Evidence for heterogeneous forms of the snake venom metalloproteinase jararhagin: a factor contributing to snake venom variability

The reprolysin subfamily of metalloproteinases includes snake venom metalloproteinases (SVMP) and mammalian disintegrin/metalloproteinase. These proteins are synthesized as zymogens and undergo proteolytic processing resulting in a variety of multifunctional proteins. Jararhagin is a P-III SVMP isolated from the venom of Bothrops jararaca. In crude venom, two forms of jararhagin are typically found, full-length jararhagin and jararhagin-C, a proteolytically processed form of jararhagin that is composed of the disintegrin-like and cysteine-rich domains of jararhagin. To better understand the structural and mechanistic bases for these forms of jararhagin in the venom of B. jararaca and the source of venom complexity in general, we have examined the jararhagin forms isolated from venom and the autolysis of isolated jararhagin under the conditions of varying pH, calcium ion concentration, and reducing agents. From our results, jararhagin isolated from venom appears as two forms: a predominant form that is stable to in vitro autolysis and a minor form that is susceptible to autolysis under a variety of conditions including alkaline pH, low calcium ion concentrations, or reducing agent. The autolysis site for production of jararhagin-C from isolated jararhagin was different from that observed for jararhagin-C as isolated from crude venom. Taken together, these data lead us to the conclusion that during the biosynthesis of jararhagin in the venom gland at least three forms are present: one form which is rapidly processed to give rise to jararhagin-C, one form which is resistant to processing in the venom and autolysis in vitro, and one minor form which is susceptible to autolysis under conditions that promote destabilization of its structure. The presence of these different forms of jararhagin contributes to greater structural and functional complexity of the venom and may be a common feature among all snake venoms. The biological and biochemical features in the venom gland responsible for these jararhagin isoforms are currently under investigation.     

Nevoid basal cell carcinoma syndrome (Gorlin syndrome)

Brachydactyly ("short digits") is a general term that refers to disproportionately short fingers and toes, and forms part of the group of limb malformations characterized by bone dysostosis. The various types of isolated brachydactyly are rare, except for types A3 and D. Brachydactyly can occur either as an isolated malformation or as a part of a complex malformation syndrome. To date, many different forms of brachydactyly have been identified. Some forms also result in short stature. In isolated brachydactyly, subtle changes elsewhere may be present. Brachydactyly may also be accompanied by other hand malformations, such as syndactyly, polydactyly, reduction defects, or symphalangism. For the majority of isolated brachydactylies and some syndromic forms of brachydactyly, the causative gene defect has been identified. In isolated brachydactyly, the inheritance is mostly autosomal dominant with variable expressivity and penetrtance. Diagnosis is clinical, anthropometric and radiological. Prenatal diagnosis is usually not indicated for isolated forms of brachydactyly, but may be appropriate in syndromic forms. Molecular studies of chorionic villus samples at 11 weeks of gestation and by amniocentesis after the 14^th week of gestation can provide antenatal diagnosis if the causative mutation in the family is known. The nature of genetic counseling depends both on the pattern of inheritance of the type of brachydactyly present in the family and on the presence or absence of accompanying symptoms. There is no specific management or treatment that is applicable to all forms of brachydactyly. Plastic surgery is only indicated if the brachydactyly affects hand function or for cosmetic reasons, but is typically not needed. Physical therapy and ergotherapy may ameliorate hand function. Prognosis for the brachydactylies is strongly dependent on the nature of the brachydactyly, and may vary from excellent to severely influencing hand function. If brachydactyly forms part of a syndromic entity, prognosis often depends on the nature of the associated anomalies.     

Identification of molecular forms of cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital and 3-methylcholanthrene

Eight electrophoretically homogeneous forms of cytochrome P-450 were isolated from liver microsomes of phenobarbital (PB)- and 3-methylcholanthrene (MC)-induced male Wistar rats, using chromatography on 1.8-diaminooctyl-Sepharose, SEAE-Sephacel and hydroxylapatite. These cytochrome forms were compared to those described in literature in terms of their ability to metabolize androstenedione (AD), benzphetamine (BP) and 7-ethoxyresorufin (7-ER). Cytochrome P-450b capable of catalyzing with a high specificity the 16-hydroxylation of AD and N-demethylation of BP, and cytochrome P-450e immunologically related to P-450b but incapable of catalyzing these reactions were isolated from PB-microsomes. Besides, a male-specific cytochrome P-450h catalyzing the 16 alpha-hydroxylation of AD was isolated from PB-microsomes. Cytochrome P-450c possessing a high 7-ER-O-deethylase activity, and a high spin cytochrome P-450d as well as cytochrome P-450a specifically catalyzing the 7 alpha-oxidation of AD were isolated from MC-microsomes. Two forms of cytochrome P-450 isolated from PB-microsomes possessed no such activities. Data from immunochemical analysis suggest that one of these forms can be identified as cytochrome P-450k. It is concluded that the specificity of metabolism and the molecular activity of Wistar rat liver cytochrome P-450 forms are comparable with the corresponding parameters of hemoproteins isolated from other rat species. At the same time, data from metabolic analysis are suggestive of differences in the levels of certain cytochrome P-450 forms, in particular P-450a.     

Genetic properties of the Salmonella enteritidis R404 plasmid aggregate I. Conjugational separation of different derivative plasmid aggregates and their genetic properties.

Conjugational transfer of R404 factor, found in Salmonella enteritidis strain, was examined. Six derivative forms differing in resistance pattern were isolated. The manner of conjugational separation during transfer of R404 factor from E. coli to E. coli strain was the same as from the original strain of S. enteritidis to E. coli strain. During following conjugation, all isolated forms except one, could give forms differing in a resistance pattern from the parenteral ones. This one form, from which different conjugational forms could not be separated, was assumed to be a single plasmid and was designated pCK2. All other forms, including the original R404 factor were assumed to be plasmid aggregates.     

Multiple forms of alkaline phosphatase from Escherichia coli cells with repressed and derepressed biosynthesis of the enzyme.

Isolation of multiple forms of alkaline phosphatase from Escherichia coli cells with repressed and derepressed biosynthesis of the enzyme is reported. Three enzyme forms were isolated from cells with derepressed synthesis, and one form was isolated from cells with repressed enzyme synthesis. The multiple enzyme forms did not differ in pH optimum, thermostability, or the degree of inhibition with orthophosphate; however, they did differ in the relative rate of hydrolysis of different substrates. The addition of substrates to the cells during enzyme derepression resulted in changes of the ratio of the multiple forms.     

Isolation and characterization of the affinity chromatography forms of human Glu- and Lys-plasminogens and plasmins.

Affinity chromatography forms, 1 and 2, were each isolated from human Glu- and Lys-plasminogens by gradient elution from a L-lysine-substituted Sepharose column with a linear gradient of epsilon-aminocaproic acid. Although each of the two zymogen forms contains two affinity chromatography forms, the relative concentrattions of these forms in each of the zymogen preparations depended upon the plasma sample or enriched plasma fraction used for the preparation of the zymogen. Specific analytical acrylamide gel electrophoretic systems were used for the characterization of the zymogen and enzyme forms, and their component affinity chromatography forms, 1 and 2. The four zymogen affinity chromatography forms, Glu-1-plasminogen, Glu-2-plasminogen, Lys-1-plasminogen, and Lys-2-plasmingoen, show distinct stepwise differences in their molecular size and charge. The Glu-1-form is the largest in molecular size and the most acidic, and the Lys-2-form is the smallest in molecular size and the most basic. The proteolytically altered Lys-1- and Lys-2- forms appear to be specifically df the zymogen affinity chromatography forms showed a different distribution of isoelectric forms. The major isoelectric forms isolated from Glu-plasminogen with pI values of 6.2, 6.3, 6.4, and 6.6, and the major isoelectric forms isolated from Lys-plasminogen with pI values of 6.7, 7.2, 7.5, 7.8, and 8.1, (Summaria, L., Arzadon, L., Bernabe, P., Robbins, K. C., and Barlow, G. H. (1973) J. Biol. Chem. 248, 2984-2991) were shown to be mixtures of the Glu-1- and Glu-2- forms, or the Lys-1- and Lys-2- forms, respectively. Although the sialic acid contents of the Glu- and Lys- forms appear to be similar, the isolated affinity chromatography forms show distinct differences. The sialic acid contents of the Glu-1- and Lys-1- forms are identical, and are substantially higher than the sialic acid contents of the Glu-2- and Lys-2- forms which are also identical to each other. It is possible that the charge difference between the zymogen-1- and -2- forms may be related to the differences in their sialic acid content. Each of the four zymogen affinity chromatography forms, when activated by urokinase in the presence of the plasmin inhibitor, Trasylol, was converted to an apparently unique and different enzyme form. The four enzyme forms show distinct stepwise differences in molecular size; Glu-1-plasmin is the largest in size whereas Lys-2-plasmin is the smallest in size. Each plasmin-derived carboxymethyl heavy(A) chain was found to be different in molecular size, but the two carboxymethyl light(B) chains found in each of the four enzyme forms appeared to be identical and of the same molecular sizes. The four heavy(A) chains show a stepwise difference in molecular size; the Glu-1-heavy(A) chain is the largest in size whereas the Lys-2-heavy(A) chain is the smallest in size...     

Isoenzymes de l'anhydrase carbonique d'un poisson euryhalin: Variations en relation avec l'osmoregulation

A chemical study of carbonic anhydrase (EC 4.2.1.1) from the red blood cells and the gills of an euryhaline fish (Anguilla anguilla) is presented. Animals adapted to fresh water were compared to those adapted to sea water.The physicochemical constants of the various molecular forms isolated by chromatograhy and isoelectric focusing were determined; isoeleletric pH, molecular weight, and the K_m and V/E of the enzyme dehydration activity were compared.In both red cells and gills of fish adapted in either media various forms were isolated, characterized by different enzymatic kinetics (high- and low-activity forms) but having the same molecular weight (27 250). Some isoenzymes isolated from the gills differed significantly from those isolated from the red cells.Adaptation to fresh water or sea water is accompanied by modifications in the distribution of yhe isoenzymes in both red cells and gills: adaptation to sea water is characterized by a shift of the molecular forms towards an isoelectric pH higher than pH = 6.The role of these enzymes is discussed under both a physiological and biochemical point of view in relation to the electrolyte exchange across fish gill. The origin of the different molecular forms of the carbonic anhydrase is discussed in relation to the prevailing theories on this subject.     

At least 60 ADP-ribosylated variant histones are present in nuclei from dimethylsulfate-treated and untreated cells.

The level of histone adenosine diphospho (ADP) ribosylation was studied in isolated nuclei from mouse myeloma cells in culture. The cells were treated with dimethylsulfate (DMS), a DNA-methylating agent, and histones were analyzed using two-dimensional gel electrophoresis. Seventeen or more bands probably representing mono-to heptadeca (ADP-ribosylated) histones could be visualized for each major variant histone. DMS treatment, by increasing the number of chromatin sites undergoing repair, greatly enhanced histone ADP-ribosylation. When histones were labeled in a cell lysate rather than in isolated nuclei, mono- and oligo(ADP-ribosylated) histone forms prevailed. The presence of approximately 87 ADP-ribosylated variant histone forms in cell lysates and of approximately 170 in isolated nuclei is shown for the first time in this work. Previous studies show multiple ADP-ribosylated forms for only histone H1. The theoretical number of variegated nucleosomes is thus much higher than previously thought, provided that histone-histone contacts are not disrupted at up to a certain level of histone ADP-ribosylation.     

Biological characteristics of Streptococcus pneumoniae strains isolated from children with pneumococcal diseases

The biological properties of 97 S. pneumoniae strains isolated from 92 children with purulent meningitides, meningoencephalitides, pneumonia and otitis, hospitalized at the Leningrad Research Institute of Childhood Infections, were studied. The data obtained in this investigation were indicative of the formation of atypical forms of pneumococci (R-forms, unbalanced growth forms and L-forms) in all clinical forms of pneumococcal infection in children. In purulent meningitides and meningoencephalitides serovars 1 and 6, in pneumonia serovars 1, 3 and 6 and in otitis serovars 6 and 19 played the leading role. The determination of sensitivity to antibiotics showed that the forms under study retained high sensitivity to a number of antibiotics. The appearance of strains resistant to benzylopenicillin was registered. (10%). The isolated strains either possessed low virulence or were avirulent in bioassay on white mice.     

The antilysozyme activity of Mycobacterium tuberculosis L forms

The method for the detection of antilysozyme activity (ALA) in M. tuberculosis L forms was developed. The level of ALA in M. tuberculosis L forms isolated from patients with different clinical forms of the disease varied within 1-5 micrograms. M. tuberculosis L forms with the ALA level > 4 micrograms were isolated from patients with the progressing course of the disease. The method for the prognostication of the course of the tuberculous process in the lungs by the results of the antilysozyme test was proposed.     

Characterization of multiple forms of prostatropin (prostate epithelial cell growth factor) from bovine brain

Two molecular forms of prostatropin distributed among five chromatographic peaks have been isolated from bovine brain by heparin-Sepharose affinity and reverse phase high performance liquid chromatography. One form has an apparent molecular weight of 16000 and an amino terminal sequence of asn-tyr-lys-lys-pro-lys-leu-leu-tyr-x-ser-asn-gly. The other form has an apparent molecular weight of 18000 and a blocked amino terminus. Both forms are similar in amino acid composition. The sequence of a proteolytic fragment from the blocked form overlaps the NH2-terminal sequence of the unblocked form, therefore, the smaller form may be derived from the larger form through proteolytic processing. Both forms contain regions identical in sequence to brain-derived, heparin-binding growth factors that have been isolated on the basis of mitogenic activity for fibroblasts and endothelial cells. Both forms of prostatropin exhibit potent mitogenic activity for normal and tumor prostate epithelial cells.     

Role of carbohydrate content on the properties of galactose oxidase from Dactylium dendroides

The stability of intracellular, extracellular, and deglycosylated forms of galactose oxidase was compared with respect to the denaturing effects of heat, pH, and guanidine hydrochloride. The highly glycosylated forms were found to be more stable to pH and thermal inactivation. All forms were reversibly denaturated by guanidine hydrochoride, but the extent was dependent on the carbohydrate content. Deglycosylation did not affect the affinity of the enzyme for dihydroxyacetone and galactose. Exposure of different forms of galactose oxidase to proteases like pronase and trypsin resulted in a rapid degradation of the glycoenzymes with the formation of stable products. After pronase digestion of intra- and extracellular forms of galactose oxidase catalytic species were isolated by gel filtration. The species (61 and 42 kDa) isolated from pronase-digested extracellular enzyme lost their ability to oxidize primary alcohols. Species (67 and 46 kDa) obtained from the intracellular enzyme kept the specificity of the original enzyme. Active pronase-derived peptides (42 and 46 kDa, respectively) had a higher carbohydrate content than the inactive ones.     

Isoenzymes of carbonic anhydrase of an euryhaline fish. Variations related to the osmoregulation]

A chemical study of carbonic anhydrase (EC 4.2.1.1) from the red blood cells and the gills of an euryhaline fish (Anguilla anguilla) is presented. Animals adapted to fresh water were compared to those adapted to sea water. The physiochemical constants of the various molecular formsisolated by chromatography and isoelectric focusing were determined; isoelectric pH, molecular weight, and the Km and V/E of the enzyme dehydration activity were compared. In both red cells and gills of fish adapted in either media various forms were isolated, characterized by different enzymatic kinetics (high- and low- activity forms) but having the same molecular weight (27 250). Some isoenzymes isolated from the gills differed significantly from those isolated from the red cells. Adaptation to fresh water or sea water is accompanied by modifications in the distribution of the isoenzymes in both red cells and gills: adaptation to sea water is characterized by a shift of the molecular forms towards an isoelectric pH higher than pH equals 6. The role of these enzymes is discussed under both a physiological and biochemical point of view in relation to the electrolyte exchange across fish gill. The origin of the different molecular forms of the carbonic anhydrase is discussed in relation to the prevailing theories on this subject.     

Oestrogen sulfotransferase: isolation of a high specific activity species from bovine placenta

During the course of a study of the control of expression of steroid-binding proteins in human mammary cancer oestrogen sulfotransferase was isolated from bovine placenta. By a combination of salt precipitation and ion-exchange and gel-permeation chromatography two forms of the enzyme were isolated. The forms, which apparently differ only in charge, have specific activities 100-300 times greater than has previously been reported for the enzyme. Partial peptide sequences of these enzymes are presented.     

Endochironomus tendens (F.) (Chironomidae,Diptera) an example of stasipatric speciation

Endochironomus tendens has been studied from different localities and geographically isolated populations. On the basis of the detailed karyotaxonomic analysis the investigated material can be divided into two forms. These forms are distinguished by their karyotype and phenotype. The results of the karologica and hybridization analysis showed that the differences between these forms lie only on the basis of microevolution differentiation of species, without being reproductively isolated. This is an example of stasipatric speciation-chromosomal aberrations having an adaptive value in a homozygous state are fixed in definitive localities of species range.     

Atypical behaviour and survival of Streptococcus pyogenes L forms during intraperitoneal infection in rats

Groups of rats were injected intraperitoneally with cell wall-deficient (L) forms of Streptococcus pyogenes, with their parental (S) forms, as well as with a combined inoculum of both forms (S+L). Peritoneal exudate samples were harvested on days 1, 3, 7, 15 and 30 after challenge and were investigated by microbiological, electron microscopic, cytometric and biochemical methods. Parental S forms were isolated from peritoneal exudate samples up to day 15 post infection, while L form cultures were isolated until the end of the examined interval. Electron microscopic examination revealed continuous adhesion of L forms on the macrophage surface as well as intracellular persistence inside them. It was demonstrated that the intraperitoneal inflammatory response to L form infection was higher than to the other infections and the monocyte-macrophage populations were predominant. The established atypical behaviour and long survival of S. pyogenes L forms in the rat's peritoneum could explain some of the mechanisms of the pathogens' persistence as well as the reasons for chronic streptococcal infections.     

Multiple forms of human phosphoglycerate kinase.

Phosphoglycerate kinase was isolated by affinity chromatography from human skeletal muscle and erythrocytes. As in the tissue extracts, the purified enzyme showed in Cellogel electrophoresis one major and two minor bands with phosphoglycerate kinase activity. The multiple forms were separated by chromatography on CM-Sepharose. From the three separated forms, A, B, and C, the latter was not detectable in electrophoresis of tissue extracts or in the purified unresolved phosphoglycerate kinase. The faintest, most anodically migrating form observed in the tissue extracts could not be isolated in pure form by chromatography on CM-Sepharose. The electrophoretic mobility of the phosphoglycerate kinase forms depended strongly on the buffer systems used. The different forms had identical molecular weight, substrate affinity, and heat stability and were inhibited to the same extent by antibody. They could also not be separated by column affinity chromatography. Small differences were found in thiol group content and in the specific activity, the latter being a consequence of diminished free sulfhydryl residues. Exposure to either reductive or oxidative conditions changed the specific activity, but did not result in interconversion among the pure forms. The multiple forms probably arise as a result of epigenetic factors occurring after the primary polypeptide chain has been synthesized.     

Insulin-induced dissociation of its receptor into subunits: possible molecular concomitant of negative cooperativity

The detergent-solubilized avian insulin receptor retains negative cooperativity and other binding properties of the membrane bound form. On gel filtration the receptor elutes as a single peak with a Stokes radius of 72 A. Preincubation of the receptor with low levels of insulin leads to the formation of a second, smaller form with a Stokes radius of 40 A. The percent of receptor in this second peak is proportional to the insulin concentration and correlates well with the insulin-induced increase in dissociation rate (negative cooperativity). Both the isolated high molecular weight and the isolated low-molecular-weight forms of the receptor re-equilibrate in the presence of insulin and, upon refiltration of either isolated peak, both forms of the receptor are obtained. These results are compatible with a model of the insulin receptor in which a tetrameric form can dissociate to a monomeric form as a concomitant of negative cooperativity.     

Characterization and purification of polymorphic arylalkylamine N-acetyltransferase from the American cockroach, Periplaneta americana.

We separated two forms of arylalkylamine N-acetyltransferase (AANAT) from various organs of the American cockroach, Periplaneta americana. Both forms of the enzyme had an equivalent molecular mass of 28 kDa. One form isolated from the testicular accessory glands had high enzyme activity at acidic pHs. The isoelectric point was 5-6 and the substrate specificity was wider than the other type. The other isolated form from female midguts had a higher level of enzyme activity at basic pHs. These findings suggested that P. americana contains polymorphic AANAT, as is the case in Drosophila melanogaster. These forms differed not only in pH specificity, and substrate specificity but in chromatographic behavior and kinetic properties. Most of the organs we examined contained a mixture of the two forms since two types of AANAT activity were separated in different chromatographic fractions when two pH conditions were used for activity measurement.