The chloroplast-targeting domain of plastocyanin transit peptide can form a helical structure but does not have a high affinity for lipid bilayers.

Conformational properties and interactions with lipid membranes were studied for the chemically synthesized peptides PC(1-37) and PC(1-43), corresponding to the N-terminal 37 and 43 residues, respectively, of the transit peptide of the precursor to plastocyanin of Silene pratensis. PC(1-43) covers the entire chloroplast targeting domain of the transit peptide. CD spectra of PC(1-37) and PC(1-43) showed that both peptides have little ordered structure in aqueous solutions but form partially helical conformations in the presence of detergent micelles or in methanol. Vesicle disruption and direct-binding experiments revealed, however, that neither PC(1-37) nor PC(1-43) had a high affinity for lipid membranes. Since in the intact plastocyanin transit peptide the chloroplast-targeting domain is followed by a hydrophobic thylakoid-transfer domain, the plastocyanin precursor may well be transported to the chloroplast surface first with the aid of the thylakoid-transfer domain. The chloroplast-targeting domain may then form a helical structure in the lipid environments, and a chloroplast-specific motif displayed on the helical structure may be recognized by a receptor protein located at the chloroplast envelope membranes.     

PHOTOREGULATION OF PHYCOBILISOME STRUCTURE DURING COMPLEMENTARY CHROMATIC ADAPTATION IN THE MARINE CYANOPHYTE PHORMIDIUM SP. C861

Changes in the molecular structure of phycobilisomes during complementary chromatic adaptation were studied in the marine cyanophyte Phormidium sp. C86. This strain forms phycoerythrin (PE)-less phycobilisomes under red light but synthesizes PE-rich phycobilisomes under green light. Analysis of phycobiliprotein composition and electron microscopic examination of phycobilisomes in ultra-thin sections of cells and of isolated phycobilisomes were performed for cells acclimated to red and green light, respectively. The structure of phycobilisomes formed under red light conditions was typically hemidiscoidal. Phycobilisomes in cells acclimated to green light were twice as large in size as those in cells acclimated to red light. This increase in phycobilisome size was a result of the increase in the molar ratio of antenna pigment (PE and phycocyanin) to allophycocyanin, from 3.5 to 11.3. Pigment composition and fine structure of phycobilisomes formed under green light were similar to those of “nonhemidiscoidal” phycobilisomes reported in Phormidium persicinum. These results suggest that changes occur not only in the molecular species of peripheral rods but also in the structure of rods and probably of cores in relation to their connection with rods during chromatic adaptation of Phormidium sp. C86.     

ISOZYME ANALYSIS OF MATING POPULATIONS OF GONIUM PECTORALE (CHLOROPHYTA)1

Intraspecific variation among 36 strains of the freshwater alga Gonium pectorale Müller (Chlorophyceae) isolated from three geographically different locations in Tibet, Nepal, and Japan was investigated by isozyme analysis. Variation in isozyme patterns of eight enzyme systems (malate dehydrogenase, glutamate dehydrogenase, tetrazolium oxidase, lactate dehydrogenase, octanol dehydrogenase, xanthine dehydrogenase, phosphoglucomutase, and malic enzyme) of axenic and clonal cultures was revealed by polyacrylamide gel electrophoresis. Unweighted average linkage clustering, based on Jaccard's similarity coefficient, illustrated the high similarity between most strains from Nepal and all strains from the Ryukyu Islands (Japan). However, there was relatively low similarity between strains from Tibet and those from Nepal and Japan. Strains from the Ryukyu Islands (Japan) grouped into two clusters, and most Nepalese strains formed a single cluster, but Tibetan strains were heterogenous.     

An Evidence Indicating That a Weak Orange Light Absorbed by Phycobilisomes Causes Inactivation of PSII in Cells of the Red Alga Porphyridium cruentum Grown under a Weak Red Light Preferentially Exciting Chl a

Changes in the PSII fluorescence upon shift of light qualitywere studied with the red alga Porphyridium cruentum IAM R-1and supplementarily with P. cruentum ATCC 50161, the cyanophytesSynechocystis spp. PCC6714 and PCC6803 and Synechococcus sp.NIBB1071. When Porphyridium cruentum grown under a weak redlight (PSI light) preferentially absorbed by Chl a was illuminatedwith a weak orange light (PSII light) mainly absorbed by phycobilisomes(PBS), a change of PSII fluorescence at room temperature wasinduced. The ratio of Fvm (Fm— Fo) to Fm was reduced rapidlyaccompanying the increase in Fo (T^1/2 ca. 3 min). The effectsof DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinoneindicated that the fluorescence change is induced when plastoquinonepool is highly reduced. The fluorescence change after a shortPSII light illumination was reversible; it rapidly recoveredin the dark (T ^1/2 ca. 3 min). The reversibility was graduallyreduced and disappeared after 40 h under PSII light accompanyingdecrease in PSII activity per PBS down to almost 50%. Sincethe pattern of the fluorescence change resembles that observablewhen PSII is photoinactivated, PSII light probably induces thephotoinactivation of PSII, possibly reversibly at first andirreversibly after prolonged illumination. Such a rapid fluorescencechange was insignificant in Synechocystis sp. either PCC6714or PCC6803. Only a slow and small decrease in Fvm/Fm level appearedafter prolonged PSII light illumination (the reduction of PSIIactivity per PBS was around 20%). In Porphyridium, shift fromPSII light to PSI light caused a rapid and chloramphenicol-sensitiveFvm/Fm elevation during the first 10 h while the increase inPSH activity per PBS was only 10% of that before the light shift.Then, a gradual elevation followed up to the level at the steadystate under PSI light. A similar rapid increase in Fvm/Fm wasobserved with Synechocystis PCC6714, in which the synthesisof PSII is not regulated, suggesting that a rapid increase inFvm/Fm does not reflect the acceleration of the synthesis ofPSII. Results were interpreted as that (1) PSII light causesphotoinactivation of PSII. Such a photoinactivation is markedin Prophyridium cells grown under PSI light. (2) In Porphyridium,changes in the abundance of PSII upon shift of light qualityare largely attributed to the photoinactivation of this type. (Received February 19, 1999; Accepted June 14, 1999)     

An increase in apparent affinity for sucrose of mung bean sucrose synthase is caused by in vitro phosphorylation or directed mutagenesis of Ser11

A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli, in which two different acidic amino acid residues (Asp or Glu) were introduced at Ser11 (S11D, S11E). Only the wild-type enzyme (Ser11) was phosphorylated in vitro by a Ca(2+)-dependent protein kinase from soybean root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase. The apparent affinity for sucrose was increased in this phosphorylated enzyme and also in the S11D and S11E mutant enzymes, although the affinities for UDP-glucose and fructose were similar in the wild-type, phosphorylated wild-type, and mutant enzymes. These results suggest that a monoanionic (1-) side chain at position 11 mimics the Ser11-P2- residue to bind and cleave sucrose for the synthesis of UDP-glucose. Since the S11E mutant enzyme showed the lowest K(m) (sucrose) and the highest catalytic efficiency of the recombinant proteins, the enzymic properties of this S11E mutant were further characterized. The results showed that replacement of Ser11 with Glu11 modestly protected the sucrose synthesis activity against phenolic glycosides and altered the enzyme nucleotide specificity. We postulate that the introduction of negative charge at Ser11 is possibly involved in the enzymatic perturbation of sucrose synthase.     

Electron Microscopy of Measles Virus Replication

Replication of measles virus in HeLa cells was examined by electron microscopy with ultrathin sectioning and phosphotungstic acid negative staining methods. The cytoplasmic inclusion bodies consisted of masses of helical nucleocapsid which was similar in structure to the nucleocapsid found in measles virions. The cytoplasmic helical nucleocapsid appeared to align near the HeLa cell membrane, and the membrane differentiated into the internal membrane of the viral envelope and the outer layer of the short projections. The viral particles were released by a budding process involving incorporation into the viral envelope of membrane which was contiguous to but morphologically altered from the membrane of the HeLa cells. The intranuclear inclusion bodies were composed of tubular structures similar to those found in the cytoplasmic inclusion bodies. These structures aggregated to crystalline arrangement. The relationship between nuclear inclusion body and replication of measles virus was not clear.     

THE FINE STRUCTURE OF NORMAL AND NEOPLASTIC MELANOCYTES IN THE SYRIAN HAMSTER, WITH PARTICULAR REFERENCE TO CARCINOGEN-INDUCED MELANOTIC TUMORS

The dermal melanocyte system of the Syrian hamster is particularly responsive to the melanogenetic and tumor-inducing effects of 7,12-dimethylbenz(a)anthracene (DMBA). The melanocytes of the hair follicles appear to be susceptible to the melanogenetic effect of DMBA but not to its tumor-inducing effect. The epidermal melanocytes are non-pigmented and are unresponsive to both melanogenetic and carcinogenic effects of DMBA. The pigmented granules of the dermal melanocytes of both the golden and the white hamster have an identical substructure and pattern of melanization which occurs in an orderly fashion on a delicate fibrillar component. The hair melanocytes have larger pigment granules with a more complicated fibrillar substructure. The epidermal melanocytes do not possess pigment granules but are recognized by their dendritic shape, the absence of desmosomes and tonofilaments, and the presence of racket-shaped or rod-shaped organelles. The melanin granules in neoplastic melanocytes of the golden hamster differ from corresponding normal melanocytes only in their larger size. In the white hamster, however, the melanin granules in tumors produced under identical experimental conditions are so bizarre and atypical that consideration was given to the possibility that a genetic difference in the melanization pattern between the two varieties becomes apparent in carcinogen-induced melanotic tumors. No definite conclusions could be reached as to the precise origin of the melanin granules in either normal or neoplastic melanocytes.     

Purification and characterization of a chloride ion-dependent α-glucosidase from the midgut gland of Japanese scallop (Patinopecten yessoensis)

Marine glycoside hydrolases hold enormous potential due to their habitat-related characteristics such as salt tolerance, barophilicity, and cold tolerance. We purified an α-glucosidase (PYG) from the midgut gland of the Japanese scallop (Patinopecten yessoensis) and found that this enzyme has unique characteristics. The use of acarbose affinity chromatography during the purification was particularly effective, increasing the specific activity 570-fold. PYG is an interesting chloride ion-dependent enzyme. Chloride ion causes distinctive changes in its enzymatic properties, increasing its hydrolysis rate, changing the pH profile of its enzyme activity, shifting the range of its pH stability to the alkaline region, and raising its optimal temperature from 37 to 55 °C. Furthermore, chloride ion altered PYG’s substrate specificity. PYG exhibited the highest V_max/K_m value toward maltooctaose in the absence of chloride ion and toward maltotriose in the presence of chloride ion.