Selectivity enhancement induced by substitution of non-natural analogues of arginine and lysine in arginine-based thrombin inhibitors.

Seven non-natural analogues of arginine and lysine have been substituted in an established arginine-based thrombin inhibitor. Four of the new compounds exhibited significant thrombin inhibition (K(i)'s 0.53-3.95 microM) and were subsequently tested for selectivity against trypsin. The two best compounds gave selectivity ratios of 962 and 525 (trypsin/thrombin), improving upon the parent compound.     

Proinsulin precursors in catfish pancreatic islets.

The purpose of these experiments was to determine whether insulin-related peptides, larger than proinsulin, could be detected in pancreatic islet cells. Catfish pancreatic islets were incubated with radiolabeled amino acids. After 15- to 60-min incubation, two acid-alcohol-extractable peptides, larger than proinsulin, were detected which were approximately of Mr = 12,000 and 11,000 (12 K and 11K, respectively). They migrated as single polypeptide chains by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis under reducing conditions, and were therefore not aggregates of insulin or proinsulin. The 12 K protein had identical mobility with catfish preoproinsulin synthesized in a wheat germ cell-free system. On standard electrophoresis at pH 8.9, the 12 K protein migrated separately from proinsulin and was at least 65% one protein with two to three minor contaminants. The 12 K and 11 K proteins were chemically related to insulin and proinsulin as shown by tryptic peptide analysis, using cation exchange resin chromatography, and by two-dimensional tryptic peptide maps. Analysis of the tryptic digest of the 12 K protein, compared to proinsulin after leucine aminopeptidase treatment, suggested that the NH2 terminus of the larger protein was different from that of proinsulin. These peptides were specifically bound to anti-insulin antibody. The binding was only 5 to 8% of the protein added, but was specific for the 12 K and 11 K proteins when the immunoprecipitates were examined by electrophoresis and not from contaminating proinsulin. During the continuous incubation of the islets with [3H]leucine, 12 K and 11 K proteins were synthesized in the cell before proinsulin. When islets were first incubated with [3H]leucine for 30 min followed by incubation with excess unlabeled leucine, the 12 K and 11 K proteins appeared to show a precursor-product relationship to proinsulin and insulin. Even when total islet protein synthesis was inhibited by cycloheximide (100 microgram/ml), proinsulin continued to be synthesized for up to 2 h. This suggested that the conversion of the proinsulin precursors to proinsulin in the fish is a post-translational event.     

Glucokinase is concentrated in insulin-secretory granules of pancreatic B-cells

We immunohistochemically examined the distribution of glucokinase (GK) in the B-cells of pancreatic islets of normal rats. GK was stained punctately in the cytoplasm of B-cells when examined under the light microscope. By use of a double-immunostaining technique, most of the GK immunoreactivity was observed to be colocalized with insulin immunoreactivity. Electron microscopic examination by the immunogold method revealed that GK immunoreactivity was predominantly located within insulin-secretory granules of pancreatic B-cells. Accepted: 20 April 1999     

Selective uptake of alloxan by pancreatic B-cells

Alloxan rapidly binds to or accumulates in pancreatic B-cells as distinct from non-B-cells. The selective uptake of this cytotoxic agent by the insulin-producing B-cells might account for its well-known diabetogenic effect.     

Differences in glucose handling by pancreatic A- and B-cells

Glucose exerts opposite effects upon glucagon and insulin release from the endocrine pancreas. Glucose uptake and oxidation were therefore compared in purified A- and B-cells. In purified B-cells, the intracellular concentration of glucose or 3-O-methyl-D-glucose equilibrates within 2 min with the extracellular levels, and, like in intact islets, the rate of glucose oxidation displays a sigmoidal dose-response curve for glucose. In contrast, even after 5 min of incubation, the apparent distribution space of D-glucose or 3-O-methyl-D-glucose in A-cells remains much lower than the intracellular volume. In A-cells, both the rate of 3-O-methyl-D-glucose uptake and glucose oxidation proceed proportional to the hexose concentration up to 10 mM and reach saturation at higher concentrations. Addition of insulin failed to affect 3-O-methyl-D-glucose or D-glucose uptake and glucose oxidation by purified A-cells. Glucose releases 30-fold more insulin from islets than from single B-cells, but this marked difference is not associated with differences in glucose handling. The rate of glucose oxidation is virtually identical in single and reaggregated B-cells and is not altered after addition of glucagon or somatostatin. It is concluded that the dependency of glucose-induced insulin release upon the functional coordination between islet cells is not mediated through changes in glucose metabolism.     

The ATP-sensitivity of K+ channels in rat pancreatic B-cells is modulated by ADP

ATP-sensitive K+ channels in inside-out membrane patches from dispersed rat pancreatic B-cells were studied using patch-clamp methods. The dose-response curve for ATP-induced channel inhibition was shifted to higher concentrations in the presence of ADP (2 mM). In glucose-free solution, the total intracellular concentration of ATP was 3.8 mM and of ADP 1.5 mM; glucose (20 mM) increased ATP and decreased ADP by approx. 40%. These results suggest that both ADP and ATP may be involved in regulating the activity of the glucose-sensitive K+ channel in intact B-cells.     

The cardiac sodium channel is post-translationally modified by arginine methylation

The α subunit of the cardiac sodium channel (Na(v)1.5) is an essential protein in the initial depolarization phase of the cardiomyocyte action potential. Post-translational modifications such as phosphorylation are known to regulate Na(v)1.5 function. Here, we used a proteomic approach for the study of the post-translational modifications of Na(v)1.5 using tsA201 cells as a model system. We generated a stable cell line expressing Na(v)1.5, purified the sodium channel, and analyzed Na(v)1.5 by MALDI-TOF and LC-MS/MS. We report the identification of arginine methylation as a novel post-translational modification of Na(v)1.5. R513, R526, and R680, located in the linker between domains I and II in Na(v)1.5, were found in mono- or dimethylated states. The functional relevance of arginine methylation in Na(v)1.5 is underscored by the fact that R526H and R680H are known Na(v)1.5 mutations causing Brugada and long QT type 3 syndromes, respectively. Our work describes for the first time arginine methylation in the voltage-gated ion channel superfamily.     

Leishmania DNA is rapidly degraded following parasite death: an analysis by microscopy and real-time PCR

Control of human leishmaniases relies on appropriate diagnosis and reliable methods for monitoring chemotherapy. The current method used for estimation of parasite burden during chemotherapy patient follow-up as well as in pharmacological studies performed in experimental models involves PCR-based assays. Compared to time-consuming conventional methods, this type of Leishmania DNA detection-based method is extremely sensitive, but could fail in distinguishing viable Leishmania from slowly degenerating ones. We have used an in vitro model to monitor the duration of Leishmania DNA persistence in mouse macrophages following exposure to l-leucine ester, a molecule otherwise known to rapidly kill intracellular Leishmania amazonensis amastigotes. At 1h of post l-leucine ester exposure, more than 98% of amastigote-loaded macrophages harbored killed parasites and parasite remnants, as assessed by microscopy. This dramatic decrease in parasite load and the microscopic parasite follow-up over the 120 h time period studied were correlated with Leishmania DNA as quantified by real-time PCR. Our results indicate that kinetoplast and nuclear parasite DNA degradation occurs very rapidly after amastigote death. These data add further weight to the argument that PCR assays represent not only a robust method for diagnosis but can also be reliable for monitoring parasite size reduction rate post any intervention (Leishmania-targeting molecules, immunomodulators...).     

Unassembled HLA-DR beta monomers are degraded rapidly by a nonlysosomal mechanism.

We previously observed that in a mutant B lymphoblastoid cell line which has a homozygous HLA-DR alpha deletion, DR beta-chains appeared to be unstable. In the present study, we have studied the pathway that leads to degradation of unassembled DR beta-chains. Unassembled DR beta-chains are degraded rapidly in the DR alpha deletion mutant cells, compared with the assembled DR heterodimers present in non-mutant cells. Accelerated DR beta turnover in 9.22.3 cells is specific; class I molecules in these DR alpha-deficient cells turned over slowly. DR beta-chains assemble with Ii in the DR alpha deficient cell line, but this did not protect DR beta-chains from degradation. The maturation of unassembled beta-chains is arrested before their reaching the medial Golgi compartment, and this degradation proceeds by a nonlysosomal, nonendosomal pathway. Degradation of DR beta-chains is blocked when cells are cultured at 16 degrees C, a temperature known to prevent vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus. Degradation is also inhibited by carbonyl cyanide m-chlorophenylhydrazone, a drug that is also known to inhibit protein transport from the ER. The results, taken together, suggest that degradation of unassembled DR beta-chains occurs by a nonlysosomal, nonendosomal pathway which involves transport of DR beta-chains out of the ER.     

The Drosophila hsp70 message is rapidly degraded at normal temperatures and stabilized by heat shock

When heat-shocked Drosophila cells are returned to normal temperatures, heat-shock protein (HSP) synthesis is repressed and normal protein synthesis is restored. The repression of HSP70 synthesis is accompanied by the selective degradation of its mRNA. We have engineered cells to produce a modified hsp70 mRNA that behaves exactly as the wild-type message. That is, it is stable during heat shock but degraded during recovery when protein synthesis returns to normal. When this message, placed under the control of the metallothionein promoter, is induced at normal temperatures it is rapidly degraded, with a half life of 15-30 min. Apparently, the hsp70 message is inherently unstable. During heat-shock, degradation of the message is suspended; during recovery degradation is restored.     

Modification of arginine and lysine in proteins with 2,4-pentanedione.

Primary amines react with 2,4-pentanedione at pH 6-9 to form enamines, N-alkyl-4-amino-3-penten-2-ones. The latter compounds readily regenerate the primary amine at low pH or on treatment with hydroxylamine. Guanidine and substituted guanidines react with 2,4-pentanedione to form N-substituted 2-amino-4,6-dimethylpyrimidines at a rate which is lower by at least a factor of 20 than the rate of reaction of 2,4-pentanedione with primary amines. Selective modification of lysine and arginine side chains in proteins can readily be achieved with 2,4-pentanedione. Modification of lysine is favored by reaction at pH 7 or for short reaction times at pH 9. Selective modification of arginine is achieved by reaction with 2,4-pentanedione for long times at pH 9, followed by treatment of the protein with hydroxylamine. The extent of modification of lysine and arginine side chains can readily be measured spectrophotometrically. Modification of lysozyme with 2,4-pentanedione at pH 7 results in modification of 3.8 lysine residues and less than 0.4 arginine residue in 24 hr. Modification of lysozyme with 2,4-pentanedione at pH 9 results in modification of 4 lysine residues and 4.5 arginine residues in 100 hr. Treatment of this modified protein with hydroxylamine regenerated the modified lysine residues but caused no change in the modified arginine residues. One arginine residue seems to be essential for the catalytic activity of the enzyme.     

Biological significance of methylated derivatives of lysine and arginine.

It has been shown by biological trials that L-lysine and L-arginine are essential for the undisturbed growth of living organisms. These amino acids show different reactivity in the molecular processes of the cell which explains their antagonistic function. As a result of enzymatic methylation the N-? as well as N^G-methylated derivatives of lysine and arginine are produced. The biological function of the methylated basic amino acids is almost unknown. Some N-?-methylated lysines, but first of all N-?-trimethyl lysine /TML/ exhibits a proliferation promoting effect on several normal and neoplastic cell systems. N^G-methylated arginines proved to have a proliferation inhibiting effect. Thus, methylation of basic amino acids may have a special significance in the regulation of cell proliferation.     

Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity.

Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In contrast with previous studies demonstrating stability in vitro in a reticulocyte lysate-based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. While the degradation of ODC is stimulated by the presence of cotransfected antizyme, degradation of antizyme seems to be independent of ODC, suggesting that antizyme degradation does not occur while presenting ODC to the 26S proteasome. Interestingly, both species of antizyme, which represent initiation at two in-frame initiation codons, are rapidly degraded. The degradation of both antizyme proteins is inhibited in ts20 cells containing a thermosensitive ubiquitin-activating enzyme, E1. Therefore we conclude that in contrast with ubiquitin-independent degradation of ODC, degradation of antizyme requires a functional ubiquitin system.     

The ATP-sensitive potassium channel in pancreatic B-cells is inhibited in physiological bicarbonate buffer

The effects of bicarbonate buffer (HCO3-/CO2) on the activity of the two K+ channels proposed by some to control the pancreatic B-cell membrane response to glucose were studied. Single K+-channel records from membrane patches of cultured B-cells dissociated from adult rat islets exposed to a glucose- and bicarbonate-free medium (Na-Hepes in place of bicarbonate) exhibit the activity of both the ATP-sensitive as well as the [Ca2+]i-activated K+ channels. However, in the presence of bicarbonate-buffered Krebs solution, the activity of the ATP-sensitive K+ channel is inhibited leaving the activity of the K+ channel activated by intracellular [Ca2+]i unaffected. In the absence of bicarbonate (Hepes/NaOH in place of bicarbonate), lowering the external pH from 7.4 to 7.0 also has differential effects on the two K+ channels. While the K+ channel sensitive to ATP is inhibited, the K+ channel activated by a rise in [Ca2+]i is not affected. To determine whether the response of the B-cell in culture to bicarbonate is also present when the B-cell is functioning within the islet syncytium, the effects of bicarbonate removal on membrane potential of B-cells from intact mouse islets were compared. These studies showed that glucose-evoked electrical activity is also blocked in bicarbonate-free Krebs solution. Furthermore, in the absence of bicarbonate and presence of glucose (11 mM), electrical activity was recovered by lowering the pHo from 7.4 to 7.0. The ATP-sensitive K+-channel activity is greatly reduced by physiologically buffered solutions in pancreatic B-cells in culture. The most likely explanation for the bicarbonate effects is that they are mediated by cytosolic pH changes. Removal of bicarbonate (keeping the external pH at 7.4 with Hepes/NaOH as buffer) would increase the pHi. Since the activity of the [Ca2+]i-dependent K+ channels is not affected by the removal of the bicarbonate buffer, our patch-clamp data in cultured B-cells indicate an involvement of [Ca2+]i-activated K+ channels in the control of the membrane potential. For the B-cell in the islet, we propose that the burst pattern of electrical activity (Ca2+ entry) is controlled, at least in part, by the [Ca2+]i-activated K+ channel.     

Chaotic and irregular bursting electrical activity in mouse pancreatic B-cells.

The glucose-induced B-cell electrical activity was recorded in islets of Langerhans isolated from Swiss Webster albino mice originating from different suppliers. 23 out of 25 islets obtained from mice bred at the Charles River Breeding Station (CR mice) exhibited irregular or chaotic burst patterns of electrical activity, while 36 out of 40 islets isolated from mice bred locally at the National Institutes of Health displayed the typical bursting activity. The CR mice tended to recover a regular pattern after 1 mo on the National Institutes of Health mouse diet. The irregular or chaotic bursting electrical activity is proposed to result from changes in B-cell membrane composition or cellular metabolism, possibly induced by differences in diet.     

Domain-specific bias in arginine/lysine usage by protein toxins

The content of lysine and arginine residues in a number of A-B type protein toxins has been examined. It is found that the A subunit, or its equivalent, often shows a strong bias in the type of basic amino acid residue used tending towards nearly exclusive use of either arginine or lysine rather than use of both, whereas the B subunit or its equivalent shows no such bias. Although arginine codons are GC-rich and lysine codons are AT-rich, the content of GC and AT in the genes coding for the toxins does not adequately explain this bias. Other explanations are discussed, including the possibility that the bias is linked to catalytic function or membrane interaction. Understanding this bias may yield valuable insights into toxin structure and function. Furthermore, identification of bias in sequences may be a useful tool for identifying new toxins and their domains.