Purification and characterization of a small dermatan sulphate proteoglycan implicated in the dilatation of the rat uterine cervix.

Ferritin was purified from chicken liver by two different methods: gel filtration on controlled-pore glass beads, and immunoaffinity chromatography employing a chicken ferritin-specific monoclonal antibody that did not cross-react with horse spleen ferritin. This antibody recognizes intact ferritin and an oligomeric 240 kDa form of the molecule after protein transfer to nitrocellulose, but not the 22 kDa chicken ferritin subunit. Chicken liver ferritin purified by these methods exhibited reduced migration on non-denaturing polyacrylamide gels compared with horse spleen ferritin. These results were consistent with the difference in calculated isoelectric points of chicken and horse ferritin subunits. By two-dimensional gel electrophoresis, chicken ferritin 22 kDa subunits exhibited isoelectric points from 6.1 to 6.6 whereas horse spleen ferritin subunits exhibited isoelectric points of 5.8-6.3. The 240 kDa form of the chicken ferritin molecule had an isoelectric point of 6.6 whereas the 210 kDa form of the horse ferritin molecule had isoelectric points of 5.1 and 4.9. Intact chicken liver ferritin particles were 13.4 +/- 0.8 nm (controlled-pore glass-purified) and 12.5 +/- 0.9 nm (affinity-purified) in diameter when viewed by electron microscopy. Horse spleen ferritin consisted of slightly smaller particles with an average diameter of 11.0 +/- 0.7 nm. However, ferritin from chicken liver and horse spleen co-migrated with an apparent molecular mass of 470 kDa when analysed by Sepharose 4B gel filtration chromatography. These results indicate that, consistent with results from other published purification methods, the chicken ferritin purified by the methods reported here exhibits both structural similarities to, and differences from, horse spleen ferritin.     

The N-acetylneuraminic acid content of five forms of human transferrin

Five isoforms of human serum transferrin were separated by isoelectric focusing and their N-acetylneuraminic acid content was determined. The forms differed in isoelectric point by about 0.1 of a pH unit with the structural differences situated in the carbohydrate parts. Each form had one sialic acid molecule (NANA) less than the next most acidic form. GLC-MS showed that the most abundant form with isoelectric point 5.5 had two two-branched carbohydrate chains, each having the galactoses covered by terminal sialic acid. The form with isoelectric point 5.4 had one three-branched and one two-branched carbohydrate chain, and all branches terminated with a sialic acid residue. The form with isoelectric point 5.6 had a terminal galactose on one of its two two-branched carbohydrate chains. Comparison of the sialic acid content of the five transferrin forms and their carbohydrate structures showed that some of the forms expose terminal galactose without attracting the asialoglycoprotein receptors on hepatocytes.     

Isoelectric-focusing behavior of acid hydrolases in rat kidney lysosomes. Effects of the pH gradient, autolysis and neuraminidase.

Isoelectric focusing was used to study the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase in lysosomes isolated from rat kidney. The isoelectric points of the main protein and hydrolase peaks were 1-1.5 units lower when electrofocusing was done in a pH 3-10 gradient than in a pH 10-3 gradient, apparently because the lysosomal constituents aggregated strongly at their isoelectric points and tended to settle somewhat in the gradient due to gravity. In the extended pH gradient the acidic form of each hydrolase occurred as asingle, relatively discrete peak. However, when pooled acidic fractions were refocused in a restricted pH gradient (pH 6-3 or 3-5) multiple acidic enzyme and protein components were resolved with isoelectric points between 2.7 and 5.1. When autolysis was minimized by extracting lysosomal fractions at alkaline pH (0.2% Triton X-100, 0.1%p-nitrophenyloxamic acid, 0.1 M glycine buffer, pH9) and including 0.1%p-NITROPHENYLOXAMIC ACID, AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND CATHEPSIN D, in the pH gradient, arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in two forms, an acidic form with an isoelectric point of about 4.4, and a basic form with an isoelectric point close to 6.2, 6.7 and 8.0, respectively. Acid phosphatase occurred in three forms with isoelectric points of 4.1, 5.6 and 7.4. When some autolytic digestion was permitted by extracting lysosomal fractions in an acidic medium (0.2% Triton X-100, 0.1 M sodium acetate buffer, pH 5.2) AT 0-4DEGREES C and omitting p-nitrophenyloxamic acid from the gradient, the acidic form of beta-glucuronidase and the intermediate form of acid phosphatase were lost, the isoelectric points of the acidic forms of acid phosphatase, arylsulfatase and beta-N-acetylhexosaminidase were increased 0.6-1.2 units, and the isoelectric point of the basic forms of acid phosphatase, arylsulfatase and beta-glucuronidase was increased 0.5 unit. When lysosomal extracts were incubated with bacterial neuraminidase before electrofocusing, the acidic forms of acid phosphatase, arylsulfatase and beta-glucuronidase were largely lost, the isoelectric point of the acidic form of beta-N-acetylhexosaminidase was increased from 4.5 to 6.4, and the isoelectric points of the basic forms of all four hydrolases were increased 0.5-1.5 units. Autoincubation of lysosomal extracts in vitro at pH 5.2 PRODUCED SIMILAR, THOUGH LESS MARKED, effects. cont'd     

Soluble and membrane-bound neutral alpha-glucosidase from the human kidney

In human kidney cortex neutral alpha-glucosidases 1 and 2 are represented by two forms, soluble (cytosolic) and membrane-bound (brush border) ones. It has been shown that the soluble enzyme preexists in human kidney but does not derive from the membrane-bound form. Similar to the membrane-bound enzyme the soluble form is a glycoprotein. Both enzyme forms possess identical electrophoretic mobility, pH-optimum, heat sensibility and Km values for maltose (0.7 mM) and 4-methylumbelliferyl-alpha-D-glucopyranoside (0.57 mM), but differ by molecular weights as determined by gel filtration chromatography. The molecular weights of the soluble neutral alpha-glucosidases 1 and 2 are lower than those of the comparable brush border enzymes (470 000, 360 000, 520 000 and 440 000, correspondingly). Neutral membrane-bound alpha-glucosidase 1 is a sialylated enzyme with a pI of 4.10 +/- 0.02. The soluble enzyme contains no or only traces of neuraminic acid and has a pI 4.40 +/- 0.03. The soluble and membrane-bound neutral alpha-glucosidases are apparently independent forms of the enzyme, differing by the degree of sialylation and by the presence of an "anchor" in the membrane-bound enzyme. The synthesis of both forms is presumably coded by the same structural gene.     

Purification and properties of gentamicin nucleotidyltransferase from Escherichia coli: nucleotide specificity, pH optimum, and the separation of two electrophoretic variants

Gentamicin nucleotidyltransferase, AAD 2", catalyzes the transfer of a nucleotide to many aminoglycoside antibiotics, which are the drugs of choice in the treatment of gram-negative bacterial infections. The transfer is accompanied by the production of pyrophosphate, which is coupled to three other enzymes so that an increase in absorbance at 340 nm of NADPH can be monitored continuously as a quantitative assay of activity. A purification method was developed for this enzyme using all common principles of protein purification. These include selection of a desirable source of enzyme (choice of plasmid pMY 10), maximizing cellular yield of enzyme (controlled and monitored growth of Escherichia coli pMY 10/W677), selective extraction of protein (modified osmotic shock), removal of nucleic acids (precipitation with streptomycin sulfate), concentration of protein (precipitation with ammonium sulfate), removal of low-molecular-weight impurities (chromatography on Bio-Gel P-2), separation of proteins on the basis of charge (ion-exchange chromatography on DEAE-Bio-Gel A), separation of proteins according to a biospecific property (affinity chromatography on gentamicin-Affi-Gel), and separation of proteins according to size (gel filtration on Ultrogel AcA 54). Purification to near-homogeneity revealed the presence of two related forms of enzyme. The first had a specific activity of 0.134 units/mg, bound rapidly and tightly to gentamicin-Affi-Gel, eluted as a function of ionic strength from Ultrogel, and migrated faster during electrophoresis in both the presence and absence of sodium dodecyl sulfate. It has an isoelectric point of 5.7 +/- 0.2 and consists of a single polypeptide of 32,500 Da. Kinetic characterization showed a pH optimum of 9.5 and Michaelis constants of 2.76 +/- 0.35 microM for tobramycin, 404 +/- 28 microM for Mg-ATP, 2008 +/- 260 microM for Mg-CTP, 30 +/- 3 microM for Mg-dATP and Mg-dGTP, and 90 +/- 7 microM for Mg-dCTP and Mg-dTTP. The second form had a specific activity of 0.274 unit/mg. It also bound tightly to gentamicin-Affi-gel but the onset of binding was time dependent. This form migrated slower during polyacrylamide gel electrophoresis in both the presence and absence of sodium dodecyl sulfate. It has an isoelectric point of 6.0 +/- 0.2 and consists of a single polypeptide of 31,500 Da. The exact relationship between the two forms has not been elucidated. It is probable that they have a recent common ancestor or are the same polypeptide because the amino acid compositions and polypeptide chain lengths are essentially identical.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resolution of simian virus 40 proteins in whole cell extracts by two-dimensional electrophoresis: heterogeneity of the major capsid protein.

The major capsid protein (VP1) of simian virus 40 (SV40) has been analyzed by two-dimensional electrophoresis. This system separates protein according to isoelectric point by isoelectric-focusing, and according to molecular weight by sodium dodecylsulphate electrophoresis (O'Farrell, 1975). VP1 synthesis in infected CV-1 cells can be monitored directly by analysis of unfractionated whole cell extracts; the resolution of VP1 from cellular proteins allows its detection as early as 13 hr after infection. The two-dimensional separation of VP1 reveals that it is heterogeneous, consisting of one major protein (molecular weight 47,000 daltons and isoelectric point of approximately pH 6.8) and five minor protein components. The minor forms of VP1 are 10% of the total VP1 and differ from the major form of VP1 both in molecular weight (by approximately 500 daltons) and isoelectric point (ranging from approximately pH 6.7 to pH 6.9). Evidence is presented to show that two of the minor forms are phosphorylated derivatives of VP1, and it is further suggested that all the different forms of VP1 are the result of modifications of the primary product of translation. A temperature-sensitive mutant of the BC complementation group (BC11) of SV40 results in the synthesis of VP1 with an altered electrophoretic mobility; both the major form of VP1 and the minor forms are shifted in their isoelectric points. In addition to the specific case of SV40, two aspects of these studies should be generally significant to investigators studying eucaryotic gene expression by two-dimensional gel electrophoresis: first, the genetic origin of a protein can be determined by a temperature-sensitive mutation which causes a charge change in the resultant protein; and second, two or more protein spots on a two-dimensional separation may be the products of a single gene.     

An increase in high-molecular weight renin substrate associated with estrogenic hypertension

We have previously reported that estrogens have the potential to induce new forms of renin substrate in addition to elevating the major circulating form of this protein. One of these estrogen-induced forms had a molecular weight in excess of 150,000. In this study we have compared the plasma concentration of the high-molecular-weight renin substrate in normotensive women receiving estrogen therapy and women with estrogenic hypertension. A statistically significant elevation of this protein was associated with estrogenic hypertension and normotensive pregnant women at term. This form of renin substrate differed from the major form with respect to electrophoretic mobility, isoelectric point, and immunologic cross-reactivity. In addition, kinetic analysis indicated that this high-molecular-weight substrate has a significantly higher affinity for the enzyme renin than the major circulating form (Km = 1800 +/- 290 versus 3520 +/- 260 ng angiotensin I equivalents/ml). These results suggest that in addition to renin substrate concentration, substrate composition may play an important role in blood pressure regulation.     

Cytochrome b from Escherichia coli nitrate reductase. Its properties and association with the enzyme complex

Membrane-bound nitrate reductase purified from Escherichia coli was resolved into two separate forms. The majority of the enzyme complex had a subunit composition of 2A:2B:4C, exhibited cytochrome b spectra, and was found to be stable after purification. A second form of nitrate reductase activity was a modified complex with a subunit composition of 2A:2B and lacked cytochrome. The subunit B from this complex was altered in its mobility on sodium dodecyl sulfate-polyacrylamide gels. The cytochrome-containing enzyme had 28 +/- 2 atoms of iron and 1.35 atoms of molybdenum whereas iron and molybdenum in cytochromeless enzyme were 24 +/- 2 atoms and 1.18 atoms/molecule, respectively. Besides cytochrome-containing nitrate reductase, two other cytochrome b-containing fractions were also resolved. These were cytochrome b associated with formate dehydrogenase and a novel cytochrome b with reduced absorption maxima at 430, 529.5, and 560 nm. Nitrate reductase cytochrome b (subunit C) was isolated from subunits A and B as a partially denatured form and its renaturation was accomplished by dialyzing against hemin. The renatured cytochrome yielded absorption spectra similar to the holoenzyme. The pure cytochrome aggregated upon heating, even in the presence of sodium dodecyl sulfate. It had a high isoelectric point (pH greater than 9.5) and had 45% hydrophobic amino acids.     

Cellular localization and substrate specificity of isoelectric forms of human liver neuraminidase activity.

The four major isoelectric forms of human liver neuraminidase (with pI values between 3.4 and 4.8) have been isolated by preparative isoelectric focusing and characterized with regard to their substrate specificity using glycoprotein, glycopeptide, oligosaccharide and ganglioside natural substrates. All forms exhibited a rather broad linkage specificity and were capable of hydrolyzing sialic acid glycosidically linked alpha 2-3, alpha 2-6 and alpha 2-8, although differential rates of hydrolysis of the substrates were found for each form. The most acidic form 1 (pI 3.4) was most active on sialyl-lactose, whereas form 2 (pI 3.9) and 3 (pI 4.4) were most active on the more hydrophobic ganglioside substrates. Form 4 (pI 4.8) was most active on the low-Mr hydrophilic substrates (fetuin glycopeptide, sialyl-lactose). Each form was less active on the glycoprotein fetuin than on a glycopeptide derived from fetuin. Organelle-enriched fractions were prepared from fresh human liver tissue and neuraminidase activity on 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid was recovered in plasma membrane, microsomal, lysosomal and cytosolic preparations. Isoelectric focusing of the neuraminidase activity recovered in each of these preparations resulted in significantly different isoelectric profiles (number, relative amounts and pI values of forms) for each preparation. The differential substrate specificity of the isoelectric forms and the different isoelectric focusing profiles of neuraminidase activity recovered in subcellular-enriched fractions suggest that specific isoelectric forms with broad but defined substrate specificity are enriched at separate sites within the cell.     

Multiple molecular forms of human plasma butyrylcholinesterase. I. Apparent molecular parameters and broad pattern of the quaternary structure (author's transl)]

Apparent molecular parameters (molecular weights, sedimentation constants, partial specific volumes, free electrophoretic mobilities and isoelectric points) of the four molecular forms C-1, C-2, C-3 and C-4 of human plasma butyrylcholinesterase (EC 3.1.1.8) have been demonstrated by polyacrylamide gel electrophoresis methods and centrifugation in sucrose gradient. The C-1 component is the monomeric form of the enzyme )Mr = 84 800 +/- 5800). All the forms are partially interconvertible and C-1, C-3, C-4 are size isomers corresponding to the monomer, dimer and tetramer of the enzyme. An estimation of the general shape of these forms attempted from electrophoretic and hydrodynamic parameters suggests that they are prolate ellipsoids. The C-4 component in which the axial ratio is at least equal to 8 appears to be arranged as a dimer of dimers (C-3)2 in which the two units are associated in a quasi-linear fashion. The C-2 component is composed of C-1 associated with an inactive smaller subunit, which is responsible for its specific electrical properties (mobility and isoelectric point).     

Cobra venom acetylcholinesterase. Purification and molecular properties.

Acetylcholinesterase from cobra (Naja naja oxiana) venom has been purified by affinity chromatography to an homogeneous state, as ascertained by sodium dodecylsulfate/polyacrylamide gel electrophoresis and sedimentation analysis. The specific activity of the preparation was 5000 IU/mg with acetylcholine as substrate. Unlike acetylcholinesterases from insoluble cell structures, the native molecule of the cobra venom enzyme consists of a single polypeptide chain of molecular weight 67,000 +/- 2000. At high enzyme concentrations (greater than 0.2 mg/ml, greater than 1 microM) and ionic strength 0.1 M, it reversibly tends to form higher-molecular-weight 7.1-S aggregates. Despite the apparent structural simplicity of the venom acetylcholinesterase, the disc electrophoresis and isoelectric focusing experiments revealed that the enzyme exists in a number of forms with a common molecular weight but with different isoelectric points. Neuraminidase treatment did not reduce the number of the forms.     

Cellulase and Abscission in the Red Kidney Bean (Phaseolus vulgaris)

Cellulase (β-1, 4-glucan-glucanohydrolase EC 3.2.1.4) activity in the abscission zone of red kidney bean (Phaseolus vulgaris) was previously shown to exist in at least two different molecular forms. The form of the enzyme which has an isoelectric point of 4.5 is present in both abscising and nonabscising tissue and requires grinding for extraction. Another form of the enzyme which has an isoelectric point of 9.5 is present only in tissue in which the abscission process has been induced. Further, much of this form of cellulase can be removed from the tissue by vacuum infiltration with buffer. Time course studies indicate that while the increase in measurable cellulase activity in tissue which is actively undergoing abscission was due primarily to the appearance of cellulase 9.5, this form of the enzyme cannot be removed by vacuum infiltration until after the breakstrength of the abscission zone has decreased nearly to zero. The intracellular localization of these two forms of cellulase is discussed.     

A family of repetitive nucleotide sequences (Sau 3A family) in the genome of 2 forms of the sockeye salmon Oncorhynchus nerka

A family of DNA sequences (Sau3A repeat sequences) has been revealed in genomes of two forms of O. nerka. A DNA fragment belonging to this "family" has been cloned in bacterial plasmid. Its copy number (in genomes of normal (570 +/- 33) and dwarf (66 +/- 12) forms) has been counted. The structural organization of sequences of Sau3A family in genomes of males and females of normal form is not the same. The organization of Sau3A sequences is the same in genomes of dwarf males and dwarf females of normal form, but the quantity of Sau3A sequences copies is smaller. It is supposed that the DNA sequences of Sau3A family bear the sex determination of dwarf form of O. nerka. The fragment of rRNA gene of nerka has been cloned and the number of rRNA gene copies in genomes of normal (200 +/- 62) and dwarf (1690 +/- 24) forms has been defined.     

EPR spectroscopy of semi-methemerythrin

EPR spectra of semi-met forms of octameric hemerythrin from Themiste zostericola, prepared by one electron reduction of methemerythrin or by one electron oxidation of deoxyhemerythrin, have been visualized at liquid helium temperatures. The spectrum of that prepared by one electron reduction has principal g-values of 1.96 +/- 0.01, 1.88 +/- 0.01, and 1.67 +/- 0.02 while that obtained by one electron oxidation has g = 1.95 +/- 0.01, 1.72 +/- 0.01, and 1.68 +/- 0.02. The amplitude of either spectrum decreases with time on incubation at room temperature according to a first order rate with t 1/2 = 5-8 min, apparently because of an intramolecular disproportionation. Similar EPR spectra have been obtained with semi-metmyohemerythrin of T. zostericola and with the octameric semi-met form of Phascolopsis gouldii. However, these forms disproportionate to a much lesser degree. The azide adduct of the octameric semi-met form of T. zostericola has g-values of 1.94 +/- 0.01, 1.85 +/- 0.01, and 1.57 +/- 0.02. Its EPR spectrum differs somewhat from those of the azide adducts of the octamer of P. gouldii and the monomer of T. zostericola although all are resistant to disproportionation. Methemerythrin and deoxyhemerythrin have no EPR spectra even at liquid helium temperature.     

Purification and analysis of lung and plasma angiotensin I-converting enzyme by high-performance liquid chromatography

We purified angiotensin I-converting enzyme (ACE) from pig and human lung and plasma for comparison of some physicochemical properties between the endothelial membrane-bound form and the soluble form of the enzyme. After affinity chromatography on Sepharose CL-4B/lisinopril, gel-filtration HPLC on Superose 12 achieved homogeneity for both forms as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Whatever the source of ACE, the molecular weight was 300 +/- 40 kDa after calibration of Superose 12 with standard globular proteins and 172 +/- 4 kDa by SDS-PAGE, with or without reduction, a result suggesting interactions between the glycopolypeptide chain and the chromatographic gel possibly related to the overall shape and sugar content of the enzyme. Ion-exchange HPLC analysis on TSK-DEAE showed that the membrane-bound and soluble forms of ACE are not isoenzymes, although isoelectrofocusing did show that the isoelectric point of soluble ACE was lower than those of tissue ACE, suggesting a different glycosylation. No significant difference between porcine and human ACE appeared. HPLC methods seem to be of particular interest for the purification of ACE with a high yield and for the analysis of its putative differently glycosylated isoforms.     

Further resolution of adult chick hemoglobins by isoelectric focusing in polyacrylamide gel.

Evidence is presented that adult chick hemoglobins exist in four types separable by isoelectric focusing on polyacrylamide gels instead of the two hemoglobin types previously resolved by other methods. These are hemoglobin A1 (HbA1), hemoglobin A2 (HbA2), hemoglobin D1 (HbD1), and hemoglobin D2(HbD2). Their pI values are 7.53 +/- 0.02, 7.37 +/- 0.02, 6.92 +/- 0.04 and 6.72 +/- 0.05, respectively, constituting about 63, 14, 18 and 5% of the total hemoglobin from adult chick erythrocytes, respectively. HbA1 and HbA2 ar identical in size, as determined on sodium dodecyl sulfate gels and similar in their amino acid composition and tryptic peptides. The molecular weight and amino acid composition of HbD1 and HbD2 are also identical although there are differences in their tryptic peptides. Experiments were done to show that the existence of four hemoglobin types is not due to genetic heterogeneity of the experimental animal, nor to artifacts of oxidation of carboxyhemoglobin to methemoglobin tetramers. Care was exercised to eliminate deamination and modification of side chain amino groups by using freshly prepared hemolysates and to minimize the "plateau phenomenon" peculiar to isoelectric focusing by controlling the duration of electrophoresis. The use of cyanmet form of (thus liganded) hemoglobin in this study reduced the chance of heterotetramer formation. Furthermore, consideration was given to possible anomalies caused by ampholytes. In the face of negative evidence for artifacts, it is concluded that adult chicken has more than the two hemoglobin types previously reported.     

Characterization of purified human liver acid beta-D-galactosidases A2 and A3.

1. Human liver acid beta-galactosidase A2 and A3 were isolated by chromatography on concanavalin A-Sepharose 4B, Sepharose 6B, and Sepharose 4B-6-aminohexyl 1-thio-beta-D-galactopyranoside. beta-Galactosidase A2 and A3 were purified to final specific activities of 45.5 and 20.6 mumol/min per mg respectively with 4-methylumbelliferyl beta-D-galactopyranoside as substrate. 2. Form A2 had a mol.wt. of 150000 +/- 15000 (gel filtration) and appeared as a single band of protein (mol.wt 65000 +/- 1000) on electrophoresis in the presence of sodium dodecyl sulphate. 3. Form A3 had a mol.wt. (gel filtration) of 660000 +/- 66000. On electrophoresis in the presence of sodium dodecyl sulphate, form A3 appeared as a major band of protein (72% of total) of mol.wt. 65000 +/- 1000 and minor protein bands of mol.wt. 44000 +/- 1000 and 26,000 +/- 1000 and 22000 +/- 1000. 4. Gel-filtration chromatography of purified beta-galactosidase A3 generated approximately equal amounts of forms A3 and A2. beta-Galactosidase A1 was not detected by gel-filtration chromatography of partially or highly purified preparations of forms A2 and A3. 5. Both forms A2 and A3 had identical isoelectric points of 4.42 +/- 0.02. The data suggest that forms A2 and A3 are dimeric and multimeric forms of beta-galactosidase A1. 6. Amino acid analysis of beta-galactosidase A2 gave a ratio of acidic to basic amino acids of 2.6:1. 7. beta-Galactosidase A2 contained 7.5% carbohydrate by weight and sialic acid, D-galactose, D-glucosamine and D-mannose were present in the molar proportions 1.1:1.0:1.7:2.7.     

Characterization of active and inactive forms of the phenol hydroxylase stimulatory protein DmpM.

The stimulatory protein DmpM of phenol hydroxylase from methylphenol-degrading Pseudomonas sp. strain CF600 has been found to exist in two forms. DmpM purified from the native strain was mostly active in stimulating phenol hydroxylase activity, whereas an inactive form accumulated in a recombinant strain. Both forms exhibited a molecular mass of 10 361.3 +/- 1.3 Da by electrospray mass spectrometry, but nondenaturing gel filtration showed molecular masses of 31 600 Da for the inactive form and 11 500 Da for the active form. Cross-linking and sedimentation velocity results were consistent with the inactive form being a dimer. Partial thermal or chemical denaturation, or treatment with trifluoroethanol, readily activated dimeric DmpM. A combination of circular dichroism and fluorescence spectroscopies, activity assays, and native and urea gel electrophoresis were used to further characterize reactivation with urea. These results showed that dissociation of the dimeric form of DmpM precedes denaturation at low protein concentrations and results in activation. The same concentration of urea that effects dissociation also converts the monomeric form to a different conformation.     

Genetic Analysis of Chromomere 3d4 in DROSOPHILA MELANOGASTER . II. Regulatory Sites for the Dunce Gene

Chromomere 3D4 of the X chromosome of D. melanogaster contains two genes, dunce (dnc) and sperm amotile (sam). Mutations in dnc cause defects in memory formation and female fertility and reduce or eliminate the activity of a cAMP-specific phosphodiesterase designated form II. A fine structure map of this region has been constructed showing the locations of two sam mutations, five dnc mutations and a newly identified locus designated control of fertility (cf) that acts in cis to regulate the female sterility phenotype of dnc. The two sam mutations are separated by 0.02 +/- 0.01 cM, the rightmost being located 0.08 +/- 0.02 cM to the left of the null mutation dncM11. A cluster of null and form II-defective dnc mutations is located 0.04 +/- 0.01 cM to the right of dncM11. The cf locus is 0.06 +/- 0.02 cM to the right of this cluster. The location of the dnc and cf sites identify a region of approximately 0.10 cM that is required for proper expression of dnc+. The dncCK mutation, associated with a reciprocal translocation between 3L and the X, exhibits reduced form II activity and female sterility. This translocation breakpoint has been mapped to the left of the dnc+ gene and is near the breakpoint of Df(1)N64j15 which also reduces expression of dnc+. The effect of these independent chromosomal breaks on the dnc+ gene suggests the existence of a site to the left of dnc+ that is also required for proper expression of the gene.     

MEMBRANE AFFINITIES AND SUBCELLULAR DISTRIBUTION OF THE DIFFERENT MOLECULAR FORMS OF CHOLINE ACETYLTRANSFERASE FROM RAT

Choline acetyltransferase from rat brain is present in three different molecular forms with isoelectric points at pH 7·4-7.6, 7·7-7·9 and 8·3. The three forms were identified in a highly purified enzyme preparation, in a preparation of synaptosomes and in a cyto-plasmic preparation from disrupted axons and perikarya (fraction S₃). The three molecular forms differed in their affinities for synaptosome membranes and for a cation exchange resin (CM-Sephadex C-50). The positive surface charges of the different molecular forms and their affinities for membranes correlated well with their isoelectric points. The molecular form with jsoelectric point 8·3 had the largest positive surface charge and the highest membrane affinity. On isoelectric focusing of an extract from rat brain synaptosomes, the molecular form with isoelectric point 8·3 formed a complex with a negatively charged compound, presumably a protein. A method was developed to remove this compound by treatment with DEAE-Sephadex or by precipitation with vinblastine. These procedures are similar to methods known to remove the neurotubular protein. The complex formation did not occur in fraction S₃.