Occurrence of NI and NII type protein kinases in the nuclei from various tissues of the rat

Cyclic nucleotide-independent protein kinases that preferentially phosphorylated casein and phosvitin as substrate were surveyed in various tissue nuclei of the rat. Enzymes were extracted from the isolated nuclei of liver, kidney, spleen, brain, heart, or testis tissue with a buffer solution containing 0.4 m NaCl, and analyzed by DEAE-Sephadex, phosphocellulose, and Bio-Gel A-1.5m column chromatographies. The chromatographic study together with characterization of the enzymes demonstrated that all the tissues contained in their cell nuclei commonly two protein kinases, the NI and NII types, and that these were exclusively found as main nuclear casein kinases. NII enzyme activity was stimulated by polyamines and strongly inhibited by heparin. By contrast, the NI enzymes were little influenced by these compounds. We interpret the present results as suggesting that NI and NII type protein kinases may be found in the cell nuclei from many tissues of rat, and have distinct functions in the cell nuclei.     

Sodium-dependent activation of intestinal brush-border sucrase: Correlation with activation by deprotonation from pH 5 to 7

The activation of rabbit intestinal brush-border sucrase in the pH range 4.8 to 9.2 was studied as a function of sucrose concentration and temperature in a metal-free, n-butylamine universal buffer, both in the absence and in the presence of sodium. When sodium was absent, enzyme activation involved the simultaneous loss of two key protons (pK₁ of about 5.6), thus yielding a high-affinity, catalytically active enzyme conformation. Inactivation followed when a third key proton (pK₂ of about 8.4) was lost. When sodium was present, kinetic analysis in the pH range 4.8 to 7.2 revealed that sodium activation involves distinct effects on the two kinetic parameters, V_m and K_m. The V_m parameter seemed to conform to the classical rules of pH-dependent enzyme activation and implicated the release of a single proton whose apparent pK (pK_1y, about 5.6) was little affected by sodium. On the contrary, the K_m parameter was strongly influenced by sodium. Here, activation of rabbit sucrase seemed to involve release of a different proton whose apparent pK (pK_1x also of about 5.6 in the absence of sodium) was strongly shifted to more acid values by saturating sodium concentrations. The functional distinction between the above two protons explains the existence of strong affinity-type activating effects of sodium on rabbit sucrase, previously shown to be pH independent (F. Alvarado and A. Mahmood, 1979, J. Biol. Chem.254, 9534–9541).     

Neutral salt effects on the velocity and activation volume of the lactate dehydrogenase reaction: Evidence for enzyme hydration changes during catalysis

The effects of different neutral salts on the maximal velocity (V) and activation volume ($\text{Δ}\text{V3}$) of the M₄-lactate dehydrogenase reaction were studied to determine the mechanistic basis of the inhibitory effects of these salts. For salting-in salts (which increase protein group solubility), increasing salt concentrations led to reductions in V and increases in $\text{Δ}\text{V3}$, with the order of salt effectiveness following the Hofmeister (lyotropic) series: KSCN > KI > KBr. A 50% reduction in V was associated with an approximately 17 cm³ mol^?1 increase in $\text{Δ}\text{V3}$ for different concentrations of the same salt and for equal concentrations of different salting-in salts. Salting-out salts were also inhibitory, but no uniform correlation between changes in V and $\text{Δ}\text{V3}$ was observed. The strongly salting-out salt KF decreased $\text{Δ}\text{V3}$ at all concentrations. The weaker salting-out salt K₂SO₄ increased $\text{Δ}\text{V3}$ at concentrations below 0.1 m and decreased $\text{Δ}\text{V3}$ at higher concentrations. KCl increased $\text{Δ}\text{V3}$ as the salt concentration was raised to approximately 0.2 m; further increases in KCl concentration were without effect on $\text{Δ}\text{V3}$. The rate and volume effects of these neutral salts, especially the highly regular covariation in V and $\text{Δ}\text{V3}$ found for salting-in salts, seem difficult to explain in terms of salt-induced changes in the geometry of the active site. We propose instead that these salt effects can all be explained in terms of the energy and volume changes which accompany transfers of protein groups (amino acid side chains and peptide backbone linkages) between the hydrophobic interior of the enzyme and the enzyme-water interface during catalytic conformational changes.     

Mechanisms of polyclonal B-cell activation in autoimmune B6-lpr/lpr mice

The influence of the lpr gene on spontaneous and lipopolysaccharide (LPS)-induced immunoglobulin production was studied in B6 mice homozygous for the mutant lpr gene (B6-lpr/lpr). Male and female mice of this congenic strain were followed for 1 year and sera serially tested by the enzyme-linked immunosorbent assay (ELISA) for the production of antibodies to single-stranded DNA (anti-sDNA), immunoglobulin (anti-IgG), and keyhole limpet hemocyanin (anti-KLH), models of autoantibody and non-autoantibody responses, respectively. Female B6-lpr/lpr mice demonstrated marked spontaneous responses to all three antigens; the responses of male B6-lpr/lpr mice were significantly lower but still exceeded those of the congenic B6-+/+ controls. These results demonstrate a generalized influence of sex on lpr associated responses. To determine whether this sex difference could be demonstrated with other forms of B-cell activation, young B6-+/+ and B6-lpr/lpr male and female mice were immunized with lipopolysaccharide and the induced responses determined. This immunization caused significant increases in the IgM response only. The levels of the induced responses produced after LPS treatment were comparable for +/+ and lpr/lpr mice. These results indicate that the enhanced responsiveness of female mice to lpr action is not reflected in the polyclonal response to LPS, which, furthermore, was unaffected by the presence of lpr. The differential influence of sex on lpr and LPS-induced responses and their apparent independence suggests that lpr and LPS promote B-cell activation by dissimilar mechanisms.     

The cholinergic system in rat striatum during morphine tolerance and dependence

Male Sprague-Dawley rats were used in the present study to assess the effects of chronic treatment of morphine on the striatal cholinergic system. The results demonstrate that neither short nor long-term morphine treatment had an effect on choline acetyltransferase (ChAT) activity or ³H- quinuclidinylbenzilate (³HQNB) binding in discrete striatal regions of the rat brain.     

DNA Replication by a membrane-DNA complex from rat liver mitochondria

The results presented here indicate that mitochondrial DNA (mtDNA) synthesis occurs on the inner mitochondrial membrane and that a membrane-DNA complex, enriched in newly synthesized DNA, can be isolated. The complex is able to synthesize DNA in vitro. Enrichment studies demonstrated that mtDNA synthesis occurs on an intact membrane-DNA complex in vitro and that pulse-labeled mtDNA could be chased from the membrane-DNA complex to the top fraction of the discontinuous sucrose gradient. The membrane-DNA complex was also shown to carry out replicative synthesis of mtDNA in vitro. Replication was shown to be asynchronous with heavy-strand synthesis preceding light-strand synthesis. The progression of mtDNA replication by the membrane-DNA complex was shown to be from small fragments (<13 S) to larger fragments (14–24 S) liberated from closed circular molecules, to a heat-stable 27 S molecule, and finally to a 38 S heat-stable molecule. The time estimated to progress from small fragments to the 38 S molecule is 120 min.     

Cell lineage relationships in Drosophila melanogaster: The relationships of cuticular to internal tissues

We have studied cell lineages within several internal structures and have also studied the relationship of such internal lineages to cuticular structures. Such observations were carried out by coupling various cuticular markers with enzyme or morphological markers for internal tissues and using these in both somatic recombination and Minute-gynandromorph manipulations. We have further explored the relationships among cuticular and internal tissues by studying the impact of mutations of the bithorax gene complex on cuticular structures and the internal organs which underly them. In brief, we are unable to demonstrate the existence of “compartments” in the internal tissues examined; we have shown that the cuticular and internal structures examined in this study are, in most cases, not demonstrably related in a clonal sense. Additionally, we show that changes in the internal tissues examined here in response to mutations in the bithorax complex are either not detected by our methods of analysis or are very different from the well-characterized responses of the imaginal disc derivatives.     

The synthesis and subcellular distribution of pyridoxal phosphate in rat and bovine retinas

In the absence of any known studies dealing with status of vitamin B₆ metabolism in mammalian retinas, the concentration of pyridoxal phosphate and the activity of its synthesizing enzyme pyridoxal kinase were determined in rat retina and bovine retina and its subcellular compartments. In bovine retina, the highest concentration of pyridoxal phosphate (148 pmol/mg protein) was present in pellet 2 fraction containing synaptosomes comparable to those isolated from brain. The second highest concentration of pyridoxal phosphate (91 pmol/mg protein) was present in pellet 1 fraction containing large synaptosomes resembling photoreceptor cell terminals. The concentrations of pyridoxal phosphate in pellets 1 and 2 fractions were approx 3- to 6-fold higher than that found in the whole retina. The concentration of pyridoxal phosphate and the activity of pyridoxal kinase in the rat retina were considerably higher than those observed in the bovine retina. In general, no apparent correlation existed between the concentrations of pyridoxal phosphate and the activities of pyridoxal kinase in bovine retina and its subcellular compartments.     

Participation of mast cell 5-hydroxytryptamine in the vasoconstrictor effect of neurotensin in the rat perfused hindquarter

Neurotensin (NT) (1 X 10(-8) - 1.5 X 10(-6) g ml-1) caused a transient, dose-dependent increase in perfusion pressure in the rat perfused hindquarter. The vasoconstrictor effect of NT was associated with a short-lived, dose-dependent release of histamine and 5-hydroxytryptamine (5-HT) in the hindquarter effluent. Compound 48/80, a classical mast cell secretagogue, also elicited a vasoconstrictor effect in, and release of histamine from, the rat hindquarter. The vasoconstrictor effect and the release of histamine and 5-HT evoked by NT were much smaller in hindquarters derived from rats pretreated with compound 48/80 for 4 days to cause mast cell depletion than in hindquarters derived from control rats. The mast cell inhibitor cromoglycate (4 mg ml-1) inhibited by about 50% the histamine releasing effect and vasoconstriction produced by the lowest concentrations of NT utilized. The histamine releasing effect of compound 48/80 was more sensitive to blockade by cromoglycate than that of NT. The steroidal antiinflammatory and antiallergic drug dexamethasone did not affect the histamine and 5-HT releasing effect of NT. The vasoconstrictor effects of NT, compound 48/80 and 5-HT were markedly reduced by the 5-HT receptor antagonist methysergide (1 X 10(-7) g ml-1). Histamine (1 X 10(-6) - 10(-4) g ml-1) evoked a decrease in perfusion pressure in hindquarters pre-exposed to noradrenaline. The results suggest the participation of mast cell 5-HT in the vasoconstrictor effect of NT in the rat perfused hindquarter.     

Induced neurotransmitter release from primary cultures of rat brain neurons

Neural cells from fetal rat brain were grown in tissue culture in the absence of serum and maintained for 4–5 weeks without medium renewal. Over 80% of the embryonic cells in the culture had a neuronal appearance and formed intercellular synaptic connections. When mature, a definite population of the neuronal cells accumulated ³H-dopamine in a sodium-dependent, benztropine inhibited process. The mature cells were also able to release ³H-dopamine in a potassium evoked, calcium-dependent process, with half maximal dopamine release achieved at a Ca²⁺ concentration of 120μM. In the maturing cells the capacity for potassium evoked, calcium-dependent dopamine release increased from an undetectable level in the first three days to a plateau level after 10–11 days $\text{in vitro}$. The fully expressed release capacity (20–30% of the neurotransmitter retained in the cells) was maintained thereafter. These results demonstrate that primary brain neurons develop a functional neurosecretion apparatus in a chemically defined medium in the absence of animal serum. This extends the utility of primary cultures of brain neurons for developmental structural and biochemical studies of neurotransmission.     

Adrenalectomy-induced deficits in maternal retrieval in the rat

Previous studies have indicated that a functioning adrenal gland is not necessary for the expression of maternal behavior in the rat. In the present study, adrenalectomized mothers were found to take longer to initiate retrieval and to retrieve a smaller percentage of their litters on postpartum Days 1–4 than did control mothers. Possible causes of these deficits are discussed.     

Hepatic microsomal N-oxidation and N-demethylation of N,N-dimethylaniline in red-winged blackbird compared with rat and other birds.

Hepatic microsomes prepared from red-winged blackbirds ($\text{Agelaius phoeniceus}$) and albino rats were incubated with $\text{N}$,$\text{N}$-dimethylaniline (DMA)_in complete incubation mixtures at pH 7.9 and 37°C for 10 min. Formaldehyde and $\text{N}$,$\text{N}$-dimethylaniline-$\text{N}$-oxide produced from DMA were measured. Redwings were found to have significantly lower $\text{N}$-demethylation activities than rats, and redwings had only marginal or no $\text{N}$-oxidation activities. Hepatic microsomes from redwings did not further metabolize the $\text{N}$-oxide. The $\text{N}$-oxidation and $\text{N}$-demethylation activities of brown-headed cowbirds ($\text{Molothrus ater}$), common grackles ($\text{Quiscalus quiscula}$), and starlings ($\text{Sturnus vulgaris}$) were similar to those of redwings.     

Purification of S-2-hydroxyacylglutathione hydrolase (glyoxalase II) from rat erythrocytes.

Glyoxalase II, a specific glutathione thiolesterase, has been purified 9100-fold from rat erythrocytes using a purification scheme which employs Affi-Gel blue as a hydrophobic affinity column and also employs a glutathione-affinity column prepared by coupling S-(p-chlorophenacyl)glutathione to Affi-Gel 202. This procedure offers a convenient method for the preparation of highly purified glyoxalase II. Also described is a convenient method for the preparation of S-lactoyl-glutathione, a substrate for glyoxalase II.     

Use of liquid chromatography with electrochemistry to measure effects of varying intensities of white light on DOPA accumulation in rat retinas

Exposure of dark-adapted rats to light enhances the activity of the retinal dopamine (DA) neurons. The purpose of this study was to determine if the response of these neurons to light varies with different intensities of light. The accumulation of dihydroxyphenylalanine (DOPA) after inhibition of L-aromatic amino acid decarboxylase with NSD-1015 was used as a measure of the $\text{in vivo}$ activity of these DA neurons. Retinal DOPA accumulation was significantly increased in dark-adapted rats that had been exposed to light for only 5 min. The activation of the retinal DA neurons by cool white fluorescent lighting was dependent upon the light intensity. Light intensities of 0.1 and 0.5 lux did not stimulate the retinal DA neurons. There was a significant, but submaximal, activation of the neurons by 5.0 lux, and intensities of 32.2 lux or more maximally stimulated the neurons. The method involving liquid chromatography (LC) with electrochemistry (EC) which was used in these experiments to measure retinal DOPA and DA concentrations is also described in detail.     

Evidence for the lack of direct phosphorylation of bovine caudate tyrosine hydroxylase following activation by exposure to enzymatic phosphorylating conditions.

Highly purified bovine caudate tyrosine hydroxylase can be activated by exposure to enzymatic phosphorylating conditions. This activation is due to both a decrease in the K_m for the pterin cofactor and to some increase in V_max. The K_m for the enzyme's substrate, tyrosine, is unchanged by activation. After tyrosine hydroxylase was activated in the presence of [γ-³²P]-ATP, no incorporation of ³²P into the enzyme was observed by either immunoprecipitation studies or by sucrose gradient studies.     

Metabolism of zinc and copper in the neonate: Effect of cadmium administration during late gestation in the rat on the zinc and copper metabolism of the newborn

Rats that have been treated with Cd (1.0 mg/kg body wt., i.v.) on the 18th day of gestation give birth to young, the livers of which are low in Zn, but not in Cu. With increasing age after birth the hepatic concentrations of total and thionein-bound Zn in these animals increase rapidly to maxima at about 7 days, approx. 6 days later than in the newborn of normal dams, whereas the liver Cu concentration reaches a higher maximum at an earlier age than in the control neonate. This rapid uptake of Cu into the liver of the newborn of the Cd-treated dam is not accompanied by a concomitant increase in the concentration of soluble thionein-bound Cu.Cadmium-treatment of the dam retards the weight gain of the liver and, at least during the first 6–8 days postpartum, the increase in body wt. of the newborn. When the hepatic concentrations of thionein-bound Zn is expressed relative to liver wt. instead of age, there is no significant difference between the newborn from normal and Cd-treated dams.The Zn concentrations in blood, brain, stomach, duodenum, pancreas, spleen, kidney and muscle of newborn rats either remain constant, or increase only slightly with age after birth and are not affected significantly by the administration of Cd to the dam in late gestation. This treatment, which probably increases the demand for Zn in the newborn, delays the deposition of Zn in bone and causes a reduction in the Zn concentration of the skin. The Cu concentrations in skin and bone, as well as in other organs of the newborn during the first 24 days postpartum, seem to be unaffected by Cd-treatment of the dam.It is suggested that hepatic Zn-thionein has an essential function in the Zn metabolism of the liver, but is unlikely to control the supply of Zn to other organs in the newborn rat.     

Resolution and reconstitution of soluble components of rat liver microsomal vitamin D3-25-hydroxylase

Solubilized components of the vitamin D₃-25-hydroxylase, isolated from intact rat liver microsomes known to catalyze the C-25 oxidation of vitamin D₃in vitro, have been separated into two submicrosomal fractions enriched in detergent-solubilized NADPH-cytochrome c reductase and cytochrome P-450 or P-448. The P-450 hemoprotein-containing fraction was obtained by solubilization with cholic acid followed by treatment with the nonionic detergent, Emulgen 911, yielding a final preparation with a specific content of 7.25 nmol/mg microsomal protein. The reduced triphosphopyridine nucleotide-dependent cytochrome P-450 reductase activity, as detected by its ability to reduce the artificial electron acceptor, cytochrome c, was isolated free of cytochromes b₅ or P-450 by solubilization with deoxycholate and chromatography on DEAE-cellulose. The reductase component was found to exhibit kinetic properties with Michaelis constants: K_m(NADPH) = 3.14 μM, K_m(NADH) = 31.25 μM, and K_{m(cyt c)} = 12.34 μM. The NADPH-cytochrome c reductase activity was sensitive to NADPH-reversible inhibition by NADP, but not rotenone or cyanide. When the isolated components were incubated in the presence of an NADPH-generating system and carbon monoxide under anaerobic conditions, enzymatic reduction of the P-450 hemoprotein was measured by the appearance of characteristic absorbances at 420 and 450 nm of the reduced carbon monoxide vs. reduced difference spectrum. Furthermore, when the soluble submicrosomal components were reconstituted with excess reduced triphosphopyridine nucleotide, ³H-labeled vitamin D₃, and soluble cytosolic supernatant, full vitamin D₃-25-hydroxylase activity was restored at rates of up to 7.68 pmol/h/mg protein, with an apparent turnover number of cytochrome P-450 of 1.16 to 1.20 under conditions where the concentrations of the hemoprotein were rate limiting for net product formation. These results strongly support the hypothesis that the rat liver microsomal mixed-function oxidase, vitamin D₃-25-hydroxylase, consists of at least two membrane-bound protein components, NADPH-cytochrome c reductase and a cytochrome P-450 terminal oxidase, for the catalytic conversion of vitamin D₃ to 25-hydroxyvitamin D₃.