Biosynthesis of the multiple forms of rabbit cardiac cathepsin D

Rabbit cardiac cathepsin D exists as multiple isomeric forms of Mr = 48,000 within cardiac tissue. Their mechanism of formation and their functional role in cardiac protein degradation are unknown. We have previously demonstrated that cathepsin D is initially synthesized as an Mr = 53,000 precursor that is processed by limited proteolysis within cardiac lysosomes to the Mr = 48,000 active forms of the enzyme. To determine if the multiple forms of active cathepsin D originate from a common precursor, isolated perfused Langendorff rabbit hearts were labeled in pulse (15 or 30 min) and pulse-chase (30 or 150 min) experiments with [35S]methionine. Newly synthesized cathepsin D was isolated by butanol/Triton X-100 extraction and immunoadsorption with anti-cathepsin D IgG-Sepharose, and the isomeric forms were separated by two-dimensional electrophoresis and fluorography. After 15- and 30-min pulse perfusions, 35S-labeled cathepsin D appeared as a single precursor form (Mr = 53,000, pI = 6.6). After 30-min pulse and 30-min chase, the precursor was modified to yield multiple precursor forms, all with molecular weight 53,000, but with differing pI values (6.6-6.0). After 30-min pulse and 150-min chase perfusion, multiple forms of both precursor and proteolytically processed active cathepsin D (Mr = 48,000, pI = 6.2-5.6) were detected. The 35S-labeled, proteolytically processed forms of active cathepsin D co-migrated with the major cathepsin D forms present in cardiac tissue. Subcellular fractionation and perfusions in the presence of chloroquine demonstrated that the multiple precursor forms of cathepsin D originated in a nonlysosomal intracellular compartment. Thus, the multiple forms of active cathepsin D originate from a common high molecular weight precursor, and their synthesis occurs prior to the limited proteolysis of the precursor in cardiac lysosomes.     

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.     

Biochemical processes at the stage of withering during black tea production

We determined the molecular weight and some properties of multiple forms of phenol oxidase from tea leaves and four other perennial plants. It was shown that multiple high- and low-molecular forms of phenol oxidase differed in substrate specificity. Low-molecular forms of the enzyme mostly demonstrated hydroxylase activity, while high-molecular forms showed catechol oxidase activity. It was revealed that the withering stage of black tea production is accompanied by the formation of only high-molecular forms of phenol oxidase, which possess catechol oxidase activity crucial for the procurement of oxidative reactions and the quality of the product.     

Nature of the multiple forms of sweet-potato glucose phosphate isomerase.

The previously reported isoenzymes of sweet-potato glucose 6-phosphate isomerase were resolved by DEAE-cellulose chromatography. The multiple forms exhibited identical electrophoretic properties and electrofocused as a single component with an apparent isoelectric pH of 4.0. Chromatographic studies also suggest that the multiple forms do not represent true isoenzymes.     

Comparative study of the multiple molecular forms of NAD-dependent glutamate dehydrogenase in the liver and femoral muscle at different stages of swine embryogenesis[]

The multiplicity of NAD-dependent glutamate dehydrogenase (GDH) was studied in the liver and femoral muscle of 30, 60 and 108 days old pig embryos by the method of discelectrophoresis in polyacrilamide gel. The GDH was shown to be presented in the tissues under study by 3--4 multiple molecular forms. Specific ratios of the multiple GDH forms were established in the tissues under study at different stages of embryogenesis.     

Distribution of the multiple molecular forms of glucose-6-phosphate dehydrogenase in different physiological states.

The distribution of the multiple molecular forms of rat liver and mammary gland glucose-6-phosphate dehydrogenase was determined by electrophoresis on 5% polyacrylamide gels. In both of these organs, changes in the distribution of enzyme activity among the several forms was slight even when approximately 20- to 40-fold changes in enzyme specific activity were achieved by fasting-refeeding experiments (for liver) or during pregnancy and lactation (for mammary gland). It was concluded that the induction of glucose-6-phosphate dehydrogenase in these two organs occurs without any major redistribution among the multiple molecular forms of this enzyme.     

心肌细胞团搏动的整数倍节律的实验观察

实验报道了心肌细胞团自发性同步化搏动的一类新节律——整数倍节律。这种稳定的节律模式由两种相关的搏动形式的随机交替出现形成,这两种搏动形式中任何一种的出现间期具有整数倍特征。在静息状态和周期1搏动间的随机交替形成0-1整数倍节律,在周期1搏动和周期2搏动之间的随机交替形成l-2整数倍节律。0-1整数倍是居于静息状态和周期1节律之间的节律模式,1-2整数倍是居于周期1节律和周期2节律之间的节律模式,实验所见的节律转迁过程清楚地展示了静息状态、0-1整数倍节律、周期1节律、1-2整数倍节律、周期2节律等顺序地构成的一种“节律谱系”。“节律谱系”的观念可以为认识正常和异常心律的关系和其间的转迁机制提供深刻的理论启示。     

Interrelation between the charge isoforms of mammalian ornithine decarboxylase

Ornithine decarboxylase (ODC) isolated from a variety of tissues has been separated, using DEAE ion-exchange chromatography, into multiple peaks of activity that appear to be related to control of this enzyme stability. Reports of these charge isoforms in current literature are generally unclear as to whether these represent a covalent posttranslational modification or merely an alteration in structural conformation or association. In this study we investigated the relationship of this form separation to the degree of enzyme polymerization, interaction with other proteins and buffer components, and the multiple isoelectric forms of this enzyme noted in denaturing concentrations of urea. High-performance chromatography techniques were used to demonstrate that two of the major enzyme forms, ODC I and II, are really monomers of the enzyme, while minor peaks of activity frequently observed to elute after ODC II contain various dimeric enzyme states. Pyridoxal 5'-phosphate (0.05 mM) added to isolated enzyme preparations composed of I and II monomers induced the formation of I and II dimers as well as a mixed I-II dimer. All three dimer forms were observed to be natural components of freshly isolated crude cell homogenates. The charge distinction between the monomer forms I and II was found to be maintained during ion-exchange chromatography in the presence of 8 M urea, and the enzyme isoforms demonstrated distinct bands on isoelectric focusing gels run in the presence of 9 M urea. Thus, although some of the multiple ornithine decarboxylase forms identified by ion-exchange chromatography of crude mammalian cell homogenates are related to enzyme conformation, the two major forms are distinctly charged protein states that can be visualized using two-dimensional gel electrophoresis of highly purified samples.     

The heterogeneity of sorbitol dehydrogenase from the cytoplasm of mammalian brain cells

The multiple molecular forms of sorbitoldehydrogenase in cytoplasm of brain cells of bull, ground squirrel, guinea-pig, rat, hamster and mouse have been found using the method of electrophoresis in polyacrylamide gel and the subsequent specific dyeing for the fermentative activity. All revealed zones of activity are related to the slowly migrating ones. A set of multiple molecular forms from different sources is various. A form with relative electrophoretic activity 0.385 is found in all analyzed animals. The conditions for obtaining of distinct zones of activity on zymograms are chosen.     

Absence of tyrosine aminotransferase multiple forms in several mammalian animals

Tyrosine aminotransferase multiple forms occurring in rat liver are not present in all mammalian species. Among animals examined only rat and mouse liver possesses multiple forms of tyrosine aminotransferase; in guinea-pig, rabbit, bovine and sheep liver the enzyme occurs in a single form. The presence of lysosomal converting factor (cathepsin T), responsible for arising of multiple forms of tyrosine aminotransferase in rat liver, has been checked in another species lacking enzyme subforms. Lysosomal extracts of guinea-pig liver interconverts tyrosine aminotransferase from rat liver; lysosomal extracts of rat liver does not generate multiple forms of the enzyme from guinea-pig liver. It has been concluded that in some animals hepatic tyrosine aminotransferase is resistant to the proteolytic cleavage by lysosomal cathepsin T.     

Multiple forms of brain cholinesterase in phylogenesis and ontogenesis of vertebrates]

Multiple forms of the brain cholinesterase have been investigated by means of the original technique based on the extraction, ammonium sulphate salting out and gel filtration on Sephadex G-200. It was shown that the number of multiple forms increases in phylogenesis (8 in triton, 13 in rat, 18 in cat, 16 in dog and 23 in man), although their relative enzymic activity decreases. Isolated multiple forms with high molecular weight enzymatically are classified as acetylcholinesterases. In higher brain structures, multiple forms are more numerous. Butyrylcholinesterases and aliesterases are more abundant in lower brain structures. In the neural plate of the developing triton, it is possible to detect the forms with high specific activity, which exceeds that in adult animals.     

Isolation and properties of trypsin inhibitor from the leaves of pedunculate oak]

Four protein fractions inhibiting trypsin are isolated from the English oak leaves by the method of chromatography on DEAE-cellulose. Three active fractions more are found in each of them after the affinity chromatography on trypsin-agarose. Each of 12 multiple forms in the calcium-free medium contains different sets of proteins and oligopeptides possessing rather high inhibiting activity. Ca2+ being introduced to the medium, all the multiple forms somewhat increase their activity, having one peak (Mm approximately 16.5 kDa) on the column with sephadex G-50. The inhibitor possesses high indices of denaturation stability and specificity to trypsin.     

Multiple forms of amylase in leaf extracts: electrophoretic transfer of the enzyme forms into amylose-containing polyacrylamide gels

A method for the analysis of multiple forms of glucan-degrading enzymes is described. The procedure consists of the separation of the proteins by electrophoresis or isoelectric focusing in glucan-free polyacrylamide gels followed by the nondenaturing electrophoretic transfer into a second polyacrylamide layer which contains immobilized glucans. The method combines the resolving power of electrophoretic separations in glucan-free media with the sensitivity of amylase activity detection in amylose-containing polyacrylamide gels. The procedure is especially useful when samples containing low amylase activity, but a large number of multiple enzyme forms, are to be analyzed.     

Picrotoxinin and Phorbol-12-Myristate-13-Acetate Stimulate the Secretion of Multiple Forms of Somatostatin from Cultured Rat Brain Cells

The molecular forms of somatostatin (SRIF) secreted by cultured fetal rat brain cells were resolved using reverse phase high performance liquid chromatography followed by radioimmunoassay. Multiple forms of SRIF-like immunoactivity were detected in media from cells treated with either picrotoxinin, phorbol-12-myristate-13-acetate, or high potassium. For stimulated cells, elevated levels of an SRIF-28-like molecule, an SRIF-14-like molecule, and a hydrophobic SRIF-like molecule were observed compared to basal conditions. All three forms of SRIF-like molecules were also detected in acid extracts of whole cells. The data are consistent with the possibility that secretion of multiple SRIFs , including SRIF-28, may be regulated by multiple effectors and mechanisms.     

Phenoxazinone Synthetase from Streptomyces antibioticus: Multiple Activities of the Enzyme

A procedure for the preparation of relatively large quantities of highly purified phenoxazinone synthetase from Streptomyces antibioticus is described. Enzyme preparations consisted of multiple forms, as determined by polyacrylamide gel electrophoresis. Each of the electrophoretically separable forms catalyzed the oxidation of catechols, ferrocyanide, and ethylenic thiols, in addition to o-aminophenols.     

The multiple forms of brain acetycholinesterase. III. Implications for the histochemical demonstration of acetylcholinesterase

The multiple forms of acetylcholinesterase (AChE, E.C. 3.1.1.7) have been investigated with regard to their histochemical demonstrability. Their pattern is influenced by buffer treatment, fixation, and by incubation conditions causing aggregation and disaggregation as well as loss or inactivation of individual forms. The standard histochemical method for AChE preferentially demonstrates the high molecular forms. Most of the oligomer forms are washed out or inactivated. A selective demonstration of the highly aggregated forms is possible either by inhibition of the oligomers with diisopropylfluoridate (DFP) or by specifically dissolving them out. No reason could be found for the selective demonstration of the low molecular weight forms.     

Modification of the multiple forms of rat hepatic phenylalanine hydroxylase by in vitro phosphorylation

The two major forms of rat liver phenylalanine hydroxylase have been isolated and partially purified. The tetrahydrobiopterin-dependent activity of these forms can be differentially stimulated by exposure to enzymatic phosphorylating conditions. This $\text{in vitro}$ treatment is associated with incorporation of 32p into the enzymes and generates a further, chromatographically distinct, species. These results suggest that the multiple forms of rat liver phenylalanine hydroxylase are due to different degrees of phosphorylation.     

Induction of polyphenol oxidase in germinating wheat seeds

A 50- and 100-fold increase in the o-diphenolase activity was observed respectively in excised coleoptiles and roots of wheat seedlings after germination for 4–5 days. This increased activity was associated with the appearance of several new multiple forms of o-diphenolase on acrylamide gels. The embryo-less half-seeds dissected from seedlings, however, revealed only a three-fold increase in o-diphenolase activity, without any alteration in the pattern of multiple forms. Cycloheximide substantially inhibited the activity and appearance of multiple forms of o-diphenolase, whereas actinomycin D failed to bring about a similar response. Protein synthesis was probably necessary for the formation of new multiple forms. Unlike o-diphenolase activity which was present in all parts of the seedling, the monophenolase activity was confined to the embryo-less endosperm. A 5–7-fold increase in monophenolase activity was observed in the embryo-less half-seed dissected from the seedling. A single broad band of monophenolase developed on acrylamide gels. This persisted during the early period of seed germination without addition of new multiple forms. No inhibition of monophenolase activity was observed in seeds treated with cycloheximide or actinomycin D.     

Theory of Phyllotaxis. 2. Crystallographic Interpretation of the Interrelation between Lower and Superior Phyllotaxis Forms

Cross-opposite phyllotaxis forms are defined as superior with respect to the alternate ones and verticillate phyllotaxis forms as superior with respect to the opposite ones. Different phyllotaxis forms can be interpreted as a result of stretching of crystal-like structures of the embryo formed by dense packing of rudiments. Based on hypothetical concepts of the properties of plant rudiments and embryos, possible mechanisms of the formation of superior phyllotaxis forms from the lower ones have been analyzed. It was shown that the superior phyllotaxis forms can be considered as the results of additive summation of the lower forms. The theoretical conclusions are confirmed by the examples of polymorphic phyllotaxis in conspecific plants and by the facts of accidental splitting of superior phyllotaxis forms into the corresponding lower forms in nature and in experiment. The hexagonal-tetragonal type of phyllotaxis was theoretically predicted and found in nature. The mechanism underlying the formation of multiple forms of the helical phyllotaxis was considered.