Solubilization of the functional C5a receptor from human polymorphonuclear leukocytes

The C5a receptor has been extracted in an active state from the membranes of human polymorphonuclear leukocytes with the detergents digitonin and beta-dodecyl maltoside. The solubilized receptor exhibits a single class of high affinity binding sites with a Kd = 90 pM, a value similar to that found with intact membranes. Physical studies with the soluble receptor demonstrate that it exists in two forms which differ in molecular mass. Gel filtration experiments with receptor to which C5a has been bound give an apparent molecular mass for the complex of 150-200 kDa. When the experiments were repeated with nonliganded receptor, most of the C5a binding activity eluted with an apparent mass of 150-200 kDa. However, the peak had a pronounced trailing shoulder indicating that, in the nonliganded state, a portion of the receptor population exists in a smaller form, which may be converted to the larger form on binding C5a. The molecular mass of the smaller form, estimated to be 30-70 kDa, is consistent with that of the binding subunit of the receptor. These data imply that the larger form, and therefore the bulk of the solubilized receptor, is oligomeric, a conclusion which is supported by cross-linking studies. When C5a was cross-linked to the soluble receptor two specific complexes with molecular masses of 52 and 95 kDa were formed. The former is the covalent adduct of C5a and the binding subunit of the receptor and the latter appears to be a complex between the 52-kDa species and an additional polypeptide.     

Characteristics of a copper-dependent cross-linking reaction between two forms of cytochrome P-450 in rabbit-liver microsomal membranes.

alpha-Lactalbumin was purified to apparent homogeneity from mouse milk by combined use of gel filtration, chromatography on DEAE-cellulose and hydroxyapatite, and concanavalin A-Sepharose affinity chromatography. Mouse alpha-lactalbumin exists in several species with different charges and in two molecular-size forms. The smaller form, which constituted over 90% of total alpha-lactalbumin, included two major and two minor species, each of which showed different electrophoretic mobility on polyacrylamide-gel electrophoresis, but gave the same single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in two different buffer systems and over the range 10-15% acrylamide concentrations. The molecular weight was estimated as 14 100. The two major species of the smaller form had the same amino acid composition and contained no significant amount of carbohydrate. The larger form of alpha-lactalbumin, consisting of two species with different charges, was present in a small amount (less than 10%) in the milk and was isolated by its ability to interact with concanavalin A-Sepharose. Each of the two species also gave the same single band of apparent mol.w.t 18 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, this value may be anomalous, since this larger form appears to be glycosylated, and glycoproteins can behave anomalously on sodium dodecyl sulphate/polyacrylamide gels by binding less sodium dodecyl sulphate. All species of mouse alpha-lactalbumin from milk were active in the lactose synthase reaction and showed identical immunological properties, as determined by the mono-specific antibody prepared against the small major species. The presence of both the larger and the smaller forms, each in a percentage concentration similar to that found in milk, was also demonstrated in alpha-lactalbumin induced by hormones in organ cultureof pregnant-mouse mammary gland.     

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.     

Phosphoglucose isomerase from Escherischia coli K10: Purification,properties and formation under aerobic and anaerobic condition

Phosphoglucose isomerase has been purified from crude extracts of Escherichia coli K10. Two forms of the enzyme were separated during the purification procedure. The major species comprises more than 90% of the enzyme activity, has an apparent molecular weight of about 125,000 and consists of two 59,000 molecular weight subunits; the minor species has an apparent size of 230,000 and consists of (possibly four) subunits of 59,000 molecular weight. Both enzyme forms have the same N-terminal amino acid, the same pH optimum of reaction and the same kinetic constants for the substrate fructose-6-phosphate and the inhibitor 6-phosphogluconate. They differ in that the minor species has half the specific enzyme activity compared to the major one and that its subunit polypeptide carries a higher electronegative charge. Since they are both coded by the pgi gene and since they show full immunological identity it seems that the minor species is a dimer of the major enzyme form and that dimerisation is caused by subunit modification. No physiological role could be found for the existence of the two forms. — Formation of phosphoglucose isomerase is under respiratory control: under anaerobiosis the enzyme (both species) is derepressed parallely with other glycolytic enzymes.Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami.

The principal RNA species isolated from labeled preparations of the arenavirus Pichinde usually include a large viral RNA species L (apparent molecular weight = 3.2 X 10(6)), and a smaller viral RNA species S (apparent molecular weight = 1.6 X 10(6)). In addition, either little or considerable quantities of 28S rRNA as well as 18S rRNA can also be obtained in virus extracts, depending on the virus stock and growth conditions used to generate virus preparations. Similar RNA species have been identified in RNA extracted from Tacaribe and Tamiami arenavirus preparations. Oligonucleotide fingerprint analyses have confirmed the host ribosomal origin of the 28S and 18S species. Such analyses have also indicated that the Pichinde viral L and S RNA species each contain unique nucleotide sequences. Viral RNA preparations isolated by conventional phenol-sodium dodecyl sulfate extraction often have much of their L and S RNA species in the form of aggregates as visualized by either electron microscopy or oligonucleotide fingerprinting of material recovered from the top of gels (run by using undenatured RNA preparations). Circular and linear RNA forms have also been seen in electron micrographs of undenatured RNA preparations, although denatured viral RNA preparations have yielded mostly linear RNA species with few RNA aggregates or circular forms.     

Multiple forms of human dopamine beta-hydroxylase in SH-SY5Y neuroblastoma cells

Dopamine beta-hydroxylase exists as three forms in human neuroblastoma (SH-SY5Y) cells. The membrane-bound form of the hydroxylase contains three different species with apparent relative molecular weights of 73,000, 77,000, and 82,000. The intracellular soluble form of dopamine beta-hydroxylase was present as a single species with an apparent molecular weight of 73,000. Pulse-chase experiments showed that membranous dopamine beta-hydroxylase contains two subunit forms of 73,000 and 77,000 after short chase times. The soluble hydroxylase was synthesized as a single species of 73,000 at approximately the same rate as the lower molecular weight species of the membranous enzyme. A constitutively secreted third form of the enzyme with an intermediate apparent molecular weight also incorporated [35S]sulfate, whereas no significant amount of [35S]sulfate was observed in the cellular forms of the enzyme. The [35S]sulfate was incorporated on N-linked oligosaccharides. Approximately 12% of the enzyme is released constitutively within 1 h. These results demonstrate that neuronal cells have the ability to constitutively secrete a specific form of dopamine beta-hydroxylase which may contribute to the levels of this enzyme found in plasma.     

Murine progesterone receptor exists predominantly as the 83-kilodalton 'A' form

Progesterone receptors (PgR) are known to exist in two molecular forms commonly designated as 'A' and 'B' forms, and the relative ratio of these two forms has been shown to vary among species. Although the rodent systems were some of the earliest experimental systems used to examine the regulation of PgR, as yet very little is known concerning the molecular composition of PgR in this species. Accordingly, to define the relative ratio of 'A' and 'B' forms in murine PgR, we have analyzed tissue extracts from normal, ovariectomized, and estradiol treated animals by photoaffinity labeling and immunoblotting techniques using a variety of anti-PgR antibodies. Under all experimental conditions, two forms of PgR with approximate molecular weights of 115 kDa ('B' form) and 83 kDa ('A' form) were found. In all tissues examined, the 83 kDa 'A' form was predominant, and this was independent of the hormonal status of the animal and different buffers used to prepare tissue extracts. In uterus the ratio of 'A' to 'B' was 3:1, in vagina it was 2:1, and in mammary glands it more closely resembled the uterus. This leads us to conclude that murine PgR exists predominantly as the 83-kDa 'A' form which may represent a general characteristic of rodent PgR. In this species there may also be some tissue specificity with regard to the absolute ratio of the two forms of PgR.     

Alpha and beta forms of cytochrome c oxidase observed in rat heart myocytes by low temperature Fourier transform infrared spectroscopy

Carbon monoxide bound to myoglobin and cytochrome c oxidase in separated adult rat heart myocytes has been observed with Fourier transform IR spectroscopy at low temperatures. CO complexes of these two proteins can be spectrally separated through temperature manipulation of the relaxation of the photolyzed systems. Photolyzed carboxymyoglobin relaxes very rapidly above 80 K, whereas the CO photolyzed from cytochrome a3 associates with CuB and relaxes very slowly below 140 K. Cytochrome c oxidase is found to be present in two major molecular forms which we designate alpha and beta. Each form contains an a3Fe and its associated CuB which we observe by their CO complexes. The predominant FeCO band, the alpha form of cytochrome oxidase, is similar to that previously seen in beef heart mitochondria, but with a slightly larger activation enthalpy, delta H = 46 kJ/mol. At least one of the beta forms is similar, but two have not been observed in beef heart mitochondria. Upon photolysis of alpha-FeCO, the alpha-CuCO species is formed. This band splits into two at low temperature. Up to half of the FeCO band area of the intact myocytes is distributed among three or more minor species (beta forms). The beta-FeCO bands all appear to be associated with only one beta-CuCO band which does not split at low temperature. After photo-dissociation of CO, the beta forms relax considerably faster than the alpha form, achieving 50% recombination in 10% of the time required for the alpha form. In a tissue slice from an opossum heart exposed to CO, we observed alpha and beta forms of cytochrome oxidase very similar to those in the rat heart myocytes. The cause of the differences between the alpha and beta forms of the enzyme is unknown, but their possible role in the control of respiration is discussed. Carboxymyoglobin contained within intact rat heart myocytes was very similar to sperm whale carboxymyoglobin, but with a much smaller amount of the lower frequency minor component.     

Study on a Species of Thinnfeldia from Liuzhi of Guizhou Province,with Remarks on Thinnfeldia Ettingshausen in China

With a careful study of the external forms and the cuticles of the fossil leaves, a species of Thinafeldia, which was collected from Langdai, Liuzhi County of Guizhou Province, has been identified as T. rhomboidalis Ett. Compare with two other species T. alethopteroides Sze and T. laxa Sze found in Yanchang Formation, Northern Shaanxi, which showed very similar external form (no cuticle structure) of T. rhomboidalis, therefore it is appropriate to clarify them as T. rhomboidalis. As for the genus Thinnfeldia, based on the study of Chinese material, the author set forth her opinion that Thianfeldia should be a separate genus instead of merging into the genus of Pachypteris as has been clarified by many palaeobotanists.     

Eria mêdogensis,a Probably Peloric Form of Eria Coronaria,with a Discussion on Peloria in Orchidaceae

Eria mêdogensis S. C. Chen et Tsi was recently found in southeastern Tibet, several specimens of which have been collected by various botanists since 1980. This is a “normal” entity with its habit very similar to that of Eria coronaria, from which it differs by having a regular perianth and longer bracts. We think it probable that this new entity is a peloric form of Eria coronaria. Peloria (or pelory) is a type of floral abnormality, which is found in many zygomorphicflowered taxa. It was first detected by Linnaeus (1744) in Linaria vulgaris, and then by others in Labiatae, Orchidaceae, etc. However, it is still an open question how to explain it theoretically and how to treat it taxonomically. In Orchidaceae, so far as our knowledge is concerned, peloria has been encountered in no less than 21 genera. In most cases, peloric flowers are found sporadically on an occassional plant, as seen in Cypripedium reginae and Eria oblitterata. Sometimes, however, peloric form may occur coexisting with normal-flowered form in one and the same species, as seen in Dendrobium tetrodon and Epipogium roseum. They are both abnormally peloric forms. It would not result in naming or renaming a plant taxonomically, whether the appearance of abnormally regular flowers on a normal-flowered inflorescence, or of abnormal-flowered individuals in normal-flowered species. In Phragmipedium lindenii, however, the case is different. It is quite “normal” and even of wider distribution than its nonpeloric allies P. wallisii and P. caudatum, from which it has once been considered to be derived. This is a normally peloric form. Whether it is a reversal or not, the appearance of a “normally” peloric taxon may be taken for a leap in the process of evolution. Taxonomically, we had better treat it as a separate species, especially when its origin is uncertain. For example, the entity just mentioned had been treated as a peloric va riety of Phragmipedium caudatum (var. lindenii) until 1975, when Dressler & Williams recognized it as an independent species based on the fact that its nonpeloric flowers occassionally found in a peloric population in Jungurahua of Ecuador are dissimilar in lip to those in P. caudatum. Garay (1979) considered it to be a peloric form of P. wallisii but maintained it at the specific level. This is indeed a good example of taxonomic treatment of normally peloric form. On the other hand, however, most of the regular-flowered entities in Orchidaceae are not peloric but rather primitive forms, such as Neuwiedia, Apostasia and Thelymitra, of which no less than 50 species have been reported since the eighteen century. They have never been regarded as peloric forms. Unfortunately, this has been neglected by some botanists. For instance, a hypothetically primitive orchid flower designed by Pijl & Dodson (1966) has a distinctly specialized lip with a short spur. In fact, in addition to the aforementioned genera we have some more examples of normally regular-flowered orchids. Among them Archineottia is the most interesting. This is a genus of four species, two of which are regular-flowered. Of special interest is that in this genus and its ally, Neottia, one can find all steps of column evolution from a simple form with stamen and style not fully united to a most complicated form in which they have well fused. Archineottia has a very primitive column, on which neither rostellum nor clinandrium is found but a terminal and undifferentiated stigma (Fig.2: 2, 4, 6, 8). In addition, there exists on the back of the column a thick ridge with its upper end joining the filament with which it is of same texture. It is obviously the lower part of the filament which has been adnate to the style (column). In Neottia, however, the column is much more advanced and very typical among the family. It has a very large rostellum and most complicated stigma structure (Fig. 10, 12, 14, 16, 18). One of the most interesting examples is Neottia acuminata, in which the stigma even becomes lamellate and almost backwards clasps the erect rostellum, but the perianth is more or less regular with its lip entire and somewhat similar to, but shorter and wider than, the petals. In these two genera there are altogether three species, namely Archineottia gaudissartii, A microglottis and Neottia acuminata, possessing regular or nearly regular perianth (Fig. 2: 1, 3, 17). They are obviously not peloric forms. We can not imagine, indeed, that a complicated form like Neottia acuminata or its allies would degenerate step by step into a simple form, and finally into a peloric form. Archineottia belongs to the subtribe Listerinae, which is closely related to Limodorinae, a rather primitivs subtribe with some genera possessing single pollen grain, relatively few and long chromosomes and monocotyledonous habit. Apparently, there is nothing surprising in the occurrence of some normally regular-flowered taxa, such as Archineottia, Diplandrorchis, Tangtsinia and Sinorchis, in these two primitive subtribes. Another instance is Aceratorchis, a genus formerly included in Orchis, from which it is distinguished by the entire lip which is more or less similar to the petals. Strictly speaking, however, its flowers are not truly regular. Two species have been described in this genus, but they were recently considered as conspecific. Aceratorchis tschiliensis is widely distributed from Hebei through Qinghai and Sichuan to northwestern Yunnan. It is cross-pollinated and produces seeds efficiently. All these indicate its normally primitive taxon, instead of peloria. It may be noted here that Asia is rich in members of Orchidioideae, as well as its primitive representatives. The occurrence of a normally regular-flowered form in Asia, whether representing primitive form of Orchis or Orchidioideae, is imaginable. In Orchidaceae, as mentioned above, regular flowers are not only found in some primitive taxa and peloric forms, but also in a few advanced groups. For example, a close investigation by the senior author (Chen 1979) on Satyrium ciliatum revealed that this species has hermaphrodite, staminate and pistallate forms, for which no less than nine names have been published. The flowers of its pistallate form are almost regular, in which nothing is found but three similar petals and an elongate style with three stigmatic lobes at its top (Fig. 2: 19). It is interesting to note that floral reversions in Orchidaceae are not always in connection with peloria. For example, Epidendrum triandrum of North America represents another kind of reversion. It is a reversal to abnormal polymery of stamens and not to abnormal regularity of perianth. Like Phragmipedium lindenii, it is also hereditary. We may give it a new name “Polyandrism” or something else, but, in fact, there is no essential distinction of this kind of reversion from peloria. It deserves mentioning that most of the regular-flowered entities, including primitive, advanced and peloric ones, occur in Asia and Australasia, where the Orchidaceae may have originated as pointed out by some botanists. We have good reason to verify the primitiveness and normality of many regular-flowered entities, but there exists no sufficient evidence for the impossible existances of normally regular-flowered species in those like Dendrobium, Eria, Lecanorchis, etc. For instance, Lecanorchis javanica, Dendrobium atavus and the new species described here are considered to be peloric forms, but it is only a conjecture, for no reason can be given for it. It is not impossible that some so-called peloric forms may prove to be truly primitive ones in the future. Of course, a closer investigation is needed. Summarizing the above, we may come to the following conclusions: 1. Regular or nearly regular perianth is a normal characteristic of orchids. It is chiefly found in some primitive taxa and sometimes also in certain peloric forms and advanced groups. Regular-flowered entities may not necessarily be peloric forms. 2. There exist two different types of peloria in Orchidaceae. One is abnormal form, with its peloric flowers appearing at random. The other is “normal” form, with its individuals all possessing peloric flowers. The latter is inheritable and can produce seeds efficiently, It would be best to treat it as an independent species taxonomically, especially when its origin is uncertain. 3. Although peloria has been considered to he a reversal as a whole, conditions vary from plant to plant. Some peloric forms have petal-like lip, and others have labellum-like petals. Sometimes the same plant produces different kinds of peloric flowers in different years, sometimes peloric flowers do not reappear upon the same plant. A few species can produce both peloric and normal individuals, but others produce peloric forms only. Peloria is in fact a term only used to cover the phase in which lip becomes similar to the petals. It is never all-embracing. We recognize the existance of peloria in Orchidaceae, but great care must be taken to distinguish truly peloric form from normally primitive one. It must be admitted that what causes peloria and even what is peloria are still problems awaiting solution. Acknowledgments: Our heartfelt thanks are due to Dr. Leslie A. Garay, Curator of the Orchid Herbarium of Oakes Ames, Botanical Museum of Harvard University, for his valuable suggestions during the preparation of this paper. We are also indebted to the artists, Mrs. Chunrung Liu and Mr. Chao-zhen Ji of our department, for their preparing the fine drawings.     

Effect of tunicamycin on the synthesis, processing, and secretion of pro-opiomelanocortin peptides in mouse pituitary cells

Pro-opiomelanocortin (POMC) is glycosylated and proteolytically cleaved to produce a number of smaller peptide hormones including adrenocorticotropic hormone (ACTH) and endorphin in mammalian pituitary and the mouse pituitary cell line AtT-20/D16v. When glycosylation of POMC is inhibited in AtT-20 cells with the drug tunicamycin, a 26,000-dalton protein appears in place of the glycosylated 29,000- and 32,000-dalton forms of POMC. The 26,000-dalton form found in tunicamycin-treated cells has the same [35S]methionine tryptic peptides as 29,000- and 32,000-dalton POMC, indicating that the decrease in apparent mass is most likely due to loss of carbohydrate and not to changes in the peptide backbone. The 4,500-dalton form of alpha(1-39)ACTH and the 3,000- and 11,000-dalton forms of endorphin are all present in tunicamycin-treated cells. The glycosylated form of alpha(1-39)ACTH, however, is missing and the glycosylated ACTH intermediates are replaced by unglycosylated ACTH intermediates. Pulse-chase studies demonstrate that the 26,000-dalton unglycosylated POMC is the precursor of the smaller ACTH and endorphin molecules in tunicamycin-treated cells. Furthermore, all of the forms of ACTH and endorphin found in tunicamycin-treated cells are secreted. Thus, it appears that glycosylation is not an essential step for correct cleavage or secretion of POMC or its products.     

Multiple forms of carboxypeptidase Y from Saccharomyces cerevisiae. Kinetic demonstration of effects of carbohydrate residues on the catalytic mechanism of a glycoenzyme.

In the course of our further investigation of the active site titration of carboxypeptidase Y, using 4-nitrophenyl trimethylacetate, we have found that carboxypeptidase Y can be isolated in different molecular forms. Carboxypeptidase Y obtained from Fleischmann baker's yeast has a molecular weight of 53,000, as compared to 64,000 for an enzyme species isolated from Anheuser-Busch baker's yeast. The amino acid analyses of both enzymes were essentially identical and very similar to those reported by others. However, we have found that the molecular weight difference is due to a variation in carbohydrate content as determined by gas chromatography. When carboxypeptidase Y was isolated from a single source, Anheuser-Busch baker's yeast, we observed a smaller variation in carbohydrate content. In all cases, sugar analyses revealed only mannose and N-acetylglucosamine to be present. The effect of the enzyme's carbohydrate content on the "burst kinetics" of the 4-nitrophenyl trimethylacetate reaction has been examined. In general, the Anheuser-Busch enzyme, containing more carbohydrate than the Fleischmann enzyme, reacts with a larger apparent bimolecular rate constant, kcat/Km. On the other hand, the deacylation rate constant, k3, is affected only slightly.     

The oxidative response of rabbit peritoneal neutrophils to leishmanias and other trypanosomatids

Luminometry has been used to measure the respiratory burst of rabbit peritoneal neutrophils that is elicited by different forms and species of Leishmania and Herpetomonas. Mid-log phase and metacyclic promastigotes of L. major evoked large responses; that due to metacyclics was lower and slower, but they also bound in smaller numbers than mid-log phase cells. Promastigotes of L. mexicana mexicana also stimulated a large respiratory burst whereas amastigotes elicited little or none. Leishmania donovani promastigotes and culture forms of H. muscarum muscarum and H. m. ingenoplastis all evoked large responses by neutrophils. There was, however, very little response to L. mexicana mexicana promastigotes, L. donovani promastigotes or H. muscarum muscarum when they were added in large numbers. This 'inhibition' was not apparent with L. major.     

Divalent cations and the molecular state of brain acetylcholinesterase (E.C. 3.1.1.7.)

Freshly solubilized (by EDTA treatment) mammalian brain acetylcholinesterase appears as two molecular weight species of approximately 80,000 and 330,000. With time, the smaller form converts to an intermediate 230,000 apparent molecular weight species. This conversion has been shown to be prevented by low concentrations of certain divalent cations, but not influenced by other ions which also can interact with postulated peripheral anionic binding sites on acetylcholinesterase.     

Beef muscle troponin: evidence for multiple forms of troponin-T.

A method is described for the purification of troponin from beef skeletal muscle. The resultant preparation differs from the troponin of rabbit skeletal muscle in that it contains at least two forms of the tropomyosin-binding component, Troponin-T: these are designated as the 37 000 and 40 000 dalton forms of Troponin-T on the basis of sodium dodecyl sulphate gel electrophoresis. Either of these Troponin-T forms may be used to reconstitute troponin by mixing with the appropriate amounts of the calcium-binding (Troponin-C) and and actomyosin ATPase-inhibitory (Troponin-I) components. These reconstituted troponins are shown to interact with tropomyosin and also to confer full calcium sensitivity on actomyosin ATPase. Despite the existence of proteolysis in troponin preparations, the experimental evidence indicates that the smaller form of Troponin-T is not derived from the 40 000 dalton species by limited degradation. Although both species of Troponin-T have been found routinely in troponin from beef skeletal muscle, only the larger form is detected in troponin preparations from beef cardiac muscle. Further studies are required in order to clarify the functional significance and differential distribution of these multiple forms of Troponin-T.     

Cellular distribution of type I and type II receptors for transforming growth factor-beta

Affinity labeling of target cells for transforming growth factor-beta (TGF beta) by cross-linking with 125I-TGF beta via disuccinimidyl suberate or by the photoreactive analogue 4-azidobenzoyl-125I-TGF beta has revealed the presence of multiple TGF beta receptor forms. Two distinct types of TGF beta receptors can be distinguished based on structural analysis of the 125I-TGF beta-labeled species by peptide mapping. Type I TGF beta receptors include the 280-kilodalton labeled receptor form previously found to be the subunit of a disulfide-linked TGF beta receptor complex. (Massagué, J. (1985) J. Biol. Chem. 260, 7059-7066), as well as a 65-kDa labeled receptor form present in all cell lines examined, and a 130-140-kDa labeled receptor form detected only in 3T3-L1 cells. The 280-kDa form is the major TGF beta receptor species in most cell lines examined, but is apparently absent in rat skeletal muscle myoblasts. Type I TGF beta receptors bind TGF beta with an apparent Kd of 50-500 pM. Type II TGF beta receptors include an 85-kDa labeled receptor form present in all mammalian cells examined and a 110-kDa labeled receptor form present in chick embryo fibroblasts. Type II TGF beta receptors bind TGF beta with an apparent Kd of about 50 pM. Except for the 280-kDa type I TGF beta receptor form, none of the TGF beta receptor forms described here is found as part of a disulfide-linked receptor complex. All the TGF beta receptor forms described here behave as intrinsic membrane proteins exposed on the surface of intact cells.     

Mitochondrial DNA genetic differentiation of the muksun Coregonus muksun (Pallas) and related Siberian species of Coregonus (Coredonidae, Salmoniformes)

Restriction enzyme analysis of the mitochondrial DNA (mtDNA) fragment encoding subunit 1 of the NADH dehydrogenase complex (ND-1) amplified via polymerase chain reaction (PCR) has been used to obtain data on genetic differentiation of muksun Coregonus muksun (Pallas) populations. Population polymorphism with respect to the restriction sites of 18 endonucleases has been described. It has been demonstrated that the muksun is genetically related to the pidschian C. pidschian (Gmelin), its sympatric species in Siberian waters. Analysis of the median network of mtDNA haplotypes has shown that haplotypes of muksun from various Siberian basins form a common group with haplotypes of pidschian of the Arctic Ocean basin, some frequent haplotypes been found in both forms. This raises the question as to the validity of the muksun as a species. Differences within this group of haplotypes are much smaller than those typical of species of the genus Coregonus. The possibility of a hybrid origin of the muksun from a pidschian-like ancestor and species of the cisco-peled (C. sardinella-C. peled) complex is discussed.     

Polymorphic segregation in Arctic charr Salvelinus alpinus (L.) from Vatnshlidarvatn, a shallow Icelandic lake

We studied the salmonid fish Arctic charr, Salvelinus alpinus , in a small and shallow landlocked lake in NW Iceland. The lake is productive but die only fish present is Arctic charr. Despite the apparent absence of discrete benthic and limnetic habitats for fish, two forms of Arctic charr are found in the lake. They show subde differences in morphology related to swimming performance and manoeuvrability, but differences in life history such as growth, and age and size at sexual maturation are more pronounced. Both forms have benthic feeding habits with one form consuming greater number of species than the other. We suggest that the segregation of these forms is based on the evolution of a specialist from a local generalist and that this has been made possible by the absence of a common fish competitor in similar lakes, the threespined stickleback Gasterosteous aculeatus.     

Expression and intracellular localization of Pyk2 in normal and v-src transformed chicken epiphyseal chondrocytes

The expression and localization of prolin-rich tyrosine kinase 2 (Pyk2) were studied in chick embryo epiphyseal chondrocytes. Two immunoreactive bands were detected in chondrocytes, a major band with an apparent Mr of 123 kDa and a minor band with an apparent Mr of 68 kDa. The major band appears to migrate as a doublet with apparent Mr of 116/123 kDa. Increased levels of the three forms of Pyk2 were observed in v-src transformed chondrocytes as compared to control uninfected chondrocytes. Immunofluorescent staining shows that Pyk2 is clearly visible in the cytosol and in the perinuclear region of control and v-src-chondrocytes and displays a pattern very similar to the distribution of the mitochondrial marker Mito Tracker. More, immunofluorescent staining shows that Pyk2 is nuclear in most chondrocytes. By subcellular fractionation, the p116/123 Pyk2 doublet, was found to be accumulated mainly in the cytoplasm while the p68 Pyk2 form, was found to be accumulated exclusively in the nucleus. The differential nuclear/cytoplasmic distribution of the Pyk2 forms remains unchanged after v-Src-induced transformation. The p68 Pyk2 form could no longer be detected by using a N-terminus domain-specific anti-Pyk2 antibody. Consistently, Pyk2 immunoreactivity was restricted to the cytoplasm of control and v-src transformed chondrocytes. Thus it appears that the p68 Pyk2 form that accumulates in the nucleus has a deletion in the N-terminus region.     

The purification and properties of hen oviduct form B DNA-dependent RNA polymerase

Hen oviduct form B DNA-dependent RNA polymerase has been extensively purified and its properties analysed. It seems likely to consist of a mixture of two forms of the type observed in tissues from other species. Furthermore using S1 nuclease to digest single-stranded DNA, we show that although form B can transcribe double-stranded DNA template it has a very strong preference for single-stranded regions. Also the rate of elongation on native DNA in vitro was measured and is an order of magnitude slower than that reported to be operative in vivo.