A new simple enzymatic assay method for urinary polyamines in humans.

We developed a new simple enzymatic assay method for measuring urinary polyamines (total amount of putrescine, spermidine, and cadaverine), using an acylpolyamine amidohydrolase and a putrescine oxidase. First conjugated polyamines (putrescine, spermidine, and cadaverine) in urine were hydrolyzed by incubation with an acylpolyamine amidohydrolase at 30 degrees for 1 hr. then, free polyamines were separated by cation-exchange chromatography and incubated with a putrescine oxidase at 30 degrees for 30 min. Hydrogen peroxide formed in this reaction was measured spectrophotometrically (at 514 nm). Polyamine levels in urine were determined in 70 normal subjects, 124 patients with cancer, and 52 patients with diseases other than cancer. Elevation above 3 S.D.s of the normal mean was found in 90 (72.6%) of the 124 patients with cancer and in 6 (11.5%) of the 52 patients with diseases other than cancer. Serial studies in 19 patients with cancer indicated that polyamines in urine were reduced after successful surgery. Our new method is simple and rapid and therefore very useful for routine clinical application. Moreover, our data indicate that the determination of polyamine levels is useful as a marker of disease activity in patients with cancer.

cDNA cloning of a novel brain-specific protein p25.

A cDNA for a bovine brain-specific protein p25 which had been originally found as a major protein in a partially purified fraction of tau protein kinases was cloned. The deduced amino-acid sequence consists of 218 amino acids (M(r) 23,472) and has no significant homology with previously reported proteins. p25 is a basic protein and has a consensus sequence for ATP-binding in the C-terminal region.

Tissue distribution of calcium-activated neutral proteinases in rat.

A simple separation method involving a minimum number of essential steps was established to separate the two kinds of calcium-activated neutral proteinase (CANP) activity from each other and from endogenous CANP inhibitor activity. Determination of CANP activity in rat tissues using this method revealed a ubiquitous distribution of two CANPs. The level of CANP activity, however, differs between tissues. Low-calcium-requiring CANP (microCANP) is plentiful in spleen and kidney, while high-calcium-requiring CANP (mCANP) is plentiful in lung and brain. In all of the tissues examined, mCANP activity predominates over microCANP activity.

Reactions of the aminoacyl-tRNA synthetase-aminoacyl adenylate complex and amino acid derivatives. A new approach to peptide synthesis.

Several kinds of dipeptide derivative were shown to be formed by the reactions of the aminoacyl adenylate-aminoacyl-tRNA synthetase complex and amino acid ester or amide. It was shown that the peptide bond could be formed by aminoacyl-tRNA synthetases even in the absence of the ribosome.

Studies on carbohydrate binding to a lectin purified from Streptomyces sp.

The anti-B specific lectin produced by Streptomyces sp. was shown to have two carbohydrate-binding sites with binding constants of 8.3 . 10(3) M-1 (15 degrees C) and 2.2 . 10(3) M-1 (4 degrees C) for L-rhamnose and D-galactose, respectively, calculated according to Scatchard plots. The binding of specific sugars to the lectin not only induced a peculiar ultraviolet difference spectrum showing a blue shift of tryptophan absorption, but also caused crystallization of the lectin at a concentration of 1 mg per ml or more. The solvent-perturbation studies on the lectin showed that the number of solvent-exposed tryptophan (or average extent of exposure) was two in the absence of L-rhamnose, and three in the presence of the sugar. This suggests that one tryptophan residue appears outside as the result of sugar-binding to the lectin, which is reflected by the difference spectra. Oxidation of two tryptophan residues with N-bromosuccinimide led to complete loss of carbohydrate-binding activity of the lectin, indicating that these residues are important for retaining the activity.

Effect of perfringolysin O on the lateral diffusion constant of membrane proteins of hepatocytes as revealed by fluorescence recovery after photobleaching.

Perfringolysin O is a thiol-activated cytolytic exotoxin the primary receptor of which is the membrane cholesterol on the cell surface. The effect of perfringolysin O was tested in various hepatocyte preparations. (i) Smears of fresh liver exposed to a mild H2O2 (1.0 mM) injury for 10 min at 37 degrees C, develop a 'peroxide-induced autofluorescence' (PIAF) on the membrane proteins. PIAF is suitable for measuring the average lateral diffusion constant (D) of the membrane proteins by means of fluorescence recovery after photobleaching technique (FRAP). Incubation for 5 min with 600 or 2000 units/ml of the perfringolysin O resulted in a significant increase (32 and 46%, respectively) of D as compared to the controls of the same age group (13-14 months). Various tests like heat denaturation of cholesterol saturation of perfringolysin O before its application as well as thiol-activation of the smears with dithiothreitol revealed that the increase of D is a specific toxin effect due mot probably to the reaction of perfringolysin O with cholesterol. (ii) Isolated hepatocytes were exposed to perfringolysin O and their viability as well as the release of two cytosolic enzymes (lactate dehydrogenase and glutamic-pyruvic transaminase) were measured; 40-60 units/ml of perfringolysin O in 30 min reduced the viability of the hepatocytes to zero and caused a release of about 70% of both cytosolic enzymes. The significance of the results is discussed from the points of view of both the toxin-effect and the FRAP method.

Characterization of tau phosphorylation in glycogen synthase kinase-3beta and cyclin dependent kinase-5 activator (p23) transfected cells.

One of the histopathological markers in Alzheimer's disease is the accumulation of hyperphosphorylated tau in neurons called neurofibrillary tangles (NFT) composing paired helical filaments (PHF). Combined tau protein kinase II (TPK II), which consists of CDK5 and its activator (p23), and glycogen synthase kinase-3beta (GSK-3beta) phosphorylate tau to the PHF-form in vitro. To investigate tau phosphorylation by these kinases in intact cells, the phosphorylation sites were examined in detail using well-characterized phosphorylation-dependent anti-tau antibodies after overexpressing the kinases in COS-7 cells with a human tau isoform. The overexpression of tau in COS-7 cells showed extensive phosphorylation at Ser-202 and Ser-404. The p23 overexpression induced a mobility shift of tau, but most of the phosphorylation sites overlapped the endogenous phosphorylation sites. GSK-3beta transfection showed the phosphorylation at Ser-199, Thr-231, Ser-396, and Ser-413. Triplicated transfection resulted in phosphorylation of tau at 8 observed sites (Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-396, Ser-404, and Ser-413).

Possible role of tau protein kinases in pathogenesis of Alzheimer's disease.

Tau protein kinases (TPK) I and II were isolated as candidate enzymes responsible for the hyperphosphorylation observed in PHF-tau. Four phosphorylation sites of tau were identified for each kinase, accounting for most, but not all, of the major phosphorylation sites of PHF-tau. Immunostaining with anti-TPKI antibody indicated that this kinase is up-regulated in AD brain. Such up-regulation of TPKI and phosphorylatioin of tau were reproduced by treating cultured hippocampal cells with amyloid beta (Abeta) protein. In addition, we found that TPKI can phosphorylate and inactivate pyruvate dehydrogenase (PDH), which is expected to result in depletion of acetyl-CoA, a key substrate of acetyl choline synthesis. Indeed, when septum cells were treated with Abeta, the level of acetyl choline decreased dramatically.

Cloning of cDNA and expression of the gene encoding rat presenilin-2.

We have cloned the rat homologue of the presenilin-2 (PS-2) cDNA. PS-2 is responsible for chromosome 1-linked familial Alzheimer's disease. Sequence analysis predicted that the rat PS-2 encodes a 448 amino acid (aa) protein, and there was a very high degree of amino acid identity between rat and human PS-2 (95%). All the mutated codons in PS-2 and PS-1 in chromosome 1- or 14-linked familial Alzheimer's disease patients were conserved in rat PS-2. The expression of PS-2 was weaker than that of PS-1. The alternatively spliced short form of PS-2 mRNA, which was detected in human tissues was not detected in various rat tissues. During brain development, the expression level of both PS-2 and PS-1 increased but decreased in the adult. No remarkable change was observed in neural differentiation of PC12 cells.

A cdc2-related kinase PSSALRE/cdk5 is homologous with the 30 kDa subunit of tau protein kinase II, a proline-directed protein kinase associated with microtubule.

We previously reported that tau protein kinase II (TPKII) from bovine brain was composed of 30 kDa and 23 kDa subunits. The 30 kDa subunit of TPKII can be regarded as a catalytic subunit because of its ATP-binding activity. Antibodies directed against TPKII-phosphorylated tau also reacted with tau phosphorylated by cdc2 kinase obtained from starfish oocytes, indicating that TPKII and cdc2 kinase phosphorylate the same sites. We determined the amino acid sequence of the 30 kDa subunit and found it to be homologous with a cdc2-related kinase, PSSALRE/cdk5. Moreover, an antibody against PSSALRE/cdk5 reacted with the 30 kDa subunit. These results indicate that the 30 kDa subunit of TPKII is bovine homologue of PSSALRE/cdk5. Expression of the 30 kDa subunit mRNA was enhanced in juvenile rat brain. This result supports our previous hypothesis that the kinase works actively in juvenile brain.

Glycogen synthase kinase 3 beta is identical to tau protein kinase I generating several epitopes of paired helical filaments.

We previously reported that tau protein kinase I (TPKI) induced normal tau protein into a state of paired helical filaments (PHF); this is further confirmed here by immunoblot analysis using several antibodies. We also present the amino acid sequence of TPKI, which is identical to glycogen synthase kinase 3 beta (GSK3 beta). Moreover, we found that TPKI activity was inseparable from GSK3 activity throughout the purification procedure. These results indicate that TPKI is identical to GSK3 beta.

A fragment of an endogenous inhibitor produced in Escherichia coli for calcium-activated neutral protease (CANP) retains an inhibitory activity.

A C-terminal fragment of an endogenous rabbit liver inhibitor for calcium-activated neutral protease (CANP) was produced in Escherichia coli and its inhibitory activity was examined after purification. The truncated inhibitor (373 amino acid residues), which contains two internal repeat structures, inhibits 2 mol CANP whereas the native liver inhibitor (639 residues), containing four internal repeat structures, inhibits 4 mol CANP. This supports the hypothesis that the repeating unit is the functional unit of inhibition. The results also indicate that post-translational modification of the inhibitor is not essential for inhibition.

Precursor of cdk5 activator, the 23 kDa subunit of tau protein kinase II: its sequence and developmental change in brain.

Tau protein kinase II (TPKII) is shown by immunoprecipitation to be a complex composed of two subunits, a catalytic subunit, cdk5, and regulatory subunit, p23. By sequence analysis of p23 cDNA, p23 was found to occupy a region from the 99th amino acid residue to the C-terminus of a novel protein with a molecular weight of 34,000 Da, suggesting that this 34 kDa protein is a precursor of p23 (pre-p23). These findings suggest that p23 results from the processing of the precursor protein, pre-p23. The precursor mRNA was expressed most abundantly in rat brain just before and after birth. Expression of pre-p23, but not of cdk5, mRNA changed, coinciding with the developmental change of TPKII activity, suggesting that its expression controls the phosphorylation of tau by the TPKII/TPKI system in the neonatal brain. p23 appears to be a cdk5 activator in neuronal cells.

Identification of the 23 kDa subunit of tau protein kinase II as a putative activator of cdk5 in bovine brain.

Tau protein kinase II (TPKII) was reported previously to be composed of a neuron-rich cdc2-related kinase (PSSALRE/cdk5) and 23 kDa subunit. Here we show that the 23 kDa subunit is a putative activator for the kinase activity. Amino acid sequence analysis revealed that the protein was novel and included a partial similarity of amino acids to a cyclin box important for the interaction with cdc2-related kinase. These results suggest that the 23 kDa subunit, but not cyclin, activates cdk5 in neuronal cells, which no longer exhibit cell cycling but are terminally differentiated cells.

A novel brain-specific 25 kDa protein (p25) is phosphorylated by a Ser/Thr-Pro kinase (TPK II) from tau protein kinase fractions.

A novel brain-specific 25 kDa protein (p25) was purified from a bovine brain extract. The protein was phosphorylated by Ser/Thr-Pro kinase (TPK II) in tau protein kinase fractions at the Ser residues of Ser-Pro sequences. Using immunoblot analysis, the protein was found only in brain extracts, and was most abundant in the brain regions such as cerebrum and hippocampus, but less abundant in cerebellum, medulla oblongata and olfactory bulb. The protein was detected in rat, bovine and human brain extracts, indicating that this protein specifically exists in mammalian brain tissues.

Characterization of human presenilin 1 using N-terminal specific monoclonal antibodies: Evidence that Alzheimer mutations affect proteolytic processing.

The majority of cases of early-onset familial Alzheimer disease are caused by mutations in the recently identified presenilin 1 (PS1) gene, located on chromosome 14. PS1, a 467 amino acid protein, is predicted to be an integral membrane protein containing seven putative transmembrane domains and a large hydrophilic loop between the sixth and seventh membrane-spanning domain. We produced 7 monoclonal antibodies that react with 3 non-overlapping epitopes on the N-terminal hydrophilic tail of PS1. The monoclonal antibodies can detect the full-size PS1 at Mr 47000 and a more abundant Mr 28000 product in membrane extracts from human brain and human cell lines. PC12 cells transiently transfected with PS1 constructs containing two different Alzheimer mutations fail to generate the 28 kDa degradation product in contrast to PC12 cells transfected with wild-type PS1. Our results indicate that missense mutations in this form of familial Alzheimer disease may act via a mechanism of impaired proteolytic processing of PS1.

Age-estimations of rats based on the average lateral diffusion constant of hepatocyte membrane proteins as revealed by fluorescence recovery after photobleaching.

A method has been developed recently for measuring the average lateral diffusion constant of the proteins (D) in the cell membrane of hepatocytes in liver smears by fluorescence recovery after photobleaching (FRAP). A peroxide-induced autofluorescence (PIAF) of the membrane proteins was used as a fluorescent label. It has been established that D displays a significant negative linear correlation with age. The present paper describes age-estimations carried out on 12 male Fischer 344 rats (7-29 months of age) in so-called "blind experiments": the operator knew only the sex of the rat, determined D from a small piece of the freshly removed liver, and estimated the age of the rat from the age-dependent regression line for D established previously on 16 other Fischer 344 male rats of various ages. There was a strong correlation of the estimated age with the actual one (r = 0.92), the slope of the regression line was 0.98 and its intercept differed from 0 by only 0.5 months. These results indicate that D may play a decisive role in the determination of membrane functions as predicted by the membrane hypothesis of aging.

Studies on histone oligomers. V. Reconstitution of chromatin from purified DNA and acid-extracted histones.

DNA-histone complexes were reconstituted from DNA and acid-extracted core histones and the products were characterized by micrococcal nuclease digestion to examine whether proper nucleosome structure had been reconstituted. No nucleosome structure was produced starting from the mixture of acid-extracted histones and purified DNA in 2 M NaCl-5 M urea, while the reassociation of chromatin by the same procedures was successful. This was due to the inappropriate conformation of acid-extracted histones, which was preserved in 2 M NaCl even in the presence of 5 M urea. If acid-extracted histones were reannealed from the completely denatured state, such as in 5 M urea, 6 M guanidine hydrochloride or 0.6 M NaCl-5 M urea, reconstitution of nucleosome structure was always successful.

Studies on histone oligomers. II. Reconstitution of heterotype histone oligomers and pH dependency of oligomer formation.

Histone oligomers were reconstituted by annealing from dissociated calf thymus whole histones in 2 M NaCl-5 M urea. The products were fractionated in terms of solubility in ammonium sulfate solution. The oligomers formed at pH 5 were heterotype histone oligomers (H2A . H2B . H3 . H4)n at high ionic strength, and heterotype histone tetramer (H2A . H2B . H3 . H4) at low ionic strength. The pH dependency of oligomer formation was determined. Heterotype oligomers were formed at pH 4-6 and homotype oligomers at pH 7-9.

Colicin E3 is an endonuclease.

It was confirmed by polyacrylamide gel electrophoresis that isolated 16S rRNA was cleaved by the active component (protein A) or the active fragment (T2A) of colicin E3. However, the degradation was random, in contrast with the specific cleavage observed in the interaction of colicin E3 with ribosomes. Furthermore, the active component and the active fragment had low activities, and far greater amounts of these materials were required for degradation of the isolated rRNA than for ribosome inactivation. The degradation of rRNA cannot be due to contaminating ribonuclease(s), but is due to colicin E3 itself, because of the following facts. (1) Protein B of colicin E3, which specifically inhibits the ribosome-inactivating activity of colicin E3, inhibited the degradation of rRNA. (2) Protein B of colicin E2, which inhibits the action of colicin E2 but not of colicin E3, failed to inhibit the degradation of rRNA. (3) The activity appeared in the peak of protein A or fragment T2A, respectively, when they were rechromatographed on Sephadex G-75.