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Citrullination is a protein PTM of arginine residues catalyzed by peptidylarginine deiminase. Protein citrullination has been detected in the CNS and associated with a number of neurological diseases. However, identifying citrullinated proteins from complex mixtures and pinpointing citrullinated residues have been limited. Using RP LC and high-resolution MS, this study determined in vitro citrullination sites of glial fibrillary acid protein (GFAP), neurogranin (NRGN/RC3), and myelin basic protein (MBP) and in vivo sites in brain protein extract. Human GFAP has five endogenous citrullination sites, R30, R36, R270, R406, and R416, and MBP has 14 in vivo citrullination sites. Human NRGN/RC3 was found citrullinated at residue R68. The sequence of citrullinated peptides and citrullination sites were confirmed from peptides identified in trypsin, Lys-C, and Glu-C digests. The relative ratio of citrullination was estimated by simultaneous identification of citrullinated and unmodified peptides from Alzheimer's and control brain samples. The site occupancy of citrullination at the residue R68 of NRGN ranged from 1.6 to 9.5%. Compared to CID, higher-energy collisional dissociation (HCD) mainly produced protein backbone fragmentation for citrullinated peptides. CID-triggered HCD fragmentation is an optimal approach for the identification of citrullinated peptides in complex protein digests., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Myelin basic protein (MBP) is a commonly used substrate for in vitro determination of numerous protein kinase activities. Herein we describe a rapid method for isolating relatively large amounts of MBP from bovine brain with a purity greater than that currently available from commercial sources. Lipids were first extracted from the CNS tissue by homogenization in sec-butanol. Washes under neutral and mildly basic conditions were employed to remove neutral and acidic proteins from the defatted residue. MBP was subsequently extracted under acidic conditions and further purified by chromatography on CM Sephadex C-25. Potential contaminating enzyme activities were destroyed by heart treatment. This method typically yields a recovery of 1.0-1.5 mg MBP per gram of starting material with a purity of greater than 95%. The MBP prepared in this manner was suitable for determination of kinase activities by both solution and the "in gel" kinase assay systems., (Copyright 2000 Academic Press.)

The concurrent release of myelin basic protein (MBP) and extrinsic proteinases from isolated myelin membranes by aqueous solvents of high ionic strength is considered circumstantial evidence of a presumptive mutual interaction in situ. The joint release of proteins and proteinases from myelin membranes of bovine brain, depending on the ionic strength of aqueous solvents, was therefore examined; 25 mM Tris buffer released an average 1.4% of total myelin protein. It was attributable to about 25 different electrophoretic bands, but no apparent MBP. However, the extract potently mediated the limited proteolysis of added MBP at pH 4.0, 5.6, and 9.0. Because of the pH and the effects of specific inhibitors, proteolysis appears to be owing to activities of cathepsin B and D, and an alkaline metalloproteinase. The subsequent extraction of myelin membranes with buffered 300 mM NaCl released an additional 20% of total myelin protein, mainly MBP. The extracts, unlike those of untreated myelin membranes, no longer cleaved MBP at pH 5.6 and 9.0, and did so only slightly at pH 4.0. The results indicate that the bulk of soluble myelin-associated proteinases is much less tightly bound than MBP. The weak binding of the former and the prevalence of lysosomal cathepsin B- and D-like activities suggest that during their isolation, myelin membranes may adsorb soluble cellular proteins of tissue homogenates. At any rate the washing of myelin membranes with dilute buffer was found to largely remove soluble proteinase activities that are otherwise associated with salt-soluble MBP of myelin.

The multimeric clathrin assembly proteins AP-1 and AP-2 with molecular masses of approximately 270 kDa and the monomeric clathrin assembly proteins AP180 and auxilin with molecular masses of approximately 90 kDa catalyze the assembly of clathrin into artificial clathrin baskets under physiological conditions. We have now identified a much smaller approximately 20-kDa clathrin assembly protein in 0.5 M Tris, pH 7.0, extracts of bovine-brain coated vesicles and purified it to near homogeneity. A polyclonal antibody against this protein did not cross-react with any of the other assembly proteins, and sequencing data suggest that this new protein is similar or identical to myelin basic protein (MBP). At a molar ratio of 3 molecules per clathrin triskelion, MBP catalyzes polymerization of clathrin into artificial baskets that appear structurally similar to the baskets assembled by the other assembly proteins. In addition, like the other baskets, the clathrin-MBP baskets are uncoated by hsp70. MBP represents a significant fraction of the total assembly protein activity present in 0.5 M Tris, pH 7.0, extracts of coated vesicles. It is not clear if it acts as an assembly protein in vivo, but because it is well characterized and easily available, MBP will be a useful protein to investigate the mechanism of clathrin assembly and disassembly in vitro.

To study the role of HIV-1 gp120 in loss of myelin in HIV encephalopathy, the binding of gp120 to various types of neural cells and its effects on myelination were examined in rat primary brain culture. Double-staining of cultured cells with gp120 and specific antibodies for different neural cell types showed that gp120 bound to most of the galactocerebroside (GalC)-positive oligodendrocytes, a small population of type-2-like astrocytes and a few small neurons. Gp120 did not bind to type-1-like astrocytes, most neurons, or to macrophage/microglia. To assay myelination, cells were bathed in a myelination medium containing chick embryo extract and high glucose, with or without gp120. Seven days after the application, myelination in the culture was observed morphologically and by staining with anti-myelin basic protein (MBP) antibody, and was found to be significantly inhibited by the addition of gp120 (50-100 nM). The processes of oligodendrocytes were reduced in length and arborization relative to the control, but MBP production by oligodendrocytes was unaffected. These results show that gp120 can cause a functional disorder of oligodendrocytes and thus could underlie the diffuse loss of myelin sheaths of HIV encephalopathy.

The amounts of NG-methylarginine derivatives in myelin basic protein (MBP) purified from dysmyelinating mutant and different stages of normal myelinating mouse brains have been studied by using h.p.l.c. with a highly sensitive post-column o-phthaldialdehyde derivative-formation method. All three naturally occurring derivatives (NG-monomethylarginine (MeArg), NGN'G-dimethylarginine [Me2(sym)Arg] and NGNG-dimethylarginine [Me2(asym)Arg]) were found in MBP; however, their relative concentrations varied significantly with the age of the animal. The amounts of MeArg and Me2(sym)Arg in MBP increased as a function of the age of the brain, whereas that of Me2(asym)Arg decreased. MBP from early-myelinating mouse brain was shown to contain a high proportion of Me2(asym)Arg, which was hardly detectable in older brain MBP. This derivative, Me2(asym)Arg, was also absent from MBP embedded in the most compact multilamellar myelin, but was present in MBP in the least compact myelin (P3B). Comparing the extent of total methylation in vivo (sum of all three arginine derivatives), MBP extracted from less-compact myelin (P3A and P3B) showed a level approx. 40% higher than that from compact myelin. MBPs isolated from dysmyelinating mutant mouse brains, such as jimpy (jp/y) and quaking (qk/qk), contained a much higher level of Me2(asym)Arg relative to the other two methyl derivatives and also in comparison with those levels in the mother brain MBP. SDS/PAGE analysis of MBPs extracted from the mutant (both jp/y and qk/qk) as well as young normal (6-13 days old) mouse brains indicated the presence of a high-molecular-mass isoform of MBP (about 32 kDa), but this isoform was not found in adult brains. These results therefore indicate that structural integrity of myelin membrane in which MBP is embedded appears to play a pivotal role in determining the extent and the kind of Me2Arg formation in MBP at the post-translational level.

Sodium chloride extracts obtained from purified bovine brain myelin were found to contain proteolytic activity capable of degrading isolated myelin basic protein as assessed by SDS gel electrophoresis. Using gels copolymerized with gelatin as substrate, two bands at about 54 and 117-125 KDa, respectively, were detected. Activity corresponding to the 54 KDa band was inhibited by zinc. Data presented in this article suggest that proteolytic activity can be released from the myelin sheath in water-soluble form and recognize MBP as substrate.

Rat brain plasma membranes were solubilized in detergent and a glycoprotein-enriched fraction was obtained by lectin affinity chromatography. This glycoprotein fraction contained insulin receptors, as well as protein kinases capable of phosphorylating some exogenously added substrates such as MAP2 (microtubule associated protein 2) and MBP (myelin basic protein), but not ribosomal protein S6. Phosphoamino acid analysis of MAP2 and MBP showed that phosphotyrosine residues, as well as phosphoserine/phosphotheronine residues, were present in both proteins under basal conditions. Whereas the addition of insulin to the rat brain membrane glycoprotein fraction in vitro had no effect on MAP2 phosphorylation, MBP phosphorylation was stimulated 2.7-fold in response to insulin. This phenomenon was dose-dependent, with half-maximal stimulation of MBP phosphorylation observed with 2 nM insulin. Phosphoamino acid analysis of MBP indicated that insulin stimulated the phosphorylation of tyrosine residues nearly three-fold, whereas the phosphorylation of serine or threonine residues was not increased. These results identify MBP as a substrate for the rat brain insulin receptor tyrosine-specific protein kinase in vitro.

Myelin fractions with different degrees of compaction were isolated from bovine brain, and post-translational methylation of membrane-associated proteins was studied. When the purified myelin-basic-protein-specific protein methylase I and S-adenosyl-L-[methyl-14C]methionine were added exogenously, the most compact myelin fraction exhibited higher methyl-accepting activity than the less compact dense fractions. The methylated protein was identified as myelin basic protein (18.4 kDa) exclusively among the several myelin proteins from all membrane fractions, by SDS/PAGE/radioautography of methyl-14C-labelled membrane proteins. The methyl-14C-labelled amino acid residue in the basic protein was identified by h.p.l.c. as NG-methylarginine, indicating the high degree of specificity for the arginine residue as well as the myelin basic protein in the intact myelin membranes. The possibility of a charge alteration of myelin basic protein resulting from its arginine methylation was investigated by using the purified component 1 of myelin basic protein. The methylated component was shown to be less cationic than the unmethylated component by Bio-Rex 70 cation-exchange chromatography, since the former preceded the latter. However, in the presence of the denaturant (guanidinium chloride), the two species were co-eluted, indicating that the charge difference between methylated and unmethylated myelin basic protein can only be shown under the renatured condition.

The substrate specificity of bovine brain myelin basic protein (MBP)-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), which methylates arginine residues of protein, has been studied using various MBPs, several synthetic peptides and heterogeneous nuclear ribonucleoprotein complex protein (hnRNP). (1) Among MBPs from different species of brain, the carp MBP was found to be the best substrate for MBP-specific protein methylase I. This high degree of methyl acceptability is most likely due to the fact that carp MBP is not in vivo methylated at the arginine residue (Deibler, G.E. and Martenson, R.E. (1973) J. Biol. Chem. 248, 2387-2391) and that the methylatable amino acid sequence is present in this protein. (2) In order to study the minimum chain length of MBP polypeptide which functions as the methyl acceptor, several synthetic polypeptides whose sequences are identical to the region surrounding the residue 107 of bovine MBP (the in vivo methylation site) were synthesized. It was found that the hexapeptide, Gly-Lys-Gly-Arg-Gly-Leu (corresponding to residues 104-109 of bovine MBP), was the shortest methyl accepting peptide, while the tetrapeptide, Gly-Arg-Gly-Leu (corresponding to residues 106-109) was inactive as a substrate. (3) hnRNP protein is known to contain methylarginine at residue 193 (Williams, K.R., Stone, K.L., LoPresti, M.B., Merrill, B.M. and Plank, S.R. (1985) Proc. Natl. Acad. Sci. USA 82, 5666-5670) which is post-translationally modified. Thus, the RNP protein overproduced in Escherichia coli and therefore did not contain methylarginine was examined for its methyl acceptability. It was found that neither MBP-specific nor histone-specific protein methylase I could methylate this methylarginine-less RNP protein. This suggests a possible existence of a distinct protein methylase I specific for this nuclear protein.

Myelin basic proteins (MBPs) from 6-day-old, 10-day-old, 20-day-old and adult normal mouse brain were compared with those from 20-day-old jimpy (dysmyelinating mutant) mouse brain to determine the effect of reduced levels of proteolipid protein (PLP) on MBPs. Alkaline-urea-gel electrophoresis showed that 6-day-old and 10-day-old normal and jimpy MBPs lacked charge microheterogeneity, since C8 (the least cationic of the components; not be confused with complement component C8) was the only charge isomer present. In contrast, MBPs from 20-day-old and adult normal mouse brain displayed extensive charge microheterogeneity, having at least eight components. A 32 kDa MBP was the major isoform observed on immunoblots of acid-soluble protein from 6-day-old and 10-day-old normal and 20-day-old jimpy mouse brain. There were eight bands present in 20-day-old and adult normal mouse brain. Purified human MBP charge heteromers C1, C2, C3 and C4 reacted strongly with rat 14 kDa MBP antiserum, whereas the reaction with human C8 was weak. This suggested that MBPs from early-myelinating and jimpy mice did not react to MBP antisera because C8 was the major charge isomer in these animals. Purification of MBPs from normal and jimpy brain by alkaline-gel electrophoresis showed that both normal and jimpy MBPs have size heterogeneity when subjected to SDS/PAGE. However, the size isoforms in normal mouse brain (32, 21, 18.5, 17 and 14 kDa) differed from those in jimpy brain (32, 21, 20, 17, 15 and 14 kDa) in both size and relative amounts. Amino acid analyses of MBPs from jimpy brain showed an increase in glutamic acid, alanine and ornithine, and a decrease in histidine, arginine and proline. The changes in glutamic acid, ornithine and arginine are characteristic of the differences observed in human C8 when compared with C1.

MARPP(14-20), morphine- and cyclic AMP-regulated phosphoproteins of 14, 17-18, and 20 kDa, were identified originally by one-dimensional electrophoresis as a group of proteins whose state of phosphorylation was decreased by acute morphine and increased by chronic morphine in the rat locus coeruleus. We now show that MARPP(14-20) represent myelin basic proteins based on biochemical and immunochemical criteria. First, MARPP(14-20) were found to have isoelectric points of about 11 based on their migration on non-equilibrium pH gradient electrophoresis. Second, MARPP(14-20) were greatly enriched in myelin fractions of brain, and were not detectable, or present at very low levels, in other subcellular fractions of brain. Third, analysis of phosphorylated MARPP(14-20) by one- and two-dimensional gel electrophoresis demonstrated precise comigration with immunolabeled myelin basic proteins. In contrast, MARPP(14-20) were distinguished from histones, another group of low molecular weight, highly basic phosphoproteins, in these subcellular fractionation and immunochemical studies. Finally, we confirmed using two-dimensional electrophoresis, that changes observed previously by one-dimensional electrophoresis in the phosphorylation of MARPP(14-20) in response to acute and chronic morphine, and to acute forskolin, occur in myelin basic proteins. It was also found that changes in the state of phosphorylation of myelin basic proteins in response to chronic morphine occur without a change in the total amount of the proteins as determined by immunoblot analysis. The results demonstrate that the phosphorylation of myelin basic proteins is regulated by morphine in the nervous system, and raise the possibility that regulation of these proteins contributes to mechanisms underlying some of the acute and chronic actions of opiates in brain.

We report (a) that the shiverer mutation has pleiotropic phenotypic effects on myelin basic protein expression in the CNS of homozygous (shi/shi) mice and (b) that each of the effects of the shiverer allele is expressed co-dominantly with the wild-type allele in heterozygous (+/shi) animals. First, the total amount of myelin basic protein, as determined by radioimmunoassay, that accumulates in the CNS is approximately 0.1% of the wild-type amount in shi/shi animals and approximately 50% in +/shi animals. Second, the four major forms of myelin basic protein, with molecular weights of 21,500, 18,500, 17,000, and 14,000, that are present in wild-type mouse CNS are undetectable in either whole brain or purified myelin of shi/shi animals, and each of the four proteins is reduced commensurately in brain and myelin of +/shi animals. Third, the small amount of myelin basic protein-related material that does accumulate in the shi/shi brain consists of several polypeptides, with molecular weights ranging from 25,000 to 100,000, the pattern of which is different from that found in wild-type brain. The pattern of myelin basic protein-related polypeptides in +/shi brain is a composite of the wild type and the shiverer mutant. Fourth, messenger RNA from shi/shi brain, when translated in vitro, encodes a set of myelin basic protein-related polypeptides qualitatively similar to that encoded by wild-type messenger RNA, except that the 18,500 and 14,000 translation products are greatly reduced, while other myelin basic protein-related translation products are spared. The pattern of myelin basic protein-related translation products for +/shi messenger RNA is intermediate between the patterns for +/+ and shi/shi messenger RNAs. The results suggest that the genetic lesion in the shiverer mutation impinges on the structural gene (or genes) encoding myelin basic protein or on a cis-acting regulatory element controlling that gene (or genes).

Examination of 15-day-old rabbit brain myelin proteins by sodium dodecyl sulfate-slab gel electrophoresis revealed the presence of a basic protein with a molecular weight of 21,000 (21K protein) which was not previously reported in this species. This protein exhibited characteristic bluish green color with amido black and gave an amino acid composition strikingly similar to large basic protein (LBP). It formed a line of identity with LBP when diffused against antiserum to chicken brain basic protein. The concentration of LBP (18.9 micrograms/mg of dry myelin) is 6-fold greater than that of the 21K protein(3.31 micrograms/mg of dry myelin) in rabbit brain myelin. After the intracerebral injection of [32P]orthophosphoric acid, both LBP and 21K protein were found to be phosphorylated. [32P]Phosphate in the purified preparations of these proteins was covalently linked by phosphoester bonds to serine and threonine residues. The specific radioactivity of the 21K protein (84,693 cpm/mg of protein) was not significantly higher than LBP (69,797 cpm/mg of protein).

Human and rat myelin preparations were incubated with varying concentrations of trypsin and plasmin to determine the effects of these proteolytic enzymes on myelin-associated glycoprotein (MAG), basic protein, and other myelin proteins and to compare the effects with those of the neutral protease that was reported to be endogenous in myelin. Basic protein was most susceptible to degradation by both trypsin and plasmin, whereas MAG was relatively resistant to their actions. Under the assay conditions used, the highest concentrations of trypsin and plasmin degraded greater than 80% of the basic protein but less than 30% of the MAG, and lower concentrations caused significant loss of basic protein without appreciably affecting MAG. Neither trypsin nor plasmin caused a specific cleavage of MAG to a derivative of MAG (dMAG) in a manner analogous to the endogenous neutral protease. Thus the endogenous protease appears unique in converting human MAG to dMAG much more rapidly than it degrades basic protein. MAG is slowly degraded along with other proteins when myelin is treated with trypsin or plasmin, but it is less susceptible to their action than is basic protein.

Radioligand binding studies with the water-soluble cannabinoid [3H]5'-trimethylammonium delta 8-tetrahydrocannabinol ([3H]TMA) have revealed a saturable high-affinity site in brain that is specific for cannabinoids. To determine whether endogenous compounds of brain might act upon the site physiologically, we sought inhibitors in extracts of brain. An endogenous inhibitor has been purified to homogeneity by acid extraction of rat brain followed by adsorption to a reverse-phase matrix and gel filtration chromatography. The purified inhibitor has a subunit molecular mass of 14,500 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of [3H]TMA binding by the purified inhibitor occurs with a Ki of about 4 nM in a noncompetitive manner. The molecular weight, abundance, and extraction properties are the same as a species of myelin basic protein (MBP). The MBPs of rat, rabbit, pig, and cow also inhibit [3H]TMA binding noncompetitively with similar potencies. The purified inhibitor comigrates with rat MBP-small form on SDS-PAGE, has a similar amino acid composition, and is recognized by antibody directed against MBP. Studies of fragments of rabbit MBP suggest that the determinants of affinity for the [3H]TMA site are contained primarily within the C-terminal half of the rabbit MBP. Synthetic polycationic peptides such as polylysine and polyarginine mimic the effects of MBP, suggesting that the high-affinity cannabinoid binding site recognizes large polycations. The identification of the endogenous inhibitor of [3H]TMA binding as MBP suggests that MBP interacts physiologically with the high-affinity cannabinoid site.

Myelin basic protein (MBP) isolated from bovine white matter is obtained as a mixture of molecules which can be separated by cation-exchange chromatography at basic pH into three or more charge isomers. The three principal charge isomers of the microheterogeneous myelin basic protein have been isolated, and compared individually by high-resolution H NMR spectroscopy (360 and 400 MHz). In addition to confirming sources of MBP charge microheterogeneity such as fractional deamidation of Gln and loss of C-terminal Arg, NMR difference and spin-echo spectra further suggested (i) the presence of significant oxidation of (both) MBP Met residues to methionine sulfoxide; (ii) the three charge isomers contain equal ratios and absolute contents of mono- and dimethylated Arg; and (iii) the most-cationic isomer is deficient in its content of a putative extra Ala residue vs the other two isomers. Spectral analysis suggested that each MBP charge isomer is itself not a unique molecule, but more likely a mixture of molecules of equal net charge which are modified at any of the indicated functional side chains throughout the 169-residue protein. The results are discussed with respect to the possible consequences of MBP microheterogeneity to protein conformation and function.

Northern blot and "dot" blot analyses using a myelin basic protein (MBP) specific cDNA probe and in vitro translation techniques were utilized to estimate the relative levels of myelin basic protein messenger RNA (mRNA) in the brains of C57BL/6J control mice, three dysmyelinating mutants (qk/qk, jp/Y, and shi/shi), and three heterozygote controls (qk/+, jp/+, and shi+) during early postnatal development. In general, the MBP mRNA levels measured directly by Northern blot and "dot" blot analyses correlated well with the indirect in vitro translation measurements. The Northern blots indicated that the size of MBP mRNAs in quaking and jimpy brain polysomes appeared to be similar to controls. Very low levels of MBP mRNAs were observed in shi/shi brain polyribosomes throughout early postnatal development. Compared to C57BL/6J controls, accumulation of MBP mRNAs in qk/qk and qk/+ brain polyribosomes was delayed by several days. That is, whereas MBP mRNA levels were below normal between 12 and 18 days, normal levels of message had accumulated in both qk/qk and qk/+ brain polyribosomes by 21 days. Furthermore, normal levels of MBP mRNAs were observed to be maintained until at least 27 days. MBP mRNA levels remained well below control levels in jp/Y brain polyribosomes throughout early postnatal development. The levels did, however, fluctuate slightly and peaked at 15 days in both jp/Y and jp/+ brains, 3 days earlier than in normal mice. Thus, it appears that jimpy and quaking mice exhibit developmental patterns of MBP expression different from each other and from C57BL/6J control mice.

Myelin was purified from the spinal cords of normal mice and mice heterozygous for the shiverer mutation, and measurements were made of the major myelin proteins and lipids and the specific activities of three myelin-associated enzymes. The myelin purified from the spinal cords of the heterozygotes (shi/+) was deficient by 30-40% in yield and had an apparently unique composition. In particular, when compared with normal mouse spinal cord myelin, there were more high-molecular-weight protein, less myelin basic protein, a higher protein-to-lipid ratio, and higher specific activities of 2',3'-cyclic nucleotide-3'-phosphohydrolase (EC 3.1.4.37) and carbonic anhydrase (EC 4.2.1.1) in the myelin purified from the shi/+ animals. These abnormalities were reflected in the composition of shi/+ whole spinal cord, where the protein-to-lipid ratio was intermediate between the respective values for +/+ and shi/shi spinal cords. Whole brains from shi/+ mice showed deficiencies in galactocerebroside and galactocerebroside sulfate and an increase in total phospholipid, and the lipid composition in the brains of the shi/shi mice was similar to that reported for another dysmyelinating mutant, quaking. The findings provide the first values for the lipids in normal mouse spinal cord myelin and show that heterozygotes are affected by the shiverer mutation. The observations imply that there can be considerable deviation from the normal CNS myelin content and composition without apparent qualitative morphological abnormalities or loss of function and that the amount of myelin basic protein available during myelination may influence the incorporation of other constituents into the myelin membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

The charge isoforms (C1-C5) of bovine myelin basic protein (MBP) were used as substrates for the rat brain enzyme protein carboxylmethyltransferase (PM II). The objective of these experiments was to ascertain whether the kinetic behavior of the MBP isoforms reflected differences in the structures of this molecular family. Initial velocity plots as a function of the MBP-isoform concentration showed significant differences (p less than 0.05) among the assayed isoforms except for isoforms C2 and C4. Under the conditions of our experiment all the curves exhibited a consistent sigmoidicity. The kinetic data were best fitted by a model, previously described for the enzyme D-beta-hydroxybutyrate dehydrogenase, in which two independent sites must be randomly occupied before any catalytic activity can occur. This mechanism is substantially different from that proposed by other investigators for similar PM II enzymes and other substrates. The differences in the rates of isoform carboxylmethylation are largely accounted for by the different apparent dissociation constants Ks and is explained on the basis of inherent structural differences among the charge isoforms.