A Simple and Rapid Method for the Purification of Ovine Pineal Arylalkylamine N-Acetyltransferase

A two-step chromatographic procedure has been developed for the purification of ovine pineal arylalkylamine N-acetyltransferase (EC 2.3.1.87), based on the principles of disulfide exchange and anion exchange. The enzyme from 20 ovine pineal glands can be purified about 500-fold in a day; recovery is about 5%. Polyacrylamide gel electrophoretic analysis of the final preparation shows four major bands; one appears to be arylalkylamine N-acetyltransferase.     

Use of charcoal adsorption for the microassay of acetyl-CoA-hydrolase.

A sensitive and rapid radiochemical micromethod is described for measuring the activity of acetyl-CoA hydrolase (EC 3.1.2.1). [1-¹⁴C]Acetyl-CoA is incubated with tissue homogenates; unhydrolyzed [1-¹⁴C]acetyl-CoA is separated from the radiolabeled product, [1-¹⁴C]acetate, by adsorption to charcoal. The soluble [1-¹⁴C]acetate is measured by liquid scintillation techniques. This procedure makes it possible to measure as little as 0.2 to 0.4 nmol acetate generated per assay.     

Activation of pineal and brain acetyl-COA hydrolase by cystamine: an apparent case of disulfide exchange.

Rat pineal acetyl-CoA hydrolase was activated about 5-fold by cystamine treatment (30 mM) at pH 6.8 and 10-fold at pH 8.5. Six other disulfides were found to be ineffective or to produce a small activation. Cystamine activation was not reversed when free cystamine was removed, but was reversed by treatment with DTT. Analysis of other tissues indicated acetyl-CoA hydrolase from rat brain, sheep pineal gland and chicken pineal gland could also be activated by cystamine. In contrast, cystamine activation of rat liver acetyl-CoA hydrolase was not seen.     

Mutagenesis of p38alpha MAP kinase establishes key roles of Phe169 in function and structural dynamics and reveals a novel DFG-OUT state

In order to study the role of Phe169 in p38alpha MAP kinase structure and function, wild-type p38alpha and five p38alpha DFG motif mutants were examined in vitro for phosphorylation by MKK6, kinase activity toward ATF2 substrate, thermal stability, and X-ray crystal structure. All six p38alpha variants were efficiently phosphorylated by MKK6. However, only one activated p38alpha mutant (F169Y) possessed measurable kinase activity (1% compared to wild-type). The loss of kinase activity among the DFG mutants may result from an inability to correctly position Asp168 in the activated form of p38alpha. Two mutations significantly increased the thermal stability of p38alpha (F169A DeltaTm = 1.3 degrees C and D168G DeltaTm = 3.8 degrees C), and two mutations significantly decreased the stability of p38alpha (F169R DeltaTm = -3.2 degrees C and F169G DeltaTm = -4.7 degrees C). Interestingly, X-ray crystal structures of two thermally destabilized p38alpha-F169R and p38alpha-F169G mutants revealed a DFG-OUT conformation in the absence of an inhibitor molecule. This DFG-OUT conformation, termed alpha-DFG-OUT, is different from the ones previously identified in p38alpha crystal structures with bound inhibitors and postulated from high-temperature molecular dynamics simulations. Taken together, these results indicate that Phe169 is optimized for p38alpha functional activity and structural dynamics, rather than for structural stability. The alpha-DFG-OUT conformation observed for p38alpha-F169R and p38alpha-F169G may represent a naturally occurring intermediate state of p38alpha that provides access for binding of allosteric inhibitors. A model of the local forces driving the DFG IN-OUT transition in p38alpha is proposed.     

VEGETATIVE NUCLEAR DIVISION IN NEUROSPORA

A re-examination of the mode of vegetative nuclear division in Neurospora crassa was facilitated by the availability of the mutant “clock” which produces definite growth bands. Since the growth rhythm is correlated with nuclear divisions, stained mycelial mats of this mutant prepared at intervals from the beginning of a growth period provided a sequence of stages of division. In a 28-hour period the following broad features of nuclear behavior were observed: In the early part of the period during rapid mycelial growth, dividing elongated nuclei predominated. At the end of the period the mycelium contained mostly rounded resting nuclei. In the middle of a growth period nuclear forms of various degrees of annularity occurred along with elongated and rounded nuclei. Elongated and rounded nuclei completed division cycles without change in form, although the corresponding stages of the two types were similar. Elongated nuclei assumed a spiral form at the beginning of division. As division proceeded, relaxation of the nuclear gyres was accompanied by a visible duplication of the chromatin thread and the appearance of chromomere-like bodies on the daughter threads. One of the chromomere-like bodies became displaced and was interpreted to be a chromosome or a segment of a chromosome that acts as a mitotic center. All the chromosomes were found to be interconnected and to act as a unit throughout the division cycle. Only after the separation of the daughter chromatin threads could seven chromosomes be counted. Electron microscopic studies complemented the observations with the light microscope. On the basis of the evidence it was concluded that the vegetative nuclear division in Neurospora differs from the classical mitotic pattern in the following respects: (1) absence of visible centrioles, (2) the presence of interconnected chromosomes, (3) the comparatively late appearance of countable chromosomes, and (4) the frequent presence of interzonal connections between separating chromatin threads.     

Segregation and linkage analyses of dopamine-beta-hydroxylase activity.

Dopamine-beta-hydroxylase (DBH) activity in serum was measured by spectrophotometric methods in 95 persons of a large family (HGAR 2), along with 27 polymorphic markers from blood, urine and saliva. The distribution of DBH activity, after appropriate transformation and age adjustment, showed a significantly better fit to a mixture of two normal distributions than a single normal distribution. Pedigree segregation analyses showed evidence of a possible major gene governing low levels of DBH activity, segregating in this family in a recessive fashion. Linkage analyses between that major locus and the 27 polymorphic markers showed no significant lod scores favoring linkage. The highest lod score obtained was 0.81 with Lp at zero recombination fraction. In addition, published data on DBH activity measured by radiochemical assays on 22 families with 161 members were reanalyzed as a quantitative trait, with appropriate correction for ascertainment bias. The results were similar to that of HGAR 2, corroborating the existence of a major locus for DBH activity.     

Rapid and reversible activation of acetyl CoA hydrolase in intact pineal cells by disulfide exchange

Using intact pinealocytes in suspended cell culture it has been determined that acetyl CoA hydrolase activity can be rapidly increased by treatment with cystamine. Similar results are seen with diacetylcystamine, but not with GSSG, penicillamine disulfide, nor with oxidized DTT. The activation of acetyl CoA hydrolase by cystamine is reversible: after cystamine treatment is terminated, enzyme activity decreases slowly in cell culture. It is also possible to reverse the activation by treating homogenates of cystamine-treated cells with dithiothreitol. These observations are consistent with previous findings indicating that pineal acetyl CoA hydrolase activity can be regulated $\text{via}$ protein thiol: disulfide exchange. The observations presented in this report also indicate that conditions within the cell allow this type of reaction to take place, and raise the possibility that disulfide exchange mechanisms may be physiologically involved in the intracellular regulation of the activity of this and perhaps other enzymes.     

Linkage relationships of Lp and Ag serum lipoproteins with 25 polymorphic markers

Summary Linkage relations of Lp and Ag serum lipoproteins with 25 polymorphic marker systems are examined in a large kindred of over 100 persons. The results indicate that Lp and ESD are probably closely linked and so the Lp locus may also be assigned to chromosome 13. No significant linkage is detected between Ag and the other marker systems.This research was supported by: a Public Health Service Research Career Development Award (1-K3-GM-31, 732) and research grant (GM 16697) from the National Institute of General Medical Sciences.