Histidine decarboxylase in rat stomach ECL cells: relationship between enzyme activity and different molecular forms.

Mammalian HDC mRNA encodes a protein with a molecular mass of 74 kDa. The reported molecular mass for the purified HDC subunit is 53-55 kDa. Western blot analysis of extracts of rat gastric mucosa and fetal rat liver has revealed the presence of at least three different forms of HDC immunoreactivity, having molecular masses of about 74, 63 and 53 kDa. There is evidence from previous studies that full length rat HDC is enzymatically inactive and that activation requires C-terminal truncation. In the present study we examined the various immunoreactive HDC forms in rat oxyntic mucosa and their response to treatments known to affect the HDC activity. Freely fed rats and hypergastrinemic rats (treated with gastrin or the proton pump inhibitor omeprazole) had higher oxyntic mucosal HDC activity and HDC mRNA level than fasted or untreated rats. The difference in HDC activity was greater than the difference in HDC mRNA level. Western blot analysis confirmed the existence of the 74, 63 and 53 kDa HDC forms in the oxyntic mucosa. All three forms were more abundant in the oxyntic mucosa of freely fed and hypergastrinemic rats than in the mucosa of fasted or untreated rats. Of the three HDC forms, the 63 kDa form was the predominant one, the 73 kDa form was quantitatively insignificant by comparison and the 53 kDa form was at or below the limit of detection in fasted rats. The activity of HDC was well correlated to the amount of the 63 kDa HDC form. Administration of cycloheximide to hypergastrinemic rats (undergoing omeprazole treatment) resulted in a rapid decline of the HDC activity (estimated half-life 1 h and 50 min). The 63 kDa HDC form disappeared with a rate that corresponded to the decline in HDC activity. The two other HDC forms seemed to have a slower turnover. Our findings suggest that the 63 kDa form is enzymatically active. The results do not allow any conclusion as to the functional activity of the 74 and 53 kDa forms.     

Direct tissue isoelectric focusing on ultrathin polyacrylamide gels. Applications in enzyme, lectin and immunohistochemistry

Summary Application of cryostal sections directly onto ultrathin polyacrylamide gels and subsequent isoelectric focusing allows elution of proteins, glycoproteins and peptides out of the sections into the gels. The eluted compounds reveal clearly delineated band patterns in the polyacrylamide gels. The advantage of this method is that enzyme histochemical reactions can be directly performed in the gel and in the electroeluted tissue sections. Therefore, this method is suitable for specifying, in more detail, histochemical enzyme reactions and for detecting multiple forms of enzymes even from a single tissue section. Furthermore, the transfer of proteins, glycoproteins and peptides from the gel onto nitrocellulose by a modified Western blot procedure offers the possibility of checking findings obtained by lectin histochemistry and immunohistochemistry.     

Isolation of an activator of multiple forms of cyclic nucleotides phosphodiesterase of rat cerebrum by isoelectric focusing.

An isoelectric focusing technique was used to isolate multiple forms of cyclic nucleotide phosphodiesterase from a 105 000 times g soluble supernatant fraction of sonicated rat cerebrum. These separated peaks of activity had iso-electric points of 5.1, 5.6, 6.0, 6.6, 8.0, and 9.0. The activities were not stimulated by an endogenous activator of the enzyme but were inhibited by EGTA treatment. However, activator-sensitive forms of the enzyme could be separated from brain if the preparation of rat cerebrum was dialyzed against an EGTA containing buffer prior to electrofocusing. The procedure was also used to isolate a column fraction that stimulated maximum velocities of cyclic AMP and cyclic GMP hydrolysis. This fraction was itself devoid of phosphodiesterase activity and had an isoelectric point of 4.7.     

Subcellular distribution of a factor inactivating tyrosine aminotransferase. Study of its mechanism and relationship to different forms of the enzyme.

The subcellular distribution of a tyrosine aminotransferase inactivating factor in rat liver has been investigated. Most of its activity is associated with plasma membranes, with minor amounts in mitochondria and endoplasmatic reticulum. The factor is also found in kidney and inactivates the enzyme reversibly in presence of cysteine, most likely by modification of -SH groups. ATP counteracts this inactivation only, when crude enzyme extracts are inactivated by purified subcellular fractions or when the purified enzyme is inactivated in presence of liver or kidney cortex homogenates. The relationship of this inactivation to reported different forms of the enzyme has been investigated. Form I of three different forms, that can be obtained by hydroxyl-apatite chromatography, is readily inactivated, form III can be partly converted to form I by incubation in presence of purified plasma membranes. The relationship of these findings to a possible multistep mechanism in the turnover of the enzyme discussed.     

Preparative isoelectric focusing of CO hemoglobins on polyacrylamide gels and conversion to their oxy forms

Isoelectric focusing on acrylamide gels has been used to prepare 100–200 mg amounts of modified hemoglobins in a state of high purity. A method for the quantitative elution of the protein from the gel is described. A new procedure for the complete conversion of carbonmonoxyhemoglobin derivatives to their oxy or deoxy forms which fully preserves their functional integrity has also been developed.     

Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.     

Modeling the isoelectric focusing of peptides in an OFFGEL multicompartment cell

In proteomic analysis of complex samples at the peptide level (termed shotgun proteomics), an effective prefractionation is crucial to decrease the complexity of the peptide mixture for further analysis. In this perspective, the high-resolving power of the IEF fractionation step is a determining parameter, in order to obtain well-defined fractions and correct information on peptide isoelectric points, to provide an additional filter for protein identification. Here, we explore the resolving power of OFFGEL IEF as a prefractionation tool to separate peptides. By modeling the peak width evolution versus the peptide charge gradient at pI, we demonstrate that for the three proteomes considered in silico (Deinococcus radiodurans, Saccharomyces cerevisiae, and Homo sapiens), 90% of the peptides should be correctly focused and recovered in two wells at most. This result strongly suggests OFFGEL to be used as a powerful fractionation tool in shotgun proteomics. The influence of the height and shape of the compartments is also investigated, to give the optimal cell dimensions for an enhanced peptide recovery and fast focusing time.     

Thin-layer isoelectric focusing of polyphenoloxidase of Sephadex and its detection by the print technique.

Separation of polyphenoloxidase isoenzymes based on their charge properties was achieved by isoelectric focusing on Sephadex G-75 thin layers containing a mixture of ampholytes in the pH ranges 4–6 and 3–10. The separated isoenzymes can be detected as colored zones by a print technique in which a dried filter paper, previously buffered with 0.1 m phosphate buffer, pH 7,0, and impregnated with 1% substrate in methanol, is placed onto the gel layer. d-Catechin and tyramine were the best substrates for detecting the diphenolase and monophenolase activities, respectively. Using this technique, two commercial preparations of mushroom tyrosinase were found to consist of 7 and 15 isoenzymes, while enzyme preparations from two potato varleties showed 11 to 15 isoenzymes. The isoenzymes of potato and mushroom polyphenoloxidase showed marked differences in their pI values.     

Protein kinase assay on isoelectric focusing gels: evaluation of its quantitative potentials.

The conditions of the Hirsch-Rosen assay (1974, Anal. Biochem.60, 389–394) for protein kinase activity oncomplete isoelectric focusing gels have been analyzed and further developed with the consequence that the test can be easily adapted to other protein kinases in a quantitative manner. Special attention was given (i) to the breakdown of the pH gradient in relation to gel size and Ampholine concentration in order to achieve optimal pH ranges for the assay, (ii) to the introduction of histone as a substrate besides protamine, and (iii) to the distribution of the particular substrates and products throughout the gel. The results of the protein kinase assay on gels were shown to be linear for at least 1 h, and to be dependent on the amount of ATP and on the amount of protein kinase applied, thereby fulfilling the requirements necessary to yield quantitative data.     

Evaluation on the effect of different in-gel peptide isoelectric focusing parameters in global proteomic profiling

Peptide isoelectric focusing (IEF) is a common technique used in two-dimensional liquid chromatography tandem mass spectrometry (2D–LC–MS/MS) proteomic workflow, in which the tryptic peptide is first pre-fractionated based on pI values before being subjected to reverse phase LC–MS analysis. Although this method has been widely used by many research groups, a systemic study on the optimal conditions and fundamental parameters influencing the experimental outcomes has been lacking, including the effect of peptide extraction methods, the extent of pre-fractionation, and the choice of pH range. In this study, we compared the effect of different parameters on the numbers of peptides and proteins identified using two complex mouse proteomes. The results indicated that extraction of peptides from immobilized pH gradient (IPG) strips by sequential elution of increasingly organic solvents provided the highest number of peptide identification. In addition, we showed that approximately 45 more unique proteins were identified for every additional fraction collected during peptide IEF. Although narrow pH ranges provided higher resolution in peptide separation as expected, different pH ranges yielded similar numbers of peptide and protein identification. Overall, we demonstrated that the extraction solvent influenced the numbers of peptide and protein identification and quantitatively demonstrated the advantage of extensive fractionation and the performance of different pH ranges in practice.     

Detection of elastase activity with a zymogram method after isoelectric focusing in polyacrylamide gel

A zymogram method for detecting elastase activity following isoelectric focusing in polyacrylamide gel is described. After enzyme activity has been visualized, the gel itself is available for protein staining and for analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in second dimension. The zymogram method is suitable for detecting microgram amounts of elastase and has one step only. It can be used with the purified enzyme as well as with crude extracts of tissue containing elastases showing activity toward succinyl-(Ala)₃-p-nitroanilide. By this method a major component of elastase in both porcine and rat pancreas was detected. In addition, two forms of elastase with isoelectric points of 8.2 and 8.8, respectively, were identified in rat leukocyte extracts.     

Separation of isoenzymatic forms of human serum galactosyltransferase by chromatofocusing and isoelectric focusing. Application to cancerology

Chromatofocusing is used to separate the multiple isoenzyme forms of human serum galactosyltransferase. At least 11 major peaks of activity are observed in normal sera, which are eluted between pH 4.3 and 6.9; a fraction of activity is eluted above pH 7.0. The normal patterns are compared with those obtained with sera from cancer patients and with an ascitic fluid. Chromatofocusing appears as resolutive as agarose isoelectric focusing.     

Comparison of isozyme patterns between Spirometra erinacei and Spirometra mansonoides by isoelectric focusing.

No differences were observed in the isozyme patterns of 4 enzymes examined between fresh samples stored at -80 C and samples stored at room temperature for 10 days after lyophilization, which supports the validity of comparing lyophilized samples to fresh frozen tissue. Mature proglottids as well as plerocercoids of Spirometra erinacei from Japan and Australia were indistinguishable by comparison of isozyme patterns after isoelectric focusing. The isozyme patterns of acid phosphatase, glucosephosphate isomerase (GPI), and mannosephosphate isomerase from plerocercoids of Spirometra mansonoides were distinctly different from those of plerocercoids of S. erinacei. The adenylate kinase isozyme patterns of the mature proglottids of S. mansonoides were also distinctly different from those of the mature proglottids and the plerocercoids of S. erinacei. The GPI isozyme pattern of the mature proglottids of S. mansonoides was also distinguishable from the GPI patterns of those of S. erinacei. These electrophoretic data suggest that the S. erinacei from Japan and Australia are closely related, if not identical, but that S. mansonoides is genetically distinct from S. erinacei.     

On the detection of functional and structural enzyme mutants by coordinated affinity chromatography and isoelectric focusing

A method of studying structural and functional heterogeneity of enzymes has been developed and tested on chymotrypsin. The enzyme, prepared from single mouse pancreata, has been fractionated with respect to function and charge content by a combination of affinity chromatography and isoelectric focusing. By comparing chymotrypsin isolated from isogenic strains, chymotrypsinogen of strains A/Sn and NZB was found to be genetically heterogeneous, thus not revealed as different chymotrypsin forms of a single zymogen. Chymotrypsinogen originating from two loci was investigated, and structural and functional differences of the corresponding enzymes were determined. At both loci, structural allelomorphism was indicated. At one locus, the structural heterogeneity was also found to be reflected in functional heterogeneity of the corresponding enzymes. By mating the two strains and fractionating the enzyme of the cross, the differences were shown to be inherited.     

Aggregation of ampholine on heparin and other acidic polysaccharides in isoelectric focusing.

The mechanism of complexation of pI range 3.5--5 Ampholine to heparin in isoelectric focusing has been explored by the dye-binding technique at different pH values in solution. There is no significant interaction between heparin and Ampholine at pH 6.7. Weak, or selective, binding occurs at pH 5.1, and very strong interaction at pH 3.5. In the latter system, the Ampholine components appear to behave as polycations due to their ordered sequence of positive charges, each two methylene groups apart, which favors a strong binding to polyanions. In addition, there appear to be variable stoichiometries for the strong binding between heparin and Ampholine, depending on their relative amounts. It is proposed that at a low ratio of heparin to Ampholine (Ampholine excess), aggregation is perpendicular to the heparin chain, with the end ammonium charge of each Ampholine molecule neutralizing one negative charge along the heparin molecule; at higher ratios (heparin excess), the bound Ampholine segment is aligned parallel to the heparin molecule, so that on the average one Ampholine component neutralizes approx. three negative charges. The banding of heparin in isoelectric focusing in the pH range 3.0--4.5 can be explained by aggregation of the various components on heparin in amounts dependent upon the net charge on the Ampholine species at the given pH, and upon the changing stoichiometries as a function of the variation in ratio of heparin to Ampholine along the pH gradient. Binding of Ampholine to polygalacturonate was also demonstrated in excess Ampholine in a pH range dependent on the degree of protonation of the carboxyl groups of this acidic polysaccharide as well as on the net positive charge of the Ampholine. The aggregation seen at pH 4.2--4.5 led to the prediction and subsequent demonstration that polygalacturonate would also exhibit binding upon isoelectric focusing. This supports the hypothesis that aggregation of Ampholine on polyanions having sufficient charge density is a general phenomenon which can lead to spurious banding of certain polymers at appropriate pH ranges in isoelectric focusing. On the basis of their behavior in isoelectric focusing at pH 3.0--4.5, strength of aggregation of the polyanions studied appears to be heparin A = heparin B greather than polyglutamate greater than carboxyl-reduced heparin B greater than polygalacturonic acid.     

Two-step purification of Cyanophora ferredoxin and its identification in soluble protein preparations by isoelectric focusing.

Cyanophora paradoxa ferredoxin is encoded by (cyano-)plastidic DNA, in contrast to those of all other photosynthetic eukaryotes investigated so far. In the present study we report (i) the rapid purification of a chloroplast-type [2Fe-2S] ferredoxin in a two-step procedure by DEAE-Sephadex and Mono Q ion-exchange chromatography; (ii) the biochemical characterization of the purified ferredoxin by electrophoretic separation methods on a microscale; and (iii) a qualitative and quantitative ferredoxin detection method in the femtomole range that allows densitometry, semidry immunoblotting, identification of ferredoxin in soluble cell protein preparations, and analysis of protein biosynthesis from cyanoplast poly(A)- RNA in vivo and in vitro. These fast micromethods should be useful for screening phototrophic species containing ferredoxins encoded by nonnuclear DNA.     

Heterogeneity of staphylococcal enterotoxin A on isoelectric focusing and disc electrophoresis.

Heterogeneity of purified staphylococcal enterotoxin A, obtained from a culture supernatant of Staphylococcus aureus, strain 13N-2909, was demonstrated by isoelectric focusing. The toxin was composed of three immunologically identical fractions with isoelectric points of 6.5, 7.0 and approximately 8.0. Heterogeneity of the toxin was also shown by disc electrophoresis. At pH 8.0 and 9.4 two major bands and a faint minor band were observed, while at pH 4.3 only one band was observed. The faster-moving band for the anode in disc electrophoresis at pH 9.4 was found to correspond with the pI 6.5 component from isoelectric focusing, while the slower-moving band corresponded with the pI 7.0 component. From the results of the electrophoretic migration tests of the toxin, the components corresponding to the two major bands found in disc electrophoresis at pH 9.4 were considered to be charge isomers.