Core ◽  
Hbv Genotypes ◽  
Core Proteins ◽  
B Virus

The core proteins (HBc) of the hepatitis B virus (HBV) genotypes A, B, C, D, E, F, and G were cloned and expressed in Escherichia coli (E. coli), and HBc-formed virus-like particles (VLPs) were purified with ammonium sulfate precipitation, gel filtration, and ion exchange chromatography (IEX). The best VLP yield was found for the HBc of the HBV genotypes D and G. For the HBc of the HBV genotypes D, F, and G, the possibility of dissociation and reassociation maintaining the native HBc structure was demonstrated. Single-stranded (ss) and double-stranded (ds) ribonucleic acid (RNA) was successfully packed into HBc VLPs for the HBV genotypes D and G.

scholarly journals Orotate Phosphoribosyltransferase from Corynebacterium ammoniagenes Lacking a Conserved Lysine

2007 ◽  
Vol 189 (24) ◽  
pp. 9030-9036 ◽  
Author(s):  
Xing Wang ◽  
Cuiqing Ma ◽  
Xiuwen Wang ◽  
Ping Xu
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Active Sites ◽  
Transfer Reaction ◽  
Gel Filtration ◽  
Orotate Phosphoribosyltransferase ◽  
Gene Encoding ◽  
Corynebacterium Ammoniagenes ◽  
Serovar Typhimurium ◽  
Optimal Ph

ABSTRACT The pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting from Corynebacterium ammoniagenes ATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 in Salmonella enterica serovar Typhimurium OPRTase) in the C. ammoniagenes OPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinant Escherichia coli strain. The molecular mass of the purified OPRTase was determined to be 45.4 ± 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 ± 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn2+ and Co2+. The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. The K m values for the four substrates were determined to be 33 μM for orotate, 64 μM for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 μM for orotidine-5-phosphate (OMP), and 36 μM for pyrophosphate. The K m value for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.

Sign in / Sign up

Export Citation Format

Share Document