Does the inhibition of microvillus protein phosphorylation by lysine explain the activity of the latter on calcium transfer?

• 1.1. Is the activity of l-lysine on calcium absorption related to the fact that its phosphorylation is competitive with that of the microvilli proteins involved in the mineral transfer?
• 2.2. The microvilli proteins phosphorylation is not cyclic GMP-dependent but is actually inhibited by l-lysine, used in general at a 100 mM concentration.
• 3.3. The electrophoresis is followed by an autoradiograph which reveals the existence of a phosphorylated protein with a molecular weight of 140,000 daltons. Another phosphorylable protein, clearly visible in some preparations but only detectable in others, has a molecular weight close to 70,000 daltons.
• 4.4. The inhibition by lysine of the microvilli proteins phosphorylation is not specific to a given protein, but is also observed for phosphorylable cytosolic proteins.
• 5.5. A scheme for calcium transfer is proposed. It involves a protein whose phosphorylation should reduce the membrane permeability to calcium.
• 6.6. The following three attributes of the phosphorylable membrane protein—its molecular weight; the fact that another protein (probably its monomer) is also phosphorylable; its well known capacity for phosphorylation—suggest that this protein might actually be alkaline phosphatase whose correlations in calcium metabolism are well known.

     

Mechanism of the sarcoplasmic reticulum calcium pump. Fluorometric study of the phosphorylated intermediates.

After removal of calcium ions bound to the high affinity sites the sarcoplasmic reticulum calcium pump can be phosphorylated by inorganic phosphate. The intrinsic fluorescence of the protein is used to follow conformational changes of the pump and an intensity change can be observed upon addition of phosphate. This effect is activated by internal calcium ($\text{K}\text{1}\text{2}\text{= 10 mM}$) and inhibited by external calcium ($\text{K}\text{1}\text{2}\text{= 0.4 μM}$) and the apparent affinity for phosphate is high (0.2 mM). We conclude that the change observed is linked to the formation of the gradient-dependent phosphorylated intermediate. It is compared with previous results concerning the enzymatic cycle of the pump.     

Intestinal maturation: The effect of glucocorticoids on in vivo net magnesium and calcium transport in the rat

We investigated with an $\text{in vivo}$ single pass perfusion technique, the effect of glucocorticoids on net magnesium and calcium absorption from the small and large intestine of suckling and adolescent rats. In control rats, rates of net magnesium and calcium absorption were several fold greater in both small and large intestinal segments of suckling rats compared to corresponding rates in segments of adolescent rats. Methylprednisolone 30 mg/kg body weight daily for three days, suppressed significantly net magnesium and calcium absorption from the small and large intestinal segments of suckling rats only. Methylprednisolone had no effect on either net magnesium or calcium absorption in adolescent rats. The mechanism(s) responsible for the observed decrease in net magnesium and calcium absorption in the suckling period by glucocorticoids are discussed.     

Specificity in the interaction of phospholipids and fatty acids with vesicle reconstituted cytochrome P-450. A spin label study

The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome $\text{P-450}$ was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome $\text{P-450}$ in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome $\text{P-450}$ content, of the presence of type I and type II substrates for cytochrome $\text{P-450}$. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome $\text{P-450}$. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome $\text{P-450}$ was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance ($\text{τ}{}_{\mathrm{<mtext>R</mtext>}}\text{> 10}{}^{\mathrm{?8}}\text{s}$). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.     

Thermal analysis of bacteriophage T4.

The process of bacteriophage T4 morphogenesis was studied using a heat leakage scanning calorimeter. Thermograms of defective mutant 49 (am NG727) in permissive and non-permissive cells of $\text{Escherichia coli}$ showed a difference in thermal properties between packaged and non-packaged DNA molecules. $\text{In vivo}$, non-packaged DNA carried out their thermal transition at 85°C, the same temperature as that of T4 DNA melting measured in the standard saline citrate buffer, while the packaged DNA gave a sharper peak at 87°C due to some interaction with the head shell structure. Empty head shells showed a sharp heat absorption peak at 89°C both $\text{in vivo}$ and $\text{in vitro}$, indicating the high degree of cooperativity in their conformational changes.     

Regulation of energy flux through the creatine kinase reaction in vitro and in perfused rat heart: 31P-NMR studies

Fluxes catalyzed by soluble creatine kinase (MM) in equilibrium in vitro and by the creatine kinase system in perfused rat hearts were studied by ³¹P-NMR saturation transfer method. It was found that in vitro both forward and reverse fluxes through creatine kinase at equilibrium were almost equal and very stable to changes in $\text{phosphocreatine}\text{creatine}$ ratio (from 0.2 to 3.0) as well as to changes in pH (from 7.4 to 6.5 or 8.1), free Mg²⁺ concentration and 2-fold decrease of total adenine nucleotides and creatine pools (from 8.0 to 4.0 mM and from 30 to 14 mM, respectively). In the rat hearts perfused by the Langendorff method the creatine kinase-catalyzed flux from phosphocreatine to ATP was increased by 50% when oxygen consumption grew from 8 to 55 μmol/min per g of dry wt. due to transition from rest to high workload. These changes could not be exclusively explained on the basis of the equilibrium model by activation of heart creatine kinase due to some decrease in $\text{[}\text{phosphocreatine}\text{]}\text{[}\text{creatine}\text{]}$ ratio (from 1.8 to 0.8) observed during transition from rest to high workload. Analysis of our data showed that an increase in the flux via creatine kinase is correlated with an increase in the rate of ATP synthesis with a linearity coefficient higher than 1.0. These data are more consistent with the concept of energy channeling by phosphocreatine shuttle than with that of the creatine kinase equilibrium in the heart.     

Nuclease activity in preparations of creatine phosphokinase: effect on mRNA stability

Creatine phosphokinase is used to generate ATP with creatine phosphate for $\text{in vitro}$ protein synthesis. Some preparations of this enzyme contain nuclease activity, which can be demonstrated by a sensitive assay of the cleavage of poly(A)-containing RNA. These preparations of creatine phosphokinase support protein synthesis poorly in a cell-free system prepared from HeLa cells. Poly(A)-containing RNA is quite stable in this cell-free system when the phosphorylated sugar fructose 1,6-bisphosphate with no addition of enzyme is used to generate ATP.     

Formation of mutagens by heating creatine and glucose

The mutagenic activity toward $\text{Salmonella typhimurium}$ TA 98 and TA 100 was investigated by heat treatment at temperatures up to 200°C of meat with identified components such as protein, adenine, creatine and a mixture of each of the 17 amino acids or glucose. Mutagenicity of these nitrogenous compounds was detected at the temperature of 150°C by adding glucose, consequently the yield of mutagenic activity by heating creatine and glucose was remarkably high. It is assumed that mutagens would be formed by the reaction of creatine and sugars during cooking of meat.     

Phospholipid phase transitions. Effects of n-alcohols, n-monocarboxylic acids, phenylalkyl alcohols and quatenary ammonium compounds

The interactions of a series of alcohols, acids and quaternary ammonium salts with a phosphatidylcholine-water model biomembrane (dipalmitoyl phosphatidylcholine) system have been studied using differential scanning calorimetry. In particular the effects of these molecules upon the lipid endothermic phase transitions were investigated over a range of concentrations. A variety of effects was observed. (a) Those molecules which shift or broaden the main lipid transition can also remove the pretransition endotherm. (b) $\text{n-}\text{Alcohols}$ and $\text{n-}\text{monocarboxylic}$ acids containing the same number of carbon atoms have very similar effects at molar concentrations up to 40%. Those molecules containing 12 or more carbon atoms raise the main lipid phase transition whilst those molecules containing 10 or less carbon atoms lower this transition temperature. (c) The phase diagram of stearoyl alcohol in the phosphatidylcholine-water system shows the formation of lipid-alcohol complexes. (d) Alkyl trimethyl ammonium bromides showed behaviour which differs considerably from $\text{n-}\text{alcohols}$ and $\text{n-}\text{carboxylic}$ acids of the same chain length. (e) Other alkyltrialkyl and tetraalkylammonium bromides show that a variety of effects on the lipid phase transition can be obtained. (f) With the homologous series of phenylalkyl alcohols from benzyl alcohol to 4-phenyl butanol increasing the number of methylenes between the terminal OH and the benzene ring leads to greater interaction between solute and bilayer.The range of different effects obtained with the compounds studied offers a means for introducing various degrees and types of perturbation into membrane systems.     

Metabolic effects of phosphate receptors stimulating the ileal transport of calcium]

Several compounds, either metabolizable such as sorbitol and creatine or unmetabolizable such as L-xylose or ethyl-ethanolamine, increase ileal calcium transport, similarly to the well documented effect of lactose. These compounds increase the duration time but not the rate of calcium transport. They have no specific effect on intestinal calcium absorption but they prolong the presence of soluble calcium in the ileal loop, thus allowing the calcium absorption. This stimulating effect of these compounds on mineral absorption is associated with an increase of ileal mucosal ATP content.     

Structure and properties of hemocyanins. XIV. Reassociation of Helix pomatia alpha-hemocyanin

Helix pomatia α-hemocyanin dissociates within minutes into $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules, on increase of pH in the alkaline region.The rate and final state of reassociation of $\text{1}\text{10}$-size and $\text{1}\text{20}$-size molecules have been studied, by turbidity measurements and ultracentrifugation, on lowering of pH or on the addition of calcium ions.Reassociation of $\text{1}\text{10}\text{-size}$ molecules proceeds in two phases, with half-times in the order of minutes and one hour, respectively. The slow phase is linked to the disappearance of transitory intermediates, presumably dimers and tetramers of $\text{1}\text{10}$-size molecules.A hysteresis is observed in the final state of pH-dependent and Ca²⁺-dependent dissociation-reassociation.Reassociation of $\text{1}\text{20}$-size molecules depends on the initial (isomeric) state of these molecules. The F(fast sedimenting)-$\text{1}\text{20}$-size molecules reassociate almost completely to whole molecules, whereas the S(slow sedimenting)-form does not reassociate at all.     

The cation-dependence of the degree of protein phosphorylation-induced unstacking of pea thylakoids

Experiments are presented to show that the phosphorylation of the light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complex (LHC) induces structural reorganisation within the thylakoid membrane in response to the introduction of additional negative surface charges. The effect of cations of different valency on chlorophyll fluorescence measurements indicates that LHC-phosphorylation-induced reorganisation involves a change in the electrostatic screening capability of the added cation. At intermediate levels of cations (e.g., 1 or 2 mM Mg²⁺), which substantially stack non-phosphorylated membranes, it was found that membrane phosphorylation caused considerable unstacking as monitored by light scattering and electron microscopy. Concomitant with this was a large decrease in chlorophyll fluorescence indicative of randomisation of chlorophyll protein complexes which would result in an increase in energy transfer between the photosystems as well as an absorption cross-section change. At higher concentrations (e.g., above 5 mM Mg²⁺) a persistent ATP-induced decrease in chlorophyll fluorescence has been attributed to the displacement of charged phosphorylated LHC from the appressed granal to the non-appressed stromal lamellae, thus decreasing the absorption cross-section of Photosystem II. Under these circumstances only a small degree of unstacking was detected by light scattering and measurements of the percentage of thylakoid length which is stacked to form grana. However, when considered on a surface area basis, the structural changes observed can qualitatively account for the magnitude of the chlorophyll fluorescence quenching due to the lateral diffusion of LHC.     

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E₁ or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. $\text{p-}\text{Bromophenacyl}$ bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: $\text{arachidonic acid}\text{>}\text{collagen}\text{>}\text{thrombin}\text{>}\text{ionophore A}\text{23187}$. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca²⁺ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca²⁺ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.     

Interferon induction of polyamine-dependent protein kinase activity in Ehrlich ascites tumor cells

Treatment of Ehrlich ascites tumor cell cultures $\text{in}\text{vitro}$ with interferon induces a protein kinase activity that is activated by the polyamines, spermidine and spermine. Putrescine antagonizes the activation. The protein kinase yields a phosphorylated endogenous polypeptide of M_r 68,000–70,000. The polyamine-dependent protein kinase activity cofractionates with a double-stranded RNA-dependent protein kinase activity during affinity chromatography on poly (I) ·poly (C) - agarose or by chromatography on phosphocellulose. The double-stranded RNA-dependent protein kinase also phosphorylates an endogenous polypeptide of M_r 68,000–70,000. Unsuccessful attempts to discriminate between these two protein kinase activities on the bases of their respective capacities to be activated by either double-stranded RNA or spermidine/spermine, suggest that a single protein kinase enzyme may be activated by these strikingly dissimilar modifiers.     

The phosphorylation of high mobility group proteins 14 and 17 from Ehrlich ascites and L1210 invitro

The ability of the high mobility group (HMG) proteins to be phosphorylated was examined in Ehrlich ascites and L1210 cells incubated $\text{in}\text{vitro}$. HMG proteins were selectively extracted from isolated nuclei with 2% trichloroacetic acid, and electrophoretically separated on acid-urea or SDS polyacrylamide gels. Autoradiography of the gels revealed that among the HMG proteins, only HMG 14 and 17 were labeled. The specific activities of these two proteins were approximately equal to that of histone H1. Phosphorylation of HMG 14 and 17 reached a maximum in 2–3 hr and had turnover rates in pulse-chase experiments similar to that of phosphorylated histone H1.     

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes

EL4 cells were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membrane phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the $\text{P}$ value of both probes compared to the $\text{P}$ value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, $\text{P}$ values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in $\text{P}$ values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization for monitoring physical properties of membranes (such as ‘fluidity’) is discussed.     

Relationship of metabolite inhibition of growth to flow-of-carbon patterns in nature

Various genetic diseases arise from biochemical imbalances that are relatively subtle in the sense that the original mutations are not lethal, that the organism is most vulnerable to damage during certain phases of rapid development, and that in well-managed cases it may be possible to avoid damaging effects through the use of appropriate nutritional manipulations. Analogous imbalances occur in lower organisms. Data obtained with $\text{Pseudomonas}$$\text{putida}$ illustrate that susceptibility to metabolic imbalance is conditionally dependent upon the nutritional regimen.Stereoisomers of leucine, isoleucine and valine, except for L-allo-isoleucine, are metabolized as sole sources of carbon and energy by $\text{P.}$$\text{putida}$. Although the cell yields calculated following utilization of $\text{D}$-leucine and $\text{L}$-leucine were similar, the rate of growth on D-leucine was seven-fold faster than on $\text{L}$-leucine. Slower growth on the $\text{L}$-isomer is not explained as 2-ketoisocaproate limitation since 2-ketoisocaproate production from $\text{L}$-leucine appears to occur more readily than from $\text{D}$-leucine. Spontaneous mutants were obtained which grew 2–10 times more rapidly than wild type on $\text{L}$-leucine, $\text{L}$-isoleucine, or $\text{L}$-valine. It is concluded that the true growth potential (rate) of wild type on any of the branched-chain amino acids is masked by a partial, sustained inhibitory effect produced by the corresponding keto acids or their derivative metabolites. Inhibition of growth rate was only found during utilization of branched-chain amino acids as the sole source of carbon and energy, indicating that the metabolite vulnerability is unique to particular flow-of-carbon patterns during growth. The partial and sustained depression of growth rate by branched-chain amino acids in the absence of other carbon sources cannot be attributed to mis-regulation events localized within the biosynthetic pathway. It is concluded that the catabolism of branched-chain amino acids produces a generalized state of metabolic imbalance owing to the existence of abnormally high levels of degradative metabolites such as keto acids of Coenzyme-A derivatives. Such compounds could (1) interfere with keto acid (e.g. pyruvate) metabolism, (ii) cause feed-forward inhibition of rate-limiting steps in the pathways of branched-chain amino acid catabolism, (iii) perturb fatty acid composition or disrupt the biochemical integrity of membrane material, or (iv) react with substrate-ambiguous enzymes, either slowing essential biochemical reactions to rates that are growth-limiting or producing erroneous products having antimetabolite properties.These effects of branched-chain amino acids in $\text{P.}$$\text{putida}$ may be quite relevant to the molecular events that characterize maple syrup urine disease in man. Metabolite inhibition is probably more common in nature than is generally appreciated, and an appreciation of the molecular basis for anomalous inhibitions of growth in prokaryotic systems should help supply insight into various molecular diseases in man, many of them yet to be described.     

The 5'-termini of the oligonucleotides from ribonuclease T 1 digested E. coli ribosomes

Oligonucleotides remaining in the 70s $\text{Escherichia}\text{coli}$ ribosomal particles after varying degrees of digestion with ribonuclease T₁ were phosphorylated with polynucleotide kinase in the presence of γ-labeled³²P-ATP. The resulting radioactively labeled RNA molecules were further digested with pancreatic ribonuclease and analyzed by a two-dimensional finger-printing technique. The numbers of labeled oligonucleotides were proportional to the duration of T₁ digestion; most of these oligonucleotides yielded ¹pAp and/or ¹pCp as their 5′-end groups upon alkaline hydrolysis.     

Pharmacological properties of imidazole. 2. Action on the normal body temperature regulation in rats

Rats injected intraperitoneally with imidazole (100 to 400 mg/kg) showed a progressive dose-dependent decrease in rectal temperature. A decrease of 6$\text{°}$C occurred when the imidazole-treated animals were placed in a 4$\text{°}$C environment. In a 23$\text{°}$C environment, the rectal temperature declined about 2$\text{°}$C. The results show that imidazole induces a decline in body temperature in non-febrile rats, possibly due to an action on hipothalamic calcium levels.     

Interactions of lead and calcium on the synaptosomal uptake of dopamine and choline

Exposure of rodents to lead $\text{in vivo}$ has been associated with alterations in cholinergic and dopaminergic neurotransmission in the CNS. These effects have been hypothesized to result from competitive interactions between lead and calcium at sites involved in uptake and release of neurotransmitters and their precursors. These experiments reproduced the $\text{in vivo}$ observation by $\text{in vitro}$ exposure of crude synaptosomal suspensions to lead. Lead-induced inhibition of high affinity choline uptake was mimicked by reduced in vitro calcium concentrations, which suggests that lead's effects on cholinergic function are explainable by the lead-calcium hypothesis. However, inhibition of dopamine uptake was produced only by lead and not by reduced calcium; further additions of calcium did not reverse lead-induced effects on dopamine uptake. Increased calcium concentrations were shown to increase the release of dopamine; lead in the presence of normal calcium concentration did not affect dopamine release. However, more dopamine was released when increased calcium was combined with exposure to 1 × 10^?4 lead. This effect may have resulted from lead's ability to increase the uptake of calcium by synaptosomes. Thus, the interactions between lead and calcium appear to differ in terms of effects on cholinergic and dopaminergic function; in the former, the results suggest a competitive interaction similar to that shown functionally at peripheral cholinergic sites; in the latter, a different role for calcium is hypothesized which may account for the different effects of lead.