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We have found that the 14-3-3 protein, an acidic neuronal protein, is substantially identical to the 'activator' protein [(1981) J. Biol. Chem. 256, 5404-5409] that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca 2 +,calmodulin dependent protein kinase II. This finding is based on the remarkable similarity of both these proteins in physicochemical, biochemical and immunochemical properties, as welt as on detection for the 14-3-3 protein of an activator activity towards tryptophan 5-monooxygenase. The result suggests that the 14-3-3 protein plays a role in the regulation of serotonin and noradrenaline biosynthesis in brain.14-3-3 protein; Activator protein; Tryptophan 5-monooxygenase; Tyrosine 3-monooxygenase; Ca 2 +/calmodulin-dependent protein kinase; (Brain)

A cDNA encoding the homolog of the human pre-Bcell colony-enhancing factor (PBEF), a cytokine-like secreted protein, was isolated from a rat cDNA library. This protein existed in both the cytoplasm and nucleus of the cells, and the amount was higher in the cytoplasm than in the nucleus of proliferating PC-12 and Swiss 3T3 cells but higher in the nucleus than in the cytoplasm of the PC-12 cells treated with nerve growth factor and the 3T3 cells grown to a con£uent state. Thus, the so-called PBEF is not a cytokine-like secreted protein but an intracellular protein associated with the cell cycle.

Tryptophan 5-monooxygenase was purified approximately 5500-fold, to apparent homogeneity with a specific activity of 374 nmol min-' mg-' at 30 "C, from rat brain-stem using Sepharose CL-6B, DEAE-Sepharose CL-6B and pteridine-agarose chromatography. Two distinct active forms were separable by DEAE-Sepharose CL-6B and designated as form I and form I1 based on their order of elution from the gel column. The apparent molecular weight of form I was determined to be 300000 by gel filtration on Ultrogel AcA 34 and 288000 by gradient polyacrylamide gel electrophoresis. The enzyme gave a single band on sodium dodecylsulfate/polyacrylamide gel electrophoresis, the molecular weight of which was estimated to be 59 000, indicating that the enzyme might be composed of four identical subunits. The tetrameric structure of the enzyme was further suggested by crosslinking studies using dimethyl suberimidate as a bifunctional reagent. The enzyme activity was stimulated approximately 3.5-fold by the addition of Fe2+. Kinetic studies revealed that this activation was associated with an increase of V value. The purified enzyme had an activity of phenylakdnine hydroxylation but not an activity of tyrosine hydroxylation.Tryptophan 5-monooxygenase [L-tryptophan, tetrahydropteridine : oxygen oxidoreductase (5-hydroxylating)] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the initial and rate-limiting step in the biosynthesis of the neurotransmitter serotonin [l, 21. Although much effort has been devoted to purification and characterization of tryptophan 5-monooxygenase [3 -1 I], relatively little progress has been made because of the extreme lability of the enzyme with the standard purification procedure. Tong and Kaufman [I 11 reported that they obtained an almost homogenous (85 -90 % pure) preparation of tryptophan 5-monooxygenase from rabbit hindbrain with a 25 yield. However, the specific activity of their final preparation, 2.1 nmol min-' mg-' at 37 "C, was only 58-fold higher than that of the crude enzyme. Recently we purified the rabbit brain tryptophan 5-monooxygenase to a specific activity of 15.9 nmol min-' mg-' at 30 "C with a 29 % yield but it was still far from a homogenous preparation [12], suggesting that much higher purification was required to attain homogenous enzyme.In the present study tryptophan 5-monooxygenase was purified to a specific activity of 374nmol min-' mg-' at 30°C from rat brain-stem and some physical and catalytic properties of the enzyme were examined. This is the first demonstration of the purification of mammalian tryptophan 5-monooxygenase to an apparent homogeneity. EXPERIMENTAL PROCEDURE MaterialsEscherichia coli P-galactosidase, rabbit muscle aldolase rabbit muscle lactate dehydrogenase, beef liver catalase and horse Abbreviations. Me~PteH4, 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine; MePteH4, 2-arnino-4-hydroxy-6-methyltetrahydropteridine; Hepes, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid.Enzyme. Tryptophan 5-monooxygenase or tryptophan hydroxylase (EC ...

A synthetic peptide corresponding to the autophosphorylation site of Ca 2؉ /calmodulin-dependent protein kinase II (CaMKII) (residues 281-289) was conjugated to paramagnetic particles, and phosphorylated by a constitutively active CaMKII fragment. Using this phosphopeptide conjugate as a substrate, a calyculin A-insensitive, Mn 2؉ -dependent, and poly-L-lysine-stimulated protein phosphatase activity was detected in the crude extract of rat brain. The protein phosphatase (designated as CaMKII phosphatase) (CaMKIIPase) was purified to near homogeneity from rat brain. CaMKIIPase showed apparent molecular weights of 54,000 and 65,000, on SDS-polyacrylamide gel electrophoresis and gel-filtration analysis, respectively. It was not inhibited by 100 nM calyculin A or 10 M okadaic acid. Mn 2؉ , but not Mg 2؉ , was absolutely required for activity. CaMKIIPase was potently activated by polycations. Autophosphorylated CaMKII was dephosphorylated by CaMKIIPase, whereas phosphorylase kinase, mixed histones, myelin basic protein, and ␣-casein (which had been phosphorylated by cAMP-dependent protein kinase) and phosphorylase a (phosphorylated by phosphorylase kinase) were not significantly dephosphorylated. No other proteins than CaMKII in rat brain extract which had been phosphorylated by CaMKII were dephosphorylated. The stimulated Ca 2؉

The whole genome scan for quantitative trait loci (QTLs) specifying peak bone mass was performed with the F2 intercrosses of SAMP6, an established murine model of senile osteoporosis, exhibiting a significantly lower peak bone mass, and SAMP2, exhibiting a higher peak bone mass. Cortical thickness index (CTI), a parameter of bone mass of femurs, was measured in 488 F2 progeny at 4 months of age, when the animals attained peak bone mass by microphotodensitometry. Genetic markers were typed at 90 loci spanning all chromosomes except the Y. By interval mapping of 246 male F2 mice, two loci were identified with significant linkage to peak bone mass, one on Chromosome (Chr) 11 and another on Chr 13, with a maximum lod score of 10.8 (22.2% of the total variance) and 5.8 (10.0%), respectively. Another locus on the X Chr was suggestive of a QTL associated oppositely with a low peak bone mass to the SAMP2 allele. This association was consistent with the distribution of peak bone mass in the F1 and F2. These findings should be useful to elucidate the genetics of osteoporosis.