Characterization of a soluble form of dipeptidyl peptidase IV from pig liver

Summary Soluble dipeptidyl peptidase IV (EC 3.4.14.5) was purified from the 100,000×g supernatant fraction of pig liver homogenate. The purified enzyme had the same properties as, and immunological identity with, the membrane-bound enzyme which was described previously. However, the purified enzyme had a pattern of molecular heterogeneity different from the membrane-bound enzyme; this was shown by isoelectric focusing. Carbohydrate analysis revealed that the soluble enzyme contained glucose, which is not found in the membrane-bound one, and less fucose, mannose, and sialic acid than the latter. From these results, we conclude that the soluble form of dipeptidyl peptidase IV in pig liver is closley related to the membrane-bound enzyme, but is not simply a proteolytically solubilized product of it.We would like to thank Miss S. Fukushima for technical assistance.     

Increased activity of dipeptidyl peptidase IV in serum of hepatoma-bearing rats coincides with the loss of the enzyme from the hepatoma plasma membrane

The specific activity of dipeptidyl peptidase IV (DPP IV E.C. 3.4.14.-) in the plasma membrane of Morris hepatoma 9121 or hepatoma 7777 was 3.5% and 2.9%, respectively, of that in the plasma membrane of rat liver. The enzyme activity in the serum of hepatoma-bearing rats was 141% (hepatoma 91219) and 162% (hepatoma 7777) of the normal value. Cytochemical investigation showed that the DPP IV activity was almost completely absent from the hepatoma cell plasma membrane and was not sequestered within these cells. Indirect immunofluorescence staining with a polyclonal antibody directed against DPP IV indicated that the loss of activity was due to the absence of DPP IV molecules in the plasma membrane. The possibility that the enzyme is transferred from the membrane into the serum as a result of structural alterations is discussed.     

Increased activity of dipeptidyl peptidase IV in serum of hepatoma-bearing rats coincides with the loss of the enzyme from the hepatoma plasma membrane

Summary The specific activity of dipeptidyl peptidase IV (DPPIV E.C. 3.4.14.-) in the plasma membrane of Morris hepatoma 9121 or hepatoma 7777 was 3.5% and 2.9%, respectively, of that in the plasma membrane of rat liver. The enzyme activity in the serum of hepatoma-bearing rats was 141% (hepatoma 91219) and 162% (hepatoma 7777) of the normal value. cytochemical investigation showed that the DPP IV activity was almost completely absent from the hepatoma cell plasma membrane and was not sequestered within these cells. Indirect immunofluorescence staining with a polyclonal antibody directed against DPP IV indicated that the loss of activity was due to the absence of DPP IV molecules in the plasma membrane. The possibility that the enzyme is transferred from the membrane into the serum as a result of structural alterations is discussed.     

The prolyl oligopeptidase family

A group of serine peptidases, the prolyl oligopeptidase family, cannot hydrolyze peptides containing more than about 30 residues. This group is unrelated to the classical trypsin and subtilisin families, and includes dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B, in addition to the prototype prolyl oligopeptidase. The recent crystal structure determination of prolyl oligopeptidase (80 kDa) has shown that the enzyme contains a peptidase domain with an α/β hydrolase fold, and its catalytic triad is covered by the central tunnel of an unusual seven-bladed β-propeller. This domain operates as a gating filter, excluding large, structured peptides from the active site. The binding mode of substrates and the catalytic mechanism differ from that of the classical serine peptidases in several features. The members of the family are important targets of drug design. Prolyl oligopeptidase is involved in amnesia, depression and blood pressure control, dipeptidyl peptidase IV in type 2 diabetes and oligopeptidase B in trypanosomiasis. Received 8 August 2001; received after revision 19 September 2001; accepted 21 September 2001     

Deficiency of membrane-bound dipeptidyl aminopeptidase IV in a certain rat strain

The activity of membrane-bound dipeptidyl aminopeptidase IV (EC 3.4.14.5) was found to be markedly reduced in the Fischer 344 rat strain compared with that in the Wistar strain. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Ouchterlony immunodiffusion revealed the specific absence of this enzyme molecule in the Fischer 344 strain.     

Deficiency of membrane-bound dipeptidyl aminopeptidase IV in a certain rat strain

Summary The activity of membrane-bound dipeptidyl aminopeptidase IV (EC 3.4.14.5) was found to be markedly reduced in the Fischer 344 rat strain compared with that in the Wistar strain. Analysis of membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Ouchterlony immunodiffusion revealed the specific absence of this enzyme molecule in the Fischer 344 strain.     

Studies on substrate specificity of X-prolyl dipeptidyl-aminopeptidase during new chromogenic substrates, X-Y-p-nitroanilides

Substrate specificity of X-prolyl dipeptidyl-aminopeptidase (dipeptidyl aminopeptidase IV) was examined by using newly synthesized 8 chromogenic substrates, X-Y-p-nitroanilides. Homogeneous enzyme from human submaxillary gland hydrolyzed glycylproline p-nitroanilide almost specifically, except alanylalanine p-nitroanilide which had 11% activity.     

Studies on substrate specificity of X-prolyl dipeptidyl-aminopeptidase using new chromogenic substrates,X-Y-p-nitroanilides

Summary Substrate specificity of X-prolyl dipeptidyl-aminopeptidase (dipeptidyl aminopeptidase IV) was examined by using newly synthesized 8 chromogenic substrates, X-Y-p-nitroanilides. Homogeneous enzyme from human submaxillary gland hydrolyzed glycylproline p-nitroanilide almost specifically, except alanylalanine p-nitroanilide which had 11% activity.     

Immunochemical studies on Rhodotorula gracilis D-amino acid oxidase.

Polyclonal antibodies were prepared from rabbit sera after immunization with holo- and apo-D-amino acid oxidase purified from R. gracilis. Both anti-holo- and anti-apoenzyme IgG fractions (as well as affinity-purified IgG) were highly specific: in blot-transfer analyses after SDS-PAGE only a 39 kDa band, corresponding to enzyme monomer, was recognized even in the partially purified yeast extract. No cross-reaction was detected with pig kidney D-amino acid oxidase. As a difference from the mammalian enzyme, yeast D-amino acid oxidase anti-holo- and anti-apoenzyme IgGs had different properties in inactivation and precipitation experiments, indicating the existence of different antigenicity sites related to the FAD-binding domain in the enzyme.     

Immunochemical identity of dipeptidyl aminopeptidase IV from pig serum, liver, submaxillary gland and kidney.

The enzymes which were extracted by autodigestion from the microsomal fractions of the pig kidney, liver and submaxillary gland and from the serum showed an immunochemical identity by a double immunodiffusion test. But the kidney enzyme had a different pI-value from the pI-values of the enzymes of other organs.     

Immunochemical studies onRhodotorula gracilis D-amino acid oxidase

Summary Polyclonal antibodies were prepared from rabbit sera after immunization with holo- and apo-D-amino acid oxidase purified fromR. gracilis. Both anti-holo- and anti-apoenzyme IgG fractions (as well as affinity-purified IgG) were highly specific: in blot-transfer analyses after SDS-PAGE only a 39 kDa band, corresponding to enzyme monomer, was recognized even in the partially purified yeast extract. No cross-reaction was detected with pig kidney D-amino acid oxidase. As a difference from the mammalian enzyme, yeast D-amino acid oxidase anti-holo- and anti-apoenzyme IgGs had different properties in inactivation and precipitation experiments, indicating the existence of different antigenicity sites related to the FAD-binding domain in the enzyme.     

Developmental and ageing changes in aminopeptidase activities in selected tissues of the rat

Aminopeptidase activities, assayed as arylamidase activities, were investigated in selected tissues of 1, 6, 12 and 24-month-old rats. The enzyme activities were found to have a heterogeneous distribution and age-related changes were observed. The highest levels of soluble arginyl-aminopeptidase activity were detected in brain homogenate at all the studied ages, whereas membrane-bound activity presented the highest levels in brain and kidney in the four ages tested. Aspartyl-aminopeptidase activity was detected mainly in the particulate fraction of kidney at all four ages. In 1, 6 and 12-month-old animals, soluble aspartyl-aminopeptidase activity was also higher in the kidney than in the rest of the tissues, whereas in the group of 2-year-old rats, the highest levels were found in both kidney and liver. Age-related changes were observed in all the studied tissues and for all the assayed enzymatic activities. In general, the maximal levels were detected in both the youngest and the oldest animals, and the minimal ones in 6 and 12-month-old rats. However, in the adrenals, the soluble and membrane-bound arginyl-aminopeptidase activity was higher in 6-month and 2-year-old rats than in 1-month and 12-month-old rats. These changes may reflect the functional status of the susceptible endogenous substrates of aminopeptidases.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8-7.4) and ionic strength (20-50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO4(2-) and cations (Mg2+, Ca2+) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD+ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0-37 degrees C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

Interaction between the soluble and particulate neuraminidases of chick liver.

The sum of the neuraminidase activities found in the isolated soluble and particulate fractions of chick liver was considerably higher than that observed in the cytoplasmic extract from which these fractions were obtained. Addition of increasing amounts of particulate neuraminidase to a constant amount of the soluble preparation resulted in a progressive loss of enzyme activity.     

Immunochemical identity of dipeptidyl aminopeptidase IV from pig serum,liver, submaxillary gland and kidney

Summary The enzymes which were extracted by autodigestion from the microsomal fractions of the pig kidney, liver and submaxillary gland and from the serum showed an immunochemical identity by a double immunodiffusion test. But the kidney enzyme had a different pI-value from the pI-values of the enzymes of other organs.This investigation was supported by a grant from the Ministry of Education, Japan to K. M. F. We would like to thank Dr Toshiharu Nagatsu (Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama, Japan) for helpful suggestions, Mr Moritoshi Sato (Matsumoto Meat Inspection Station, Matsumoto, Japan) for their generous supply of pig organs and Ajinomoto Co. Inc., Tokyo, Japan, for the gift of Gly-Pro-p-nitroanilide tosylate. Technical assistance of Miss K. Yanagisawa is gratefully acknowledged.     

Ambiquitous behavior of rabbit liver lactate dehydrogenase

Summary Rabbit liver mitochondrial fraction shows lactate dehydrogenase activity. The enzyme can be released from particles by increasing the pH and the ionic strength of the medium. There is a narrow range of pH (6.8–7.4) and ionic strength (20–50 mM NaCl) in which the solubilization sharply increases. It has been shown that divalent anions (SO ₄ ^2– ) and cations (Mg²⁺, Ca²⁺) are highly effective specific solubilizing agents. NADH (1.5 mM) and ATP (1.0 mM) were effective in solubilizing 50% of the enzyme bound, whereas the same concentrations of the analogs NAD⁺ and ADP had little effect. Cytosolic lactate dehydrogenase bound to the mitochondrial fraction and a saturation of particles by enzyme was observed in all experiments performed. The in vitro binding requires a short period of incubation between the enzyme and particles and the binding is independent of the temperature in the 0–37°C range. Binding was prevented by 0.15 M NaCl. The bound enzyme is approximately 20% less active than the soluble one. The results described give support to the proposal that rabbit liver lactate dehydrogenase has an ambiquitous behavior, like other glycolytic enzymes, which have not a fixed intracellular localization.     

The PREPL A protein, a new member of the prolyl oligopeptidase family, lacking catalytic activity

The PREPL (previously called KIAA0436) gene encodes a putative serine peptidase from the prolyl oligopeptidase family. A chromosomal deletion involving the PREPL gene leads to a severe syndrome with multiple symptoms. Homology with oligopeptidase B suggested that the enzyme cleaves after an arginine or lysine residue. Several PREPL splice variants have been identified, and a 638-residue variant (PREPL A) was expressed in Escherichia coli and purified. Its secondary structure was similar to that of oligopeptidase B, but differential-scanning calorimetry indicated a higher conformational stability. Dimerization may account for the enhanced stability. Unexpectedly, the PREPL A protein did not cleave peptide substrates containing a P1 basic residue, but did slowly hydrolyse an activated ester substrate, and reacted with diisopropyl fluorophosphate. These results indicated that the catalytic serine is a reactive residue. However, the negligible hydrolytic activity suggests that the function of PREPL A is different from that of the other members of the prolyl oligopeptidase family.     

Interaction between the soluble and particulate neuraminidases of chick liver

Summary The sum of the neuraminidase activities found in the isolated soluble and particulate fractions of chick liver was considerably higher than that observed in the cytoplasmic extract from which these fractions were obtained. Addition of increasing amounts of particulate neuraminidase to a constant amount of the soluble preparation resulted in a progressive loss of enzyme activity.This investigation was supported in part by U. S. Public Health Service Grant NS09176 from the National Institutes of Health.     

Affinity chromatography of glucose dehydrogenase

Porcine liver beta-D-glucose dehydrogenase, a multi-functional protein, has been purified to apparent homogeneity. The enzyme has been separated from the endoplasmic reticulum using Triton X-114 and further purified using NAD to release glucose dehydrogenase from a NADP-linked sepharose column. The purified enzyme is capable of producing both NADH and NADPH in vivo as indicated by kinetic studies.     

Purification and properties of ornithine aminotransferase from rat brain

Ornithine aminotransferase (E.C. 2.6.1.13) from rat brain was purified 100-fold by ammonium sulphate fractionation, DEAE cellulose chromatography, calcium phosphate gel and alumina C gamma gel. Pyridoxal phosphate was essential for maximum activity of the enzyme. The brain enzyme did not differ from liver and kidney enzymes in properties such as pH optimum, Km, substrate specificity and the inhibition by branched chain amino acids. Unlike rat liver enzyme, brain ornithine aminotransferase was able to catalyze the reaction between L-lysine and 2-oxoglutarate. Spermidine and spermine inhibited brain ornithine aminotransferase activity.