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Horse liver alcohol dehydrogenase (isozyme EE) in the crystalline state was alkylated with iodoacetate under conditions resulting in the single substitution of Cys-46, which is a ligand to the active-site zinc atom. Alkylation was facilitated by the prior formation of a complex with imidazole bound to the zinc atom. Extent and specificity of the reaction were determined by use of 14C-labelled iodoacetate and by analyses of radioactive peptides after cleavage with trypsin. Ternary complexes of the enzyme with coenzymes and inhibitors effectively protected the protein against alkylation. ADP-ribose, Pt(CN)42-, 1,10-phenanthroline, Au(CN)2- and AMP also prevented alkylation with decreasing effectiveness. Crystallographic studies of the alkylated enzyme show that the carboxymethylated sulfur atom of Cys-46 is still liganded to the active-site zinc atom and that the iodide ion liberated during alkylation is bound as the fourth ligand to zinc, displacing imidazole. Crystallographic analyses were also performed of the binding of AMP and Pt(CN)42- to the enzyme. It was found that Arg-47 interacts with the phosphate moiety of the nucleotide. Lys-228 and Arg-47 interact in the platinate complex with the bulky anion, the center of which coincides with the position of the nucleotide phosphate. Some of the cyano-ligands to platinum occupy a crevice between the coenzyme phosphate binding site and the active-site zinc atom. The results of the combined studies on primary and tertiary structures confirm previous suggestions that iodoacetate enters the active site via reversible binding to an anion-binding site. This site interacts with the negatively charged groups of the coenzyme as well as with ADP-ribose, Pt(CN)42- and to a lesser extent Au(CN)2- and AMP, which therefore prevent the reversible binding of iodoacetate. 1,10-Phenanthroline does not block the binding site but interferes with alkylation presumably by changing the coordination of zinc. Identification of this labelled residue in both chemical and crystallographic studies correlates the primary and tertiary structures. Characterizations of the active-site zinc region and the general anion-binding site are also presented. [ABSTRACT FROM AUTHOR]

Alkylation at N-1 of the NAD+ adenine ring with 3,4-epoxybutanoic acid, followed by chemical reduction to the alkali-stable NADH form and alkaline Dimroth rearrangement, gave the NADH derivative alkylated at the exocyclic adenine amino group. Enzymic reoxidation of the latter derivative gave nicotinamide-6-(2-hydroxy-3-carboxypropylamino)purine dinucleotide, a functionalized NAD+ analogue carrying an ω-carboxylakyl side-chain at the exocyclic adenine amino group. Carbodiimide coupling of the latter derivative to high-molecular-weight water-soluble (polyethyleneimine, polylysine) and insoluble (aminohexyl-Sepharose)polymers lave the corresponding macromolecularized NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine and polylysine analogues showed a substantial degree of efficiency relative to free NAD + with rabbit muscle lactate dehydrogenase (60 and 25% respectively) but a lower one with yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase (2-7 %). The polyethyleneimine derivative entrapped in cellulose triacetate fibres together with the lactate dehydrogenase was operationally stable during repetitive use. [ABSTRACT FROM AUTHOR]

Alkylation of ATP with iodoacetic acid at pH 6.5 yielded 1-carboxymethyl-ATP which, after alkaline rearrangement, gave N6-carboxymethyl-ATP. Condensation of this analogue with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated N6-[(6-aminohexyl)carbamoylmethyl]-ATP in an overall yield of 40% based on the parent nucleotide ATP. The coenzymic activities of both N6-adenine-substituted derivatives of ATP were tested with three kinases. Both derivatives showed coenzymic function against hexokinase with the ‘long’ derivative having highest activity (95 %) relative to unsubstituted ATP. Their activities towards the other two kinases tested was negligible except with the ‘long’ analogue against glycerokinase (20%). The latter ATP analogue, when bound to Sepharose through its terminal amino group, could be dephosphorylated to the corresponding ADP analogue with soluble hexokinase yielding glucose 6-phosphate in an enzymic ‘solid-phase’ fashion. The Sepharose-bound ADP formed could subsequently be phosphorylated back to ATP using soluble acetate kinase. Sepharose-ATP preparations were also used in preliminary affinity chromatography studies using citrate synthase. Alkylation of ADP following the above procedure yielded the corresponding ADP analogue, N6-[(6-aminohexyl)carbamoylmethyl]-ADP in an overall yield of 40%. Alkylation of AMP yielded the corresponding N6-[(6-aminohexyl)carbamoylmethyl]-AMP in an overall yield of 45%. [ABSTRACT FROM AUTHOR]

2-Amino-4-oxo-5-chloropentanoic acid inactivates specifically the homoserine dehydrogenase activity of the bifunctional enzyme, aspartokinase I - homoserine dehydrogenase I. The asparto-kinase activity remains essentially untouched and retains its threonine sensitivity. The inactivation of the dehydrogenase requires the covalent binding of one equivalent of the analogue per subunit. Alkylation does not affect the tetrameric state of the protein. The alkylating agent, a substrate analogue, meets the qualitative and quantitative requirements of an affinity label. [ABSTRACT FROM AUTHOR]

The method of scattering was applied to determine the effect of chemical modification on the conformation of the basic pancreatic trypsin inhibitor in aqueous solution. Cleavage of one of the three disulfide bonds yields no spectral changes except, of course, the manifestation of the reduction, i.e. a lower intensity of the disulfide band and the appearance of a line due to the newly formed sulfhydrylgroups. S-alkylation, however, produces several changes in the spectra as compared to the native inhibitor. In the case of the carboxymethylderivative the amide III band, shifted to lower frequency, is particularly significant. This observation suggests that the inactivity towards trypsin of the reduced carboxymethyl inhibitor is the result of structural changes near the reactive center of the molecule. [ABSTRACT FROM AUTHOR]

2-Thiocytidine incorporated into tRNAPhe from yeast by tRNA nucleotidyl transferase can be alkylated by iodoacetamide and its derivatives. Using this method a spin label was introduced specifically into tRNAPhe and the product, tRNAPhe-C-(s.l.)s²C-A, can still be aminoacylated by phenylalanyl-tRNA synthetase. Electron paramagnetic resonance spectra obtained between 10° and 70°C for tRNAPhe-C-(s.l.)s²C-A and the pentanucleotide C-A-C-(s.1.)s²C-A (a model compound) were evaluated for the correlation time, τc, of the reorientation of the spin label in its environment and for the motional anisotropy parameter, ε. Spin-labeled pentanucleotide is characterized by a spin label motion with an activation energy, EA, of 1-67 × 10-3 J × mol-1 × K-1 (7.0 kcal × mol-1 × °C-1) ) and a strongly anisotropic motion with a long axis of the rotational ellipsoid along the N-O bond of the radical. This motion is temperature independent. In comparison spin label bound to the tRNA molecule is more immobilised and its motion is more isotropic. In addition, the slope of log τc(T) and the nature of the motion changes at a critical temperature Tcr which, in the absence of magnesium, varies with ionic strength. In the presence of magnesium Tcr is 47.5 °C, whereas the midpoint temperature of the optical melting curve, Tm, is 75 °C. The melting transition monitored by the spin label is a cooperative process associated with the melting of a higher-ordered structure of the tRNAPhe molecule. [ABSTRACT FROM AUTHOR]

Alkylation of NADP+ With iodoacetic acid at pH 6.5 yielded N¹-carboxymethyl-NADP+ which, following enzymic reduction, alkaline rearrangement and enzymic reoxidation, gave N6-carboxy, methyl-NADP+. Condensation of these .analogues with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated the N¹ and N6 N-(6-aminohexyl)-acetamide derivatives of NADP+ respectively. The coenzymic activities of the N¹ and N6 adenine-substituted carboxymethyl and N-(6-aminohexyl)-acetamido derivatives of NADP+ were tested with several NADP +-dependent dehydrogenases. The carboxymethyl derivatives were more active (up to 145%) relative to unsubstituted NADP + than the corresponding N-(6-aminohexyl)-acetamido compounds (up to 10%). NADP+-N6-[N-(6-aminohexyl)-acetamide] was coupled to a soluble dextran and to Sepharose 4B by the cyanogen bromide technique. The dextran-bound derivative contained 85 µmol NADP+ analogue/g dry dextran and was reduced at approximately 35 % the rate of native NADP+ with glucose-6-phosphate dehydrogenase. The dextran-bound NADP+ cycled at a rate of about 30% relative to free NADP+ in a system comprising glucose-6-phosphate dehydrogenase and L-glutamate dehydrogenase. Sepharose-bound NADP+ proved to be an effective biospecific adsorbent for affinity chromatography and was competent for the resolution of a mixture of bovine albumin, lactate dehydrogenase and glucose-6-phosphate dehydrogenase. The latter was eluted almost quantitatively with a pulse of 0.5 mM NADP+. [ABSTRACT FROM AUTHOR]

The adenovirus type 2 hexon protein produced in excess during infection was carboxymethylated by reduction and alkylation with iodo[2-14C]acetate in 6 M guanidine hydrochloride. Only one type of polypeptide chain was detected by exclusion chromatography in dissociating buffers. Peptide mapping experiments, sequence analysis of peptides and identification of possible protein terminal residues also suggest that all subunits are similar and probably identical. Structural analysis of [14C]carboxymethylated hexon and of hexon which was labeled in vivo with [35S]cysteine revealed six unique cysteine derivatives in soluble tryptic peptides. A seventh unique cysteine derivative, only partly carboxymethylated, was present in the tryptic core material and was recovered after chymotryptic digestion. Seven unique cysteine derivatives in the hexon subunit, correspond to a minimum molecular weight of about 100 000 for the subunit. This value is consistent with values obtained from exclusion chromatography in guanidine hydrochloride, from mercurial titration of an accessible thiol group in the intact protein and from peptide mapping experiments. Peptides corresponding to more than one tenth of the total number of residues in the protein chain were characterized and, with the inclusion of other peptides studied, about one quarter of all residues are accounted for. The results exclude extensive regions of identity in primary structure within the hexon subunits and the partial resistance towards attack by proteolytic enzymes is structurally explained. [ABSTRACT FROM AUTHOR]

Of the dimeric phosphagen kinases (Mr = 80000) studied, lombricine kinase is the only one which contains only one essential thiol group and one binding site for ADP-Mg. These facts suggest a non-identity between the two subunits of this enzyme. The reversible labeling of the essential thiol group of this protein with 2,4-dinitrofluorobenzene, followed by alkylation of the remaining thiol groups, allowed the separation of the two subunits by means of a Sepharose-mercurial column. The Sepharose-mercurial was prepared by treatment of Sepharose 4B with p-aminophenyl mercuric acetate. Amino-acid analyses, N- and C-terminal determinations and fingerprintings were performed to detect the differences in the primary structure between the two subunits. Furthermore, the Sepharose-mercurial column was used to purify the tryptic peptide containing the essential thiol group of lombricine kinase. By this process, it was possible to obtain, in one step, the peptide in a pure state. It is interesting to point out the applicability of this method for the purification of peptides or proteins containing essential thiol groups which can be specifically labelled by reversible inhibitors.

Purified IgM isolated from the serum of mice bearing the transplantable plasmacytoma MOPC 104E was reduced and alkylated and then analysed by sucrose density gradient centrifugation and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. On partial reduction a mixture of IgM subunits was obtained in the absence of covalently linked 19S IgM. When examined under dissociating conditions this mixture was found to consist of disulphide-linked 7S subunits (IgMs), small amounts of HL subunits and oligomeric IgM ora size intermediate between monomeric and pentameric IgM. In the absence of a dissociating agent and on sucrose density gradient, however, the mixture resolved into a 19S and a 7S peak. The 19S peak consisted primarily of oligomeric IgM and IgMs with small amounts of HL subunits. Thus alkylated IgMs and HL subunits of IgM can associate through non-covalent forces to form a molecule sedimenting at 19S, providing oligorneric forms are present. In the absence of oligomeric forms, IgMs HL subunits and heavy and light chains sediment at about 7S. The products of partial reduction which sediment at 7S and 19S could also be isolated by preparative polyacrylamide gel electrophoresis. When this was done J chain was absent in the former and present in the latter, raising the possibility that J chain does not disulphide bond to each of the five IgMs subunits, constituting an IgM molecule. Thus, within cells secreting IgM, J chain would be expected to mediate the formation of an oligorneric form of IgM. Once the oligomeric structure has been assembled, then non-covalent forces between this and IgMs subunits will cause the formation of a 19S structure, thereby facilitating the final assembly through disulphide bonds. [ABSTRACT FROM AUTHOR]

The section of "The Chemical Reactivity and Modification of Keratin Fibres," presents information about the process of alkylation with respect to keratin fibers. When wool is reduced by tributylphosphine and is alkylated by α-haloacetic acids in a single bath concurrently, near quantitative results are given only by chloroacetic acid. S-carboxymethylcysteine, lanthionine, and lysinoalanine residues are formed by the addition of thioglycollate, cysteine residues, and lysine residues, respectively, to the double bond of the dehydroalanine residue when after repeated reduction of wool with thioglycollate and alkylation with methyl iodide.

Alkylation of NAD+ with iodoacetic acid followed by alkaline rearrangement gave N6-carboxymethyl-NAD+. Condensation of this analogue with 1,6-diaminohexane gave the analogue NAD+- N6-[N-(6-aminohexyl)-acetamide]. The coenzymic activity of the two derivatives was tested with malate dehydrogenase, alcohol dehydrogenase and lactate dehydrogenase. The efficiency relative to unsubstituted NAD+ was in the range of 50-100%. NAD+-N6-[N-(6-aminohexyl)-acetamide] was attached to Sepharose 4B by the cyanogen bromide method. The immobilized NAD+ analogue thus obtained, exhibited cofactor activity when tested in a recycling three-enzyme system (malate dehydrogenase-citrate synthase-lactate dehydrogenase). The immobilized NAD+ analogue proved to be an effective ligand in affinity chromatography. Thus a mixture of albumin, alcohol dehydrogenase and lactate dehydrogenase was resolved with good recovery. The enzymes were eluted with NAD+ and NADH, respectively. [ABSTRACT FROM AUTHOR]

After immunization of rabbits with dermatosparactic calf-skin collagen a predominantly specific response to this kind of collagen and only a weak cross-reaction with soluble skin collagen from normal calves was observed titibodies exclusively reacting with dermatosparactic collagen were obtained upon immunoadsorption. They displayed strong reactions with the constituent pα1- and pα2-chain although both chains might not be equivalent in their antigenic structure. The serologic activity was entirely recovered on a characteristic additional peptide (molecular weight 20000) obtained from pod-chain after colilagenase digestion Reduction and alkylation of this peptide, but not alkylation alone, completely destroyed the antigenic activity which, on the other hand, was quite stable towards the action of various proteases. Antigenic determinants specific for the collagen α-chain of normal calf skin could also be demonstrated m the pod-chain except the one located in the N-terminal pentapeptide sequence. In indirect immunofluorescence tests antibodies specific for dermatosparatic collagen were shown to react with collagen fibers of skin and the interstitial connective tissue of kidney and muscle of normal calves. No staining of glomerular basement membranes was observed. Strong cross-reaction with human but not with rat tissues was obtained. These results suggest that the extended collagen molecules found in dermatosparactic calves are normal tissue constituents which in all probability are identical to the biosynthetic precursor, procollagen. [ABSTRACT FROM AUTHOR]

The molecular basis of the biological effect of alkylation, mutation induction and inactivation, was studied using transforming DNA. Comparative studies of mutation induction after methylation (by methyl methanesulfonate) and ethylation (by ethyl methanesulfonate) showed that methylation was five to ten times more effective than ethylation. This observation confirms chemical studies of the reactivities of the individual bases of DNA but conflicts sharply with observations that methyl methanesulfonate is much less mutagenic than ethyl methanesulfonate in bacteriophage and some higher organisms. In each case the number of mutational events caused in transforming DNA followed single-hit kinetics. After termination of alkylation, the rates of decrease of mutants caused by methylation was twice that caused by ethylation, which fits chemical estimates of depurination rates. Reverse mutation studies with different mutagens and base analogs indicated that methyl methanesulfonate predominantly methylates guanine whereas ethyl methanesulfonate alkylates guanine and also adenine; in both cases the major product is an N7-alkyl purine. Accordingly the cause of base-pairing mistakes is unlikely to be related to stearic effects of the alkyl group but rather due to ionization of the alkylated guanine. In addition, inactivation of transforming activity was observed to be linear with treatment time of DNA with methyl or ethyl methanesulfonate in a semilogarithmic plot. Since the observed inactivation rates cannot be solely explained as inactivating mutagenic events produced by base alkylation, it is likely that phosphate alkylation is inactivating. Lethal and mutagenic DNA alterations are shown to be dissociated processes by the following observations. (a) After termination of alkylation, the number of mutants caused by alkylation of the purine bases decreased linearly with time indicating that depurination is inactivating. (b) Under identical conditions, the number of total transformants decreased three times faster than the number of mutants, indicating that in addition to depurination triester breaks must be responsible for inactivation. Therefore, base alkylations are mutagenic and depurination and triester breaks are lethal DNA alterations. [ABSTRACT FROM AUTHOR]

A method is presented to measure the alkylation of phosphates in DNA after a treatment with an alkylating agent. Using this method, we have shown that phosphate alkylation represents 15% of total alkylation when DNA is alkylated with ethyl methanesulfonate and only 1% of total alkylation when DNA is alkylated with methyl methanesulfonate. Experiments are also presented which show that phosphate triesters resulting from the alkylation of DNA by ethyl methanesulfonate are very stable, most of them remaining intact after heating at 100 °C for 90 min at pH 7.0. [ABSTRACT FROM AUTHOR]

Succinylation and a combination of succinylation with reductive alkylation and trinitrophenylation procedures were used to investigate the function of amino groups of transferrin. It was shown that apo-transferrin had two types of amino groups: those that were easily accessible to chemical modification reagents (60-61 in number) and those that were more difficult to modify (6-7 in number), which were also concerned with the maintenance of the conformation of the protein. The latter type of amino groups became even less accessible to the environment upon the binding of the iron by transferrin. However, the unreactive amino groups were not directly involved in the binding of iron by transferrin, since iron-binding properties could be partially recovered in the extensively succinylated protein. It was concluded that the function of the amino groups in question was probably to stabilize the three-dimensional structure of transferrin. [ABSTRACT FROM AUTHOR]

Quantitative light absorption data of the neutral free flavosemiquinone are reported. It is shown that the visible range of the spectrum of the radical is drastically dependent on the solvent. The spectra of the neutral blue flavosemiquinone are compared with those of the blue flavoprotein radicals and conclusions are drawn concerning the environment of the protein-bound flavosemiquinone. The stabilization of the neutral blue flavosemiquinone through N(5)­alkylation and/or π-complex formation with aromatic compounds is described. The light absorption characteristics of a tautomeric neutral, but red-coloured, flavosemiquinone are described. The results are compared with those obtained from the free anionic and protein-bound red flavosemiquinones. The two types of radicals cannot be distinguished from each other by electron spin resonance spectrometry but may be recognized by means of light absorption spectrophotometry. The possible biological significance of the neutral red flavosemiquinone is discussed. The optical properties of this radical are compared with those of free and protein-bound anionic flavosemiquinones. [ABSTRACT FROM AUTHOR]

Several laboratories have shown that the two carbon atoms C-28 and C-29 of the ethyl or ethylidene side chains of phytosterols are introduced on to a C-24 unsaturated side chain by two methylation steps. A scheme based on previous work suggests the various intermediates which can be formed by stabilisation of the carbonium ion 3. We have shown previously that in the biosynthesis of 5α-stigmastan-3β-ol (11α)and 5α-stigmast-22-en-3β-ol (12α), sterols of the myxomycete Dictyostelium discoideum, routes h and i could be excluded. In the present paper we describe the in vivo conversion, by the myxomycete, of differently labelled lanosterol (13), stigmastanol (11c), and 5α-stigmast-22-en-3β-ol (12c), into the sterols 12a and 11a, and discuss the possible biosynthetic routes for the two sterols. After incorporation of a mixture of [26,27-14C2]lanosterol and [24-³H]lanosterol (³H/14C = 5.60 ± 0.25), the isolated sterols 11a and 12a were converted into 11b (³H/14C = 5.54 ± 0,28) and 14b (³H14C = 5.61 ± 0.28). By ozonisation followed by oxidation in neutral or basic medium. 12a was converted into the esters 15 (³H/14C @ 5.54 ±0.28) and 16 (³H/14C 5.55 ± 0.3), respectively. These results show that during the C-24 methylation. H-24 is not lost: the hydrogen which is lost is located @9' neither at C-24 nor at C-23 and must therefore be at C-25. This excludes routes a and b for sterol 12. After incorporation of a mixture of 126,27-14C2]lanosterol and [23-³H]lanosterol (³H/14C = 4.36 ± 0.1) the isolated sterols 11a and 12a were converted into 11b (³H/14C = 4. 12 ± 0.2) and 14b (³H/14C = 3.32 ± 0.14); a fraction of 12b was degraded into 15 (³H/14C = 2.28 ± 0.31) and 16 (³H/14C= 3.44 ± 0.15). These results show that during the conversion of 13 into 12a, 50% of the H-23 atoms migrate from C-23 to C-24 and 25% remain on C-23 ; the other 25% are eliminated. During the conversion of 13 into 11a both H-23 atoms are retained. These results excluded route g for the two sterols. Since we have demonstrated that the conversion of 11c into 12a is possible in high yields (4 6%) with an efficiency eight times greater than the conversion of 12c into 11a, we propose that the sterol 11a is synthesized in vivo according to route e with migration of H-23 from C-23 to C-24. The sterol 12a is biosynthetized either by route d, f, the 22,23 double bond being introduced during the alkylation step, or by desaturation of the sterol 11a, the C-22 C-23 double bond being introduced after the C-24 alkylation. [ABSTRACT FROM AUTHOR]

Reaction of RNAase A with bromoacetate at pH 5.5 for 1–42 days results in multiple reactions. Alkylation of residues proceeds in the sequence: (1) N-1 of histidine-119; (2) methionine (probably methionine-30); (3) N-3 of histidine-12; (4) N-3 of histidine-105 and N-3 of 1-carboxymethyl histidine-119; and (5) lysine-1. Both histidine-12 and histidine-119 of the same active site are carboxymethylated. A derivative carboxymethylated at both active site histidines is obtained in 1 day and probably some of this derivative is obtained in short reaction times. This is contrary to the conclusions of earlier investigations. Histidine-48 undergoes little or no reaction. The results are in accord with the X-ray structure of RNAase. [ABSTRACT FROM AUTHOR]

Natural variants and chemically modified derivatives of phalloidin have been found or prepared which show all degrees of toxicity. High toxicity persists if only three of the side chains are modified. Acetylation of two of the four OH groups of phalloidin leads to less toxic products, the triacetyl derivative is nontoxic. On tosylation toxicity disappeared after reaction of only two OH groups. Alkylation of the indole nitrogen leads to equally or less toxic (N-methyl, -ethyl, carbamoylmethyl, -tert.butyl) or nontoxic (N-propyl, -pentyl) products. Toxicity is also com- pletely absent if only one of the two rings of the molecule is intact. In optical rotatory dispersion spectra all of the toxic compounds show in aqueous solution two cotton effects centered around 300 nm and 248 nm, whereas in nearly all of the nontoxic derivatives at least one of the effects is missing. Triacetylphalloidin and tribromophalloidin, both nontoxic, also exhibit “toxic” curves in water. Their conformation, however, changes on going to organic solvents like methanol or acetonitril as observed by optical rotatory dispersion studies. Thus, stability of the “aqueous” conformation in a lipophilic environment is apparently the second prerequisite for a toxic action of phallopeptides. [ABSTRACT FROM AUTHOR]

Natural variants and chemically modified derivatives of phalloidin have been found or prepared which show all degrees of toxicity. High toxicity persists if only three of the side chains are modified. Acetylation of two of the four OH groups of phalloidin leads to less toxic products, the triacetyl derivative is nontoxic. On tosylation toxicity disappeared after reaction of only two OH groups. Alkylation of the indole nitrogen leads to equally or less toxic (N-methyl, -ethyl, carbamoylmethyl, -tert.butyl) or nontoxic (N-propyl, -pentyl) products. Toxicity is also completely absent if only one of the two rings of the molecule is intact. In optical rotatory dispersion spectra all of the toxic compounds show in aqueous solution two cotton effects centered around 300 nm and 248 nm, whereas in nearly all of the nontoxic derivatives at least one of the effects is missing. Triacetylphalloidin and tribromophalloidin, both nontoxic, also exhibit “toxic” curves in water. Their conformation, however, changes on going to organic solvents like methanol or acetonitril as observed by optical rotatory dispersion studies. Thus, stability of the “aqueous” conformation in a lipophflic environment is apparently the second prerequisite for a toxic action of phallopeptides. [ABSTRACT FROM AUTHOR]

Trenimon (2,3,5-aziridine-1,4-benzoquinone) reacts with DNA by alkylation of the purine moiety, by formation of apurinic sites, and by induction of strand breaks. The presence of cross links was noted in the later stages of treatment, as was a diminished susceptibility to DNAase action. The agent causes progressive loss of priming ability in the DNA-dependent RNA polymerase system. The RNA synthesized was of shorter chain length and sedimented in the sucrose gradient for the most part with a coefficient smaller than 6 S. [ABSTRACT FROM AUTHOR]

1. At pH 7.5, inactivation of NAD kinase by bromoacetyl pyridine occurs in three independent pseudo-first-order stages. 2. Below pH 7.5, only two stages are distinguishable. 3. The final stage alkylation leads to total reactivation, the earlier stages to the formation of an enzyme species with modified properties. 4. Effects of pH on the time courses of inactivation suggest that the first stage may involve a variable number of sulphydryl groups. 5. The final stage probably involves alkylation of a sulphydryl, with abolition of NAD binding. 6. No central role m catalysis can be postulated for any of the groups modified by bromoacetyl pyridine; two groups known to participate in catalysis and the binding of ATP · Mg are apparently not alkylated. 7. Inhibition by L-malate and by free ATP may occur by attachment of these effectors at separate modifier sites. [ABSTRACT FROM AUTHOR]

1. From the pH dependence of the rate of alkylation of pig heart malate dehydrogenase by iodoacetamide the pKa of the reactive histidine residue was determined as 7.1. 2. The enzyme is not inactivated by iodoacetic acid or 3-bromopropionic acid. 3. NADU protects the enzyme almost completely against alkylation by iodoacetamide whereas NAP, ADP-ribose and N-methylnicotinamide give only partial protection. 4. Oxaloacetate and malate do not protect against alkylation but mixtures of oxaloacetate and NAB or ADP-ribose protect completely, demonstrating formation of abortive ternary complexes. [ABSTRACT FROM AUTHOR]

The total sulfhydryl content of sarcoplasmic reticulum determined in the presence of sodium dodecylsulfate is 14 sulfhydryl equivalents per 105 g of membrane protein, but in the absence of sodium dodecylsutfate only 10 sulfhydryl groups are titrateable with 5,5'-dithiobis (2-nitrobenzoic acid). The rate of ATPase inactivation by N-ethylmaleimide in the presence of ATP yields an ATP-ATPase dissociation constant of 45 µM. When the sulfhydryl group essential for ATPase activity was reacted with N-ethyl-[1-14C]maleimide after all the sulfhydryl groups unrelated to ATPase activity had been reacted previously with unlabeled reagent, only one protein band in electrophorograms was strongly labeled. The presence of ATP during trypsin proteolysis preserves ATPase activity and the band that can be specifically labeled with N-ethylmaleimide is demonstrated in gel electrophorograms. When trypsin proteolysis was carried out in the absence of ATP this band can no longer be seen. The N-ethyl-[1-14C]maleimide labeledband is tentatively identified with the ATPase. [ABSTRACT FROM AUTHOR]

The spectral course of irreversible photoreduction of the flavin nucleus by arylacetates and related compounds has been investigated in detail. The resulting benzylated dihyro flavins and their reoxidation products have been characterized spectrophotometrically, among them 5-benzyl-flavosemiquinone radical and 5-benzylflavoquinonium cation. Reversible and irreversible flavin reduction corresponding to hydride-or group-transfer from substrate to flavin are discussed with respect to their scope and limitations and possible biological implications I the action mechanism of flavin dependent dehydrogenases. [ABSTRACT FROM AUTHOR]

A method involving the use of sieves and differential centrifugation for the preparation and separation of basement membranes from human renal tubules and glomeruli is described. Electron microscopic examination of the material obtained shows the glomerular membrane as a single dense structure, while the tubular membrane is composed of two dense layers separated by a space filled with loose fibrils exhibiting the same periodicity as collagen. The carbohydrate composition of both types of membranes is the same but tubular membranes have a lower content of some specific sugars. Differences also exist in amino acid composition. The content in hydroxylysine is higher in the glomerular membrane while the content in hydroxyproline is higher in the tubular membrane. Gel electrophoresis after solubilization of the material demonstrates the presence of two, may be three, major proteins. [ABSTRACT FROM AUTHOR]

Iodoacetate alkylates and irreversibly inhibits horse-liver alcohol dehydrogenase. A reversible enzyme-iodoacetate complex also forms, as deduced from the following evidence: 1. Rate of inactivation is not proportional to iodoacetate concentration, but, rather, follows Michaelis-Menten-type kinetics. 2. The enzyme is protected competitively by other fatty-acids, and also by chloride. 3. The dissociation-constants of iodoacetate, and formate, and chloride all vary similarly with pH (increasing at high pH, and suggesting a pK = 8.9–9.1). These results are discussed in terms of a model, and it is suggested that the reversible enzyme- iodoacetate complex may be stable, rather than a necessary intermediate for alkylation of the enzyme. The great similarity of the results to those of another zinc-enzyme, carbonic anhydrase, is discussed; and it is suggested that anions bind to a zinc atom in the enzyme. [ABSTRACT FROM AUTHOR]

Fucosterol biosynthesised by Fucus spiralis from [2-14C,(4R)-4-³H1] mevalonic acid has a ³H/14C atomic ratio of 3/5. Isomerisation of the labelled fucosteryl acetate gave 3β-acetoxystigmasta-5,24- diene with a ³H/14C atomic ratio of 2/5. This demonstrated that the fucosterol had a ³H atom located at C-25 and therefore proves that hydrogen (tritium) migration from C-24 to 0-25 occurs during alkylation of the phytosterol side chain. Cycloartenol and 24-methylene cycloartanol also isolated from the Fucus spiralis were shown to be radioactively labelled, there was no evidence of radioactive lanosterol. Tritium was shown to be present at C-3 of the biosynthesised cyclo-arteriol but was absent from C-3 of the fucosterol. [ABSTRACT FROM AUTHOR]

Yeast alcohol dehydrogenase is inactivated at different rates by the antipodes of α-iodopropionic acid and its amide; the D (+) antipodes react faster. The influence of coenzyme, substrate and pH on the reaction velocity were investigated. Under all conditions second order kinetics have been observed. Activation parameters for the alkylation of the SH groups with the different antipodes were calculated from the temperature dependence of the reaction rates. Large differences in the activation enthalpies (ΔΔH = 6.8 kcal/mole and 2.3 kcal/mole, respectively) were found for the reaction of yeast alcohol dehydrogenase with the different antipodes of α-iodopropionic acid and α-iodopropionamide. These were compensated for by differences in activation entropy of —18.4 and — 5.6 entropy units, respectively. These results are interpreted by assuming highly dissymmetric surroundings of the reacting SH groups favouring the D (+) antipodes in the transition state of the alkylation reaction. The negative activation entropy indicates that this favourable transition state may be attained only by restriction of the degrees of freedom. The relationship between the stereoselective chemical reactivity and the stereospecificity of the enzyme is discussed. [ABSTRACT FROM AUTHOR]

The rate of reaction of liver alcohol dehydrogenase with iodoacetamide is independent of pH between pH 6–10. Orthophenanthroline, ADP-ribose and ADP protect the enzyme against alkylation. For iodoacetate, the rate of alkylation decreases with increasing pH over the range 7–9 and reflects the titration of a group with a pK of 7.9. Imidazote enhances the rate of alkylation by both iodoacetamide and iodoacetate and renders the rate of alkylation by iodoacetate pH independent. The interactions of ADP-ribose and imidazole with the enzyme are shown to be independent of each other. A mechanism is suggested in which the substrate and coenzyme are simultaneously bound to zinc. Mechanisms for the activation of both reaction partners are presented. [ABSTRACT FROM AUTHOR]

When undergoing nucleophilic reactions with proteins, α-iodopropionic acid and its amide show greater specificity for SH groups than do iodoacetic acid and its amide. The kinetics and stereochemistry of the reactions of the D(+) and L(-) antipodes of α-iodopropionic acid and its amide with both cysteine and papain were investigated. The antipodes of the SH reagents reacted with these asymmetric SH compounds at different rates. In the reaction with papain, the L(-) antipode of the acid reacted faster than the D(+) antipode; the stereochemical preference was inverted when the amide was used, the D(+)antipode reacting faster than the L(-)antipode. Arguing from the differing reactivities of the SH reagents and their antipodes and the pH dependence of the reaction rates, it has been suggested, that L(-)alpha;-iodopropionic acid is specifically oriented onto the reaction center through the concerted attractive and repulsive actions of a cationic and an anionic group in the protein. These have been tentatively identified as a protonated imidazole residue and a carboxylate group respectively. Parallels between these stereospecific alkylations and the catalytic action of the enzyme are discussed. [ABSTRACT FROM AUTHOR]

The reactivity and the role of sulphydryl groups in adenosine deaminase have been investigated. In the sodium dodecylsulphate-denatured enzyme 2.1 sulphydryl groups are titratable with p-mercuribenxzoate. The native enzyme is inactivated by p-mercuribenzoate or phenyl-mereuric acetate and a stoichiometric relationship exists between the equivalents of mercaptide formed and the inactivation of the enzyme. Substrate analogs protect the enzyme against p-mercuribenzoate and phenylmercuric acetate inactivation. Iodoacetamide, iodacetate, and N-ethylmaleimide, which do not affect the enzyme activity, do not react with sulphydryl groups. The results show that a sulphydryl group unreactive towards the alkylating reagents is essential in some way for the enzymatic activity. The lack of reactivity of the essential sulphydryl group with alkylating reagents suggests that the sulphydryl group may be contained in a hydrophobic region of the enzyme molecule. [ABSTRACT FROM AUTHOR]

Highly purified rabbit IgM anti-Forssman antibody, after reduction in dilute solution with 2-mercaptoethanol and alkylation with iodoacetamide, showed no diminution and often some increase in haemagglutination of sheep erythrocytes. The capacity to cause complement dependent lysis was, however, destroyed. It is shown that despite reduction and alkylation reaggregation of the subunits occurs to a variable extent producing non-covalently bound aggregates with S values ranging from 7 to more than 20. The aggregates can bind specifically to and agglutinate erythrocytes with greater effectiveness as their size increases. This spontaneous re-aggregation is not prevented by the presence of gelatin, but fails to occur when the IgM antibody is reduced in the presence of a variety of other proteins. The effect of extraneous proteins is evident only when they are present at the time of reduction or are added within 10–15 minutes. It is suggested that following rupture of the interchain disulphide bonds, the IgM molecule can undergo a complex, time-dependent rearrangement into new stable forms which retain combining site activity but do not involve covalent bonds. [ABSTRACT FROM AUTHOR]

Specific antibodies against periodate oxidized capsular polysaccharides isolated from different Klebsiella types, have been demonstrated in rabbit immune sera against five out of eleven Klebsiella types investigated. It is assumed that an oxidation similar to the periodate oxidation must occur in vivo. Antibodies specifically directed against the oxidized polysaccharide may appear early in the immunization period, and decrease or disappear again later. Reduction and alkylation of the antibodies against oxidized polysaccharide in one serum indicated that they probably belonged to the γM-immunoglobulins, whereas the antibodies against the unoxidized polysaccharide appear to be mainly γG. [ABSTRACT FROM AUTHOR]

Ovalbumin, Escherichia coli lipopolysaccharide, and a combination of metabolic and somatic antigens of Haemonchus contortus were injected into rabbits via the intravenous, intramuscular, intradermal and intraperitoneal routes. Haemagglutination titres were obtained on untreated sera and sera after reduction and alkylation. The data suggest that the route of injection most efficient in stimulating the production of circulating antibody would depend upon whether the antigen in question elicits 19S globulin, 7S globulin or a combination of the two either in the form of sequential synthesis or a prolonged response of both types of immunoglobulins. [ABSTRACT FROM AUTHOR]

Cotton fabric has been methylated under a variety of conditions. The extent of methylation has been shown to be dependent on the time of methylation and on the degree of swelling or disruption of structure. The latter is caused by the alkali pretreatment or by the swelling of the partly methylated cellulose. Fabric physical properties have been measured and the influence of the methylation treatment has been examined. [ABSTRACT FROM AUTHOR]

The article presents a subject index. Some of the subjects are: acute nephritis, immunoglobin and complement changes in children; Addition's disease and hypothyroidism, familial distribution of organ specific antibodies in blood of patients; Addison's disease, anti-adrenal cellular hypersensitivity; alkylation and reduction; amyloidosis in neonatally thymectomized and anti-lympocyte serum treated mice; anti-adrenal cellular hypersensitivity in Addison's disease; antibody--anti-lymphocyte--and autoimmune disease; antibody-producing capacity in man; Carcinoma of the post-nasal space and so on.

Purification of 7S IgM from the serum of a patient with Waldenström's macroglubulinaemia by means of gel filtration and elution from an anti-μ-chain immunoadsurbent is described. Reduction of pure 7S IgM with 2-mercaptoethanol followed by removal of the reducing agent increased slightly the sedimentation in the ultracentrifuge of a fraction of the molecules. By contrast, when 19S IgM and a small amount of 125I-labelled 7S IgM were similarly treated together, a large fraction of the iodine-labelled material sedimented with the reassociated 19S IgM. The reassociation properties of purified alkylated and non-alkylated IgM subunits made by reduction of 19S IgM with 0.2 M mercaptoethanol or 0.02 M cysteine were analysed as described for 7S IgM. [ABSTRACT FROM AUTHOR]